黑棘鲷内脏结节病病原的分离、鉴定及致病性研究

2017年4月,浙江台州某海水养殖公司跑道式养殖池黑棘鲷大量发病,病鱼活力下降、食欲减退、体表溃疡,剖检可见肝脏、脾脏、肾脏肿大且有不同程度的白色结节,同时伴随大量腹水。用胰蛋白胨大豆琼脂(TSA)从典型结节病濒死黑棘鲷器官分离到革兰阴性短杆菌。分离病原菌AS15对健康黑棘鲷的致病力结果显示,AS15腹腔注射可使健康黑棘鲷发病死亡,死亡黑棘鲷可出现自然发病症状,在(24±1)°C的条件下,(25±2) g黑棘鲷的半数致死浓度(LD_(50))为6.5×10~4 CFU/尾。经API 20E鉴定,分离菌株AS15生理生化特性与类杀鱼爱德华氏菌LADL05-105和迟缓爱德华氏菌典型菌ATCC15947相似度为86.2%,与杀鱼爱德华氏菌ET~T883的相似度为82.8%;AS15的16S rDNA与杀鱼爱德华氏菌ET~T883同源性达99%,gyrB与类杀鱼爱德华氏菌LADL05-105和杀鱼爱德华氏菌ETT883同源性分别为100%和98%;16S rDNA进化树显示,AS15与ETT883、LADL05-105聚为一簇,gyrB进化树与LADL05-105、NCIM2056聚为一簇;采用4种爱德华氏菌属种特异性引物对AS15进行PCR分析,结果显示,AS15可扩增出类杀鱼爱德华氏菌的种特异性片段,不能扩增出迟缓爱德华氏菌、鲇鱼爱德华氏菌、杀鱼爱德华氏菌的种特异性片段,表明AS15属类杀鱼爱德华氏菌成员。分析了AS15的菌毛基因、sodB等毒力基因,发现AS15具有fimA、fimB、fimC、fimD等4种菌毛基因和sodB、citC、esrB、mukF、katB等毒力基因。本实验首次从黑棘鲷上检出致病性类杀鱼爱德华氏菌,该菌对黑棘鲷的发病机制和毒力机理还需进一步研究。

Isolation, identification and pathogenicity studies on visceral granulomatous disease in Acanthopagrus schlegelii

A serious disease occurred in black seabream, Acanthopagrus schlegelii, cultivated in runway fish culture pond of mariculture farm in Taizhou, Zhejiang province, in April, 2017. The diseased fish showed sluggish swimming, anorexia and ulceration on surface. Moribund fish were dissected with symptoms of massive ascites and swelling of liver, spleen and kidney, with white granulomatous nodules, especially in spleen. The bacteria were isolated with tryptone soybean agar, and all the fish with typical symptoms showed the pure culture of grayish white colony with Gram negative rods. The bacterial strain AS15, from the spleen of fish with most serious granulomatous, was chosen for challenging test to healthy black seabream by injection intraperitoneally. Mortality was recorded with similar symptoms to native infected seabream, with the median death dose of 6.5×10⁴ CFU/ind. AS15 was identified as Edwardsiella tarda with API 20E biochemical bacteria identification system as well as 16S rDNA sequences blasted on NCBI, with 99% similarity to reference strains. With phylogenetic analysis for 16S rDNA, AS15 belonged to the same cluster with ET^T883, LADL05-105, E. piscicida according to new classification for genus E.dsiella, and same cluster to LADL05-105 and NCIM2056, E. piscicida-like, the new recommended species of the genus, with 100% similarity on gyrB phylogenetic tree. The species-specific primers for genus E. dsiella were used for PCR test, and AS15 showed positive to EPL, the species primer for E. piscicida-like, and PCR negative to all other three primers of E. tarda, E. piscicida and E. ictalur. Ten pathogenic genes, including four fimbrial genes, and sodB, citC, esrB, mukF, katB, the related virulence genes were analyzed by PCR. The strain AS15 showed positive to all four fimbrial genes and other virulence genes except gene gadB. It could be considered that AS15, E. piscicida-like bacteria, the first pathogenic strain isolated and reported from marine cultivated seabream, was the pathogen of serious outbreak case of visceral granulomatous disease in black seabream in runway pond culture system.