夏季保苗期刺参(Apostichopus japonicus)腐皮综合征病原菌的分离鉴定及其致病阈值

2013年夏季山东地区某刺参育苗场处于保苗期的刺参苗种暴发 腐皮综合征疾病，表现为附着力下降、棘刺顶端溃烂、排脏、表皮溃疡和自溶等症状。自患病个体病灶组织分离优势细菌，并利用形态学观察、生理生化测定、回接感染、16S rDNA基因序列分析等方法完成致病原的鉴定。此外，通过对发病前后养殖系统中优势菌丰度的变化，追踪确定了所分离病原菌的致病阈值。结果显示，从患病参苗体表病灶处分离的一株优势菌HP130917A-1，经回接感染证实，该菌株具有较强的致病力，其对刺参苗种的半致死浓度为1.2×106 CFU/ml。生理生化结合分子鉴定表明，该菌株为溶藻弧菌(Vibrio alginolyticus)。此外，完成了患病池保苗期间池水和附着基表面微生物菌群结构动态分析，共分离得到了6种主要优势菌，分别为溶藻弧菌、需钠弧菌(Vibrio natriegens)、副溶血弧菌(Vibrio parahaemolyticus)、枯草芽孢杆菌(Bacillus subtilis)、苏云金芽孢杆菌(Bacillus thuringiensis)和马胃葡萄球菌(Staphylococcus equorum)。溶藻弧菌一直是养殖系统中优势度最高的种类，且随着附着基在保苗池中时间的延长，其在附着基表面沉积物中的浓度越来越高。到第50天时，浓度达到8.97×106 CFU/cm2。而此时系统中参苗开始暴发腐皮综合征疾病，可将此浓度视为该病原的致病阈值。因此，在夏季保苗过程中应加强养殖系统中弧菌总数的监测，制定适宜的附着基更换频率，建立和优化刺参保苗工艺，以期为刺参苗期疾病防控和优化健康养殖管理提供理论依据和参考。

Isolation and Identification of the Pathogenic Bacteria of Skin Ulcerative Syndrome (SUS) from Apostichopus japonicus Juveniles in Summer and Study on Its Pathogenic Threshold

In the summer of 2013, there was an outbreak of skin ulcerative syndrome (SUS) in Apostichopus japonicus juveniles at a hatchery in Shandong Province. The symptoms included weak adhesion, rotten spines, viscera eversion, skin ulceration, and body autolysis. From the skin lesions of diseased juveniles we isolated a dominant strain HP130917A-1 with a median lethal concentration of 1.2×106 CFU/ml. This strain exhibited high pathogenicity and caused the same symptoms as SUS. The identification of this pathogen was verified using morphological observation, physiological and biochemical test, and 16S rDNA gene sequence analysis. The results demonstrated that HP130917A-1 was Vibiro alginolyticus and the causative pathogen associated with SUS. The microbial community structure was also analyzed using 16S rDNA gene sequencing, from which six major strains were found in the rearing system including V. alginolyticus, V. natriegens, V. parahaemolyticus, Bacillus subtilis, Bacillus thuringiensis and Staphylococcus equorum. V. alginolyticus was predominant among six major strains during the 50 days. On the 50th day of the experiment, the amount of V. alginolyticus reached the highest value of 1.31×105 CFU/ml in the substrates, which was considered as the pathogenic threshold of causing sea cucumber SUS. These results provided insights into the mechanisms of SUS and the health management of sea cucumber A. japonicus. Based on our findings we recommend healthy management strategies such as regular monitor of the amount of Vibrios, suitable frequency changing of the substrates, and the application of probiotics or herbal medicines.