Pathogenicity Research Of The Pathogen Associated With Ulceration In Cultured Sea Cucumber (Apostichopus Japonicus)

Sea cucumber(Apostichopus japonicus), which is excellent for health care and pharmacy, has been widely cultured in Shandong and Liaoning province of China in late 1990s and has become the new cultured animal that promotes the aquaculture in the North China. With the enlarging of the sea cucumber culture, diseases are increasingly serious. The ulceration disease broke out since 2003 was the most serious disease and has highly restricted the development of sea cucumber culture industry. Therefore, it is imperative to research the epidemiology and pathogenic mechanism of sea cucumber ulceration disease.In present research, two predominant bacteria strains named H1 and RH2, which were isolated from the ulcer of sea cucumber, were proved to be pathogens by virulence tests, comparing with strain RH2, strain H1 was the pathogen with high virulence, and these two strains were identified subsequently. The extracellular products (ECP) of stain H1 was prepared and the virulence, enzyme activity, protease specific activity, protein structure and immunogenicity of ECP were measured. Then a serine protease, which was considerd as the virulence factor, was purified via gel filtration chromatography and ion exchange chromatography, and the characteristic of it was analyzed later. The followings are the details.An ulceration disease causing high mortality among sea cucumber reared in Rushan, Shandong province, China broke out in March, 2002. Gross signs of diseased sea cucumber included anorexia, excessive mucus, gut disgorging, serious ulcer on the body surface and at last died with spreading of ulcer and autolyzed. Bacterial strains H1 and RH2, which were isolated from the cultured sea cucumber with skin ulceration, were proved to be pathogen by soaking with suspension of bacteria, soaking with suspension of bacteria after wound, coelom injection and muscle injection. The strain H1 can infect sea cucumber by the method of soaking with suspension of bacteria after wound and muscle injection, the LD50 does was 1.4×106CFU/per sea cucumber, while the strain RH2 can cause the sea cucumber dead only with the method of uscle injection with a LD50 of 5.7×106CFU/per sea cucumber.The strains H1 and RH2 were identified as Aeromonas salmonicida ssp. Masoucida and Vibrio alginolyticus by method of physiological and biochemical characteristic analysis, respectively. 16S rRNA gene and 60 KD heat shock protein (HSP60) gene sequence detection further proved that strain RH2 belonged to V. alginolyticus. Phylogenetic tree of Vibrio based on 16S rRNA gene and HSP60 gene both indicated that strain RH2 showed the highest level of similarity to V. alginolyticus, and it's bootstrap was 50% and 99%, respectively.The extracellular products (ECP), which was extracted from strain H1 using the cellophane plate technique, was virulent to sea cucumber, and the LD50 was 5.24μg /g weight. The ECP exhibited casinase, gelatinase, chitinase, amylase and haemolytic activity, its protease specific activity to azocasin was 470.6 Unit/mg protein at the 25℃, and the optimum temperature for ECP was 50℃. The ECP was instable to heat, it was inactivated at 70℃. The ECP was analyzed by SDS-PAGE, thirteen proteins were observed, containing nine major proteins. Seven proteins of the ECP were considered to have immunogenicity and could activate the immunoreaction of the hosts by the method of Western-blot with rats antiserum against ECP.An extracellular protease of 42 kDa was purified from the ECP via Sephadex G-100 gel filtration chromatography and DEAE Sepharose ion exchange chromatography. Stepwise virulence test to sea cucumbers indicated that the 42 kDa protease was the virulence factor of stain H1, the LD50 to sea cucumbers was 1.12μg/g, which was 4.7-fold to the ECP's. The protease was purified 21.9-fold with a total yield of 1.7%. The optimum pH and temperature for the protease were 8.0 and 40℃, and it was completely inactivated over 70℃. The protease was concluded to be a serine protease for it could be inhibited strongly by 5mM Phenylmethanesulfonyl fluoride (PMSF), a common inhibitor of serine protease. But Ca2+ and Mg2+ could increase enzymatic activity by 9% and 4%, respectively. The N-terminal 15 amino acid sequence of purified serine protease was sequenced and showed the highest level of similarity to extracellular serine protease when blast on the NCBI. 2-D electrophoresis showed that the 42 kDa protease consisted of 2 different proteins, whose isoelectric point (pI) inclines to alkalescence.