老化参池刺参腐皮综合征致病原的分离与鉴定

Isolation and identification of cultured Apostichopus japonicus pathogen associated with skin ulcer syndrome in old ponds

从患病刺参体表病灶组织分离出菌株060330B,其优势度高达90%,人工回接感染试验证实其具有较强的致病性,可导致健康刺参出现与自然患病刺参相同的症状。通过形态学观察、API半自动化鉴定和常规生理生化试验的结果表明,菌株060330B具有假单胞菌属的特征,其表型特征与恶臭假单胞菌相似。对该菌株进行16SrRNA基因序列分析和构建系统发育树,结果显示其与恶臭假单胞菌的亲缘关系最近,相似率达到99.5%。菌株060330B可鉴定为恶臭假单胞菌,并视为养殖刺参腐皮综合征的致病原之一。通过对多起不同来源患病刺参的研究表明,使用年限长、淤泥层厚呈黑色并伴有腥味的养殖池塘在该时期容易导致刺参腐皮综合征的发生。

From January to April of the year 2006,in cultured Apostichopus japonicus broke out skin ulcer syndrome in several areas nearby Qingdao,whose typical clinical signs were mouth swelling,eviscerating bowels,skin ulcer,body disjointing and then the infected sea cucumber died. Bacterium 060330B was isolated from focus tissue of Apostichopus japonicus,whose predominance degree reached 90% and was verified to be the pathogen causing skin ulcer syndrome based on artificial infection test. Morphological observation,semi-automatic identification through API system as well as traditional physiological and biochemical methods were applied in the bacterial classification. The results showed that 060330B had same character as Pseudomonas genus bacteria,its characteristics were similar with Pseudomonas putida,having short rods with several polar flagella,forming green colony on TCBS agar. Further results from 16S rRNA sequence analysis turned out that the sequence of 060330B was highly identical to Pseudomonas putida,with 99. 5% identity. Thus bacterium 060330B was identified as Pseudomonas putida,and regarded as one of the pathogens causing skin ulcer syndrome of cultured Apostichopus japonicus. In addition,epidemiology investigation and detection of several samples from different areas revealed that the ponds whose culturing history reached two years or more and having thick black bottom mud with fishy smell were likely to result in the outbreak of skin ulcer syndrome in cultured Apostichopus japonicus at that time.