3株嗜水气单胞菌弱毒株的毒力相关特性分析

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不合上述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10^8CFU／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10^5CFU／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。

Virulence-associated characterization analysis of three strains of Aeromonas hydrophila

Pathogenicity characteristics of 3 strains of Aeromonas hydrophila,NJ-4,NJ-13 and CS-13,were investigated.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 proteolytic 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 cytotoxic 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 LD50s were above 108 CFU/mL.However,the supernatant and whole bacteria of BSK-10 had higher pathogenicity for zebrafish,and the LD50 of whole bacteria was below 105 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.