Studies On The Pathogenicity And Outer Membrane Proteins Of Main Pathogenic Vibrios Of Marine Fishes

Culture of marine fish progresses to the level of high density and intensity, accompanied by a prominence of disease problem. Some strains of Vibrio species are the most serious pathogens, which can infect a wide range of fish species and cause mass death. The present study is placed upon the following aspects:Two pathogenic Vibrio anguillarum strains W-1, TL01 were originally isolated from diseased seabass (Lateolabrax japonicus) and diseased turbot (Scophthalmus maximus) in Qingdao of Shandong province, China. Pathogenicity of V.anguillarum W-l, TL01, and a pathogenic Vibrio harveyi SF-1 to turbot ((Scophthalmus maximus) and flunder (Paralichthys olivaceus) are studied by intraperitoneal injection. Vibrio anguillarum W-l and TL01 are highly pathogenic to turbot and flunder. LDso of V.anguillarum W-l to flunder and turbot are 2.06 104cfu/g and3.04 103cfu/g fish respectively; LD50 of TL01 were5.09 104cfu/g and 2.32 101cfu/g fish respectively; LD50 of Vibrio harveryi to flunder and turbot are 9.65 103cfu/g 8.63 102cfu/g fish respectively.Serological analysis show that antisera of W-l and TL01 both react with serotype O2 type strain VIB2, but not with other serotype type strains, W-l and TL01 have good cross-reaction with each other, which suggest that they both belong to serotype O2 strain.Outer membrane proteins (OMP) of 13 Vibrio type strains, 12 strains of Vibrio anguillarum, 9 Vibrio harveyi and 4 Vibrio parahaemolyticus bacteria are extracted and analyzed by SDS-PAGE.The OMP profiles of 13 Vibrio type strains are heterogeneous, four to fivemain OMPs were found, with the molecular weight ranged between 25kD and 70kD.OMP profiles of 10 serotype strains of V. anguillarum are different from each other, each strain had two to six dominant proteins of 25-70kD. One to two common protein was found among the 10 V. anguillarum strains, and of them one is 40kD in most strains. W-l and TL01 have different OMP profiles from each other, but both of them had the 40kD-55kD common proteins.The main OMPs profiles of Vibrio harveyi strains are different from each other, Each strain had three to six main proteins, and the molecular weights range from 30kD to 48kD, No common OMP is found among these strains.Vibrio parahaemolyticus have the similar OMP patterns. Each strain have three to five proteins. The molecular weights ranged from 30kD to 50kD.A 35kD protein is produced by W-land TL01 when cultured in CM9 at 37 癈. Gel filtration with Sephadex-G10(K Sephacryl S-10CK DEAE-Sepharose Fast Flow N DEAE-cellulose are used to purify the main OMP of V.anguillarum W-l but no method is efficient. The induced 35kD protein of W-l is separated by SDS-PAGE.Turbot fries were vaccinated with partly purified OMPs of V.anguillarum W-l via intraperitoneal injection. A dosage of 0.2 ml of V.anguiUarum W-l (3.7 l08cfu/ml) was intraperitoneally injected to the vaccinated fish and control group 4 weeks later. The results show that specific immune reaction can be stimulated by the proteins. Specific antibody could be detected after injection immunization and the relative percentage survival (RPS) was 45.45%.