仿刺参幼参急性口围肿胀症的细菌性病原

The etiology of acute preistome edema disease in cultured juveniles of Apostichopus japonicus

2003年春以来,山东、辽宁等地区养殖的仿刺参出现急性口围肿胀症,其症状是口围肿胀,体表溃烂,排脏,管足附着力下降,脱落沉至池底,死亡率较高。从病参口围、体表及体壁分离出优势菌10株,经人工感染试验证实其中菌株KW21、KW22、KW23、NB13和NB14是引起仿刺参出现急性口围肿胀症的病原菌。通过16SrRNA基因序列同源性及系统发育分析表明,菌株KW21为Marinomonas dokdonensis;菌株KW22和菌株NB13具有相近的亲缘关系,两者均与Vibrio splendidus有较高的相似性(99%,98%);菌株KW23为Vibrio tapetis;菌株NB14为Vibriosp.。5株病原菌均为革兰氏阴性菌,电镜下观察菌株KW21棒杆状;菌株KW22、KW23、NB13和NB14分别为杆状、弧状、卵圆形和卵圆形,且都具有单极生鞭毛。药物敏感性试验结果表明,诺氟沙星、氧氟沙星和红霉素对5株菌均有较强的抑菌作用。Vibrio tapetis,Vibrio splendidus和Marinomonas dokdonensi作为仿刺参的病原菌属首次报道。

The acute preistome edema disease in cultured juveniles of Apostichopus japonicus occurred in several hatcheries of coastal region in Shandong and Liaoning since the spring of 2003. The disease has resulted in great losses for sea cucumber farmers. The symptoms of the disease are preistome edema, skin ulceration, intestine disgorged, weak adhesion ability of the bucal podia, and finally, the holothtwoids detached from substratum to the botton of pond and died. Ten predominant bacterial strains isolated from preistome, lesions and body wall of diseased juveniles were used for artificial infection experiment. In the experiment, five healthy juveniles of A. japonicus were used as treated groups and control group and placed in 1 L beaker filled with filtered sea water. In each treated group, the holothuroids were placed in a suspension of bacteria diluted in filtered seawater （concentration of bacteria： 3.0× 10^6cell·mL^-1）. The holothuroids treated with strains KW21, KW22, KW23, NB13 and NB14 showed the same symptoms as naturally infected holothuroids and re-isolates from diseased holothuroids had same morphological characteristics. But all holothuroids in treated groups with other five strains and in control group showed no signs during 40 days＇ observation, which proved that strains KW21, KW22, KW23,NB13 and NB14 were the etiology of the disease. To identify the pathogenic bacteria, fragments of 706 bp, 571 bp, 676 bp,661 bp,643 bp sequences of strains KW21, KW22 , KW23 ,NB13 and NB14 16S rRNA genes were amplified and compared with related DNA sequences in GenBank, and molecular phylogenefic dendrogram was constructed by Neighbor-Joining method based on the genetic distance analysis, which revealed that swain KW21 can be identified as Marinomonas dokdonensis because it exhibited 99% similarity to and clustered one clade with Marinomonas dokdonensi. Swain KW22 and swain NB13 had close relation and both showed high similarity to Vibrio splendidus（99% ,98% ）. Swain KW23 can be identified as Vibrio tapetis since it exhibited 99% similarity and was clustered within same clade to Vibrio tapetis. Swain NB14 exhibited 97% similarity to three sequences of Vibrio and it was identified as Vibrio sp.. The pathogenic bacteria were all Gram negative and swain KW21 was seen rod-shaped, and swains KW22, KW23, NB13 and NB14 were seen rod-shaped, arc-shaped, ovoid and ovoid with single polar flagellum under electron microscope. Drugs sensitivity tests showed that five pathogentic bacteria were sensitive to norfloxacin, ofloxacin and erythromycin but were not sensitive to penicillum and ampicillin. This is the first time that Vibrio tapetis, Vibrio splendidus and Marinomonas dokdonensi are reported as the pathogentic bacteria of Apostichopus japonicus in China.