CHARACTERISTICS OF THE EXTRACELLAR PRODUCTS OF TWO PATHOGENIC VIBRIO HARVEYI STRAINS

Vibrio harveyi is a kind of important pathogenic bacteria for seawater animals, such as prawn, crab, clam, and ormer. Along with the development of seawater fish cage-culture in China, Vibrio harveyi also becomes a main cause of disease of cage-culture fish, including Lutianus erythopterus, Seriola dumerili, Lateoabrax japonicus, Epinephlus fario and Epinephelus coioides. Some researches indicate that Vibrio harveyi can secrete extracellular products and result in the damnification of host tissue or result in the death of host when seriously. Two Vibrio harveyi strains, EcGY020401 and SpGY020601, which were isolated from diseased Epinephelus coioides and Lutianus erythopterus in south China, were identified after morphological observation, biochemical characteristic analysis, and 16srRNA or HSP60 gene sequence detection. Both strains can cause death to freshwater fish and marine fish, such as crucian carp ( Carassius auratus) and estuary cod( Epinephelus coioides) through artificial injection. In order to understand their patho-genetic mechanism, extracellular products(ECPs) of both strains were extracted with ammonium sulfate precipitation, and some characteristics of their ECPs such as exoenzymatic, hemolytic, toxic and cytotoxic activities were analyzed. Virulence experiments showed that ECPs of the two strains had quite strong virulence to crucian carp in that their LD50 to crucian carp was about 7.1μg-protein/ g fish weight . Both strains’ECPs have the activity of amylase, protease and lipase, but have not the activity of gelatinase and urease. Except that the ECP of strain EcGY020401 has stronger haemolytic activity to the red cell of grouper, the ECPs have very weak hemolysis to erythrocytes of human, mouse, estuary cod and crucian carp. Cytotoxic activity to the fish cell line PSF was detected in both ECPs, and the lowest cytotoxic concentration to PSF is 2.0μg/ mL and 4.0μg/ mL, respectively. A main protease in ECPs produced by strain SpGY020601 was also purified following the steps of ammonium sulfate precipitation, gel filtration and anion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( SDS-PAGE) showed the estimated molecular mass of the protease is about 38KDa. The protease is lethal to crucian carp and the LD50 is 3.3μg-protein/ g. Phenylmethanesulfonyl fluoride (PMSF) and Ethylene diamine tetraacetic acid (EDTA) had a little inhibitory effects on the activity of the protease, while Ca2+ 、Zn2+ 、Mg2+ can enhance the protease’activity. These results indicated that the protease might be an alkaline cysteine protease.