3株嗜水气单胞菌弱毒株的毒力相关特性分析

Virulence-associated characterization analysis of three strains of Aeromonas hydrophila

对健康鲫鱼和水体环境中分离的3株嗜水气单胞菌NJ-4、NJ-13和CS-13的毒力相关特性进行分析。采用PCR技术检测该菌5种主要毒力基因:气溶素(aer)、细胞毒性肠毒素(act)、细胞兴奋性肠毒素(alt)、温敏胞外蛋白酶(eprCAI)和丝氨酸蛋白酶(ahp),并进行溶血性、溶蛋白性、细胞毒性、细胞黏附特性和主要外膜蛋白(MOMP)图谱分析,以及斑马鱼致病性试验。结果显示:NJ-4不含上述5种毒力基因,NJ-13只含有ahp基因,CS-13仅具备aer基因。3个菌株培养上清均不溶血、不溶蛋白;NJ-4和CS-13培养上清不能致细胞病变,NJ-13上清对细胞的毒力效价达1∶128;而强毒株BSK-10培养上清的溶血价、溶蛋白效价和细胞毒力效价分别为1∶128、1∶256和1∶256。NJ-4、NJ13和CS-13对HEp-2细胞有一定的黏附能力,但与强毒株BSK-10相比,黏附能力较弱。NJ-4、NJ-13和CS-13的培养液上清均不致斑马鱼死亡,菌体有一定致病作用,对斑马鱼的LD50超过108 CFU/mL;而BSK-10培养上清和菌体致病性均很强,菌体对斑马鱼的LD50小于105 CFU/mL。NJ-4、CS-13的MOMP条带与BSK-10相似度很高,而与NJ-13有一定差别,可能与菌株的来源有一定关系。结果表明,3株细菌均属弱毒株,特别是NJ-4的毒力最弱。

Pathogenicity characteristics of 3 strains of Aerornonas hydrophila, NJ-4, NJ-13 and CS-13, were investiga- ted. The PCR amplification results of five main virulence genes,including aer,act,alt,eprCAI and ahp, showed that NJ-4 had none of the five genes, NJ13 had the only ahp gene,and CS-13 had the only aer gene. All the three bacterial strains had no hemolytic and proteolytie activities. Both NJ-4 and CS-13 had no cytotoxicity, and the cytotoxic titre of NJ-13 was up to 1 ; 128. But in the highly virulent strain BSK-10, the hemolytic, proteolytic and cytotoxie titers were 1 ： 128,1 ： 256 and 1 ： 256 ,respectively. Compared with BSK-10,the adhesion abilities of the three bacterial strains to the HEp-2 cells were lower. The supernatant of NJ-4,NJ13 and CS-13 could not cause the zebrafish to death. The whole bacteria had lower pathogenicity for zebrafish, and their LD50 s were above 10^8 CFU/mL. However, the supernatant and whole bacteria of BSK-10 had higher pathogenicity for zebrafish,and the LD50 of whole bacteria was below 10^5 CFU/mL. The main outer membrane protein （MOMP） profiles of NJ-4 and CS-13 were similar to that of BSK-10,but not to that of NJ13. It suggested that,the three strains of A. hydrophila were lowly virulent and NJ-4 was the representative. The study provides a material basis for further investigation of the bacterial pathogenesis.