中华绒螯蟹腹水病病原分析

Analysis on pathogen producing ascitic fluid disease of Eriocher sinensis

从六安地区患腹水病的中华绒螯蟹(Eriochersinensis)体内分离到一株病原菌(HX4菌株),经API/ATB半自动化鉴定和分子遗传学鉴定,确定为拟态弧菌(V mimicus)。应用PCR技术、电镜技术、组织细胞模型和动物实验对该菌的毒素共调菌毛(ToxinCoregulatedPilus,TCP)、外毒素和内毒素等毒力因子研究的结果表明,HX4菌株为TCP+株,在pH6 7定居因子培养基中,30℃需氧培养条件下可较好表达TCP;TCP+菌对肠管的黏附率为(32 19±0 14)%、对HEp 2细胞的黏附菌数为16 03±4 66、对中华绒螯蟹的毒力(LD50)为2 08×106CFU·mL-1,均显著高于TCP-菌,其相应值分别为(0 52±0 06)%、6 8±2 06和2 09×107CFU·mL-1;外毒素具有很强的细胞毒性(TCID500 25μg)、肠毒性(皮肤蓝斑直径15mm)和中华绒螯蟹致死性(LD501 40μg),而内毒素对中华绒螯蟹基本无致病性。由此可见,TCP和外毒素是拟态弧菌HX4菌株的2种重要毒力因子,在细菌致病过程中起重要作用。

A strain of pathogenic bacteria (HX4) was isolated from Eriocher sinensis with ascitic fluid disease in the area of Liuan,Anhui Province and was classified as V.mimicus by API/ATB semiautomated idetification and molecular genetic idetification.The Toxin Coregulated Pilus (TCP) ,exotoxin and endotoxin of HX4 strain were studied by PCR technigue,electropherogram technology ,tissue or cells model and animal experiments.The results showed that HX4 strain was a TCP^(+) strain,which could express TCP very well in pH 6.5 CFA medium at 28℃ in aerobic cultivation.The intestinal adhesive rate (32.16%) ,adhesive bacterial numbers to HEp-2 cells(16.04±4.66) and virulence to E.sinensis (LD_(50) 2.08×10^(6) CFU·mL^(-1 )) of TCP^(+) strain were all markedly higher than those (0.44%,6.8±2.06 and 2.09×10^(7) CFU·mL^(-1) respectively) of TCP^(- )strain.The exotoxin produced by HX4 strain had strong cytotoxicity (TCID_(50) 0.25μg),enterotoxicity(diameter of skin blue spot 15mm)and lethality to E.sinensis(LD_(50) 1.40μg),but the endotoxin had little pathogenicity to E.sinensis.From above results,it was concluded that TCP and exotoxin were both important virulent factors of HX4 strain,which played a key role in pathogenic process.