摘要
从患溃疡病的养殖刺参(Apostichopus japonicus)病灶处分离出1株优势菌H1,以浸浴、创伤浸浴、体腔注射和体壁肌肉注射等方式进行感染实验,证实菌株H1为养殖刺参溃疡病病原菌,并证明该菌通过体表创伤侵入的方式感染刺参,以创伤浸浴和体壁肌肉注射感染的LD50(半数致死量)分别为2.26×107CFU/尾和1.80×107CFU/尾。经形态学观察、生理生化特性分析和mini API系统鉴定,确定菌株H1为杀鲑气单胞菌杀日本鲑亚种(Aeromonas salmonicida ma-soucida)。提取菌株H1的胞外产物(ECP)进行致病性实验,结果表明ECP可导致刺参死亡,其对刺参的LD50为5.24μg蛋白/g体质量。H1-ECP具有酪蛋白酶、明胶蛋白酶、几丁质酶和淀粉酶活性,并具有溶血素活性;对底物偶氮酪蛋白(Azocasin)作用的酶比活力可达到674.5活力单位/mg蛋白,最适作用温度为50℃;对热不稳定,70℃作用30 min时,酪蛋白酶活性降到0;100℃作用30 min,ECP对刺参的毒性消失;ECP酶活可被10 mmol/L EDTA完全抑制,可被5 mmol/L PMSF抑制98.8%,Ca2+和Mg2+可使酶活性分别提高约9%和4%。结论认为,该病原菌通过体表创伤侵入方式感染宿主刺参,菌株H1胞外产物是其对刺参致病的因子之一。
Bacterial strain H1,which was isolated from the diseased cultured sea cucumber Apostichopus japonicus with skin ulceration,was proved to be a pathogen by soaking with bacteria suspension,soaking with bacteria suspension after the sea cucumbers were wounded,coelom injection and muscle injection with bacteria suspension.The LD50 doses of soaking with suspension of bacteria after wound and muscle injection were 2.26×107 CFU/ind and 1.80×107 CFU/ind By methods of morphological observation,physiological and biochemical characteristic analysis and mini API systematic identification,strain H1 was identified as Aeromonas salmonicida masoucida.The lethality of the extracellulose products(ECP) of strain H1 was detected by injecting ECP to A.japonicus,the LD50 dose was 5.24 μg(protein)/g body weight.The ECP exhibited casinase,gelatinase,chitinase,amylase and haemolytic activities.The protease specific activity of ECP to azocasin was 674.5 U/mg protein when at the optimum temperature 50 ℃.The ECP was instable to heat and was inactivated at 70 ℃ and the toxicity to the sea cucumber disappeared after incubation at 100 ℃ for 30 min.The enzymatic activity could be inhibited by 10 mmol/L EDTA and 5 mmol/L PMSF,and Ca2+ and Mg2+ could increase enzymatic activity by 9% and 4%,respectively.
出处
《中国水产科学》
CAS
CSCD
北大核心
2007年第6期981-989,共9页
Journal of Fishery Sciences of China
基金
国家高技术研究发展计划项目(863)(2006AA100306)
国家自然科学基金(30271016)
中国海洋大学资助项目(1400-813764)
教育部海水养殖重点实验室开放基金(200212)
关键词
刺参
溃疡病
杀鲑气单胞菌
胞外产物
蛋白酶
Apostichepus japonicus
skin ulceration
Aeromonas salmonicida masoucida
extracellulose products(ECP)
protease
