金乌贼幼体弧菌性坏死病的病原、组织病理特征和药物敏感性
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摘要
对青岛市黄岛区人工繁育金乌贼(Sepia esculenta)苗种大规模坏死病进行了病原、组织病理、传播途径和防控药物研究。发病金乌贼幼体不摄食,生长缓慢,体色发黑;体表、肌肉、口腕、触腕及内脏团大面积溃烂,死亡率高达90%;组织病理学显示:患病金乌贼皮肤色素细胞崩解,肌丝断裂,鳃丝崩解,肝细胞大量坏死脱落,肠道微绒毛脱落;从患病金乌贼分离出3株优势菌(SE-A、SE-B和SE-C),人工感染实验表明,SE-B、SE-C对金乌贼半数致死量分别为3.98×10~6 CFU/mL和8.91×10~5 CFU/mL,有较强致病力,为该病的致病菌;根据细菌形态、生理生化和16S rDNA序列分析将SE-B和SE-C鉴定为溶藻弧菌(Vibrio alginolyticus)和塔氏弧菌(Vibrio tubiashii)。分析金乌贼幼体、育苗用水和生物饵料糠虾的病原菌来源、分布和优势度,发现致病菌主要由糠虾携带进入育苗系统。分析了2株致病菌的药物敏感性,SE-B对强力霉素和氟苯尼考等敏感,对链霉素、庆大霉素等耐药;SE-C对氟苯尼考和罗美沙星等敏感,对新霉素和克拉霉素等耐药。
The golden cuttlefish(Sepia esculenta) is a rising aquaculture species with high economic value in China. A disease with necrosis symptoms in golden cuttlefish larvae emerged in a hatchery in Huangdao District,Qingdao. In this study, the pathogen was isolated and identified, the histopathology was investigated, and its route of transmission and sensitivity to different antibiotics were tested. The symptoms included anorexia, slow growth,and a dark body. In addition,the skin, muscles,oral arm,tentacular arm, and the internal organs were extensively ulcerated, and the mortality reached as high as 90%. Histopathological analysis showed that the chromatophore and the gill filament structure were disintegrated, the myofilament was broken, a large number of hepatic lobule epithelial cells were necrotized, and the microvilli of intestinal epithelial cells were shed off. Three dominant bacteria(SE-A, SE-B, SE-C) were isolated from the diseased golden cuttlefish larvae. Artificial infection experiments illustrated that the strains of SE-B and SE-C were the causative pathogens with a median lethal dose of 3.98×10~6 CFU/mL and 8.91×10~5 CFU/mL, respectively. Based on morphological observations, and physiological, biochemical, and molecular analyses(16 S rDNA), SE-B was identified as Vibrio alginolyticus and SE-C was identified as Vibrio tubiashii. The analysis of the transmission route demonstrated that the two pathogens present during golden cuttlefish breeding were mainly carried by Mysis species. The chemotherapeutant sensitivity tests illustrated that SE-B was sensitive to doxycycline and florfenicol, etc., while it was insensitive to streptomycin and gentamicin,etc. SE-C was sensitive to florfenicol and lomefloxacin,etc.,while it was insensitive to neomycin and clarithromycin, etc.
The golden cuttlefish(Sepia esculenta) is a rising aquaculture species with high economic value in China. A disease with necrosis symptoms in golden cuttlefish larvae emerged in a hatchery in Huangdao District,Qingdao. In this study, the pathogen was isolated and identified, the histopathology was investigated, and its route of transmission and sensitivity to different antibiotics were tested. The symptoms included anorexia, slow growth,and a dark body. In addition,the skin, muscles,oral arm,tentacular arm, and the internal organs were extensively ulcerated, and the mortality reached as high as 90%. Histopathological analysis showed that the chromatophore and the gill filament structure were disintegrated, the myofilament was broken, a large number of hepatic lobule epithelial cells were necrotized, and the microvilli of intestinal epithelial cells were shed off. Three dominant bacteria(SE-A, SE-B, SE-C) were isolated from the diseased golden cuttlefish larvae. Artificial infection experiments illustrated that the strains of SE-B and SE-C were the causative pathogens with a median lethal dose of 3.98×10~6 CFU/mL and 8.91×10~5 CFU/mL, respectively. Based on morphological observations, and physiological, biochemical, and molecular analyses(16 S rDNA), SE-B was identified as Vibrio alginolyticus and SE-C was identified as Vibrio tubiashii. The analysis of the transmission route demonstrated that the two pathogens present during golden cuttlefish breeding were mainly carried by Mysis species. The chemotherapeutant sensitivity tests illustrated that SE-B was sensitive to doxycycline and florfenicol, etc., while it was insensitive to streptomycin and gentamicin,etc. SE-C was sensitive to florfenicol and lomefloxacin,etc.,while it was insensitive to neomycin and clarithromycin, etc.
引文
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[17]Li J,Feng J,Liu X,et al.Vibro alginolyticus,a pathogen of Sparus sarba cultured in Hongkong[J].Journal of Fisheries of China,1998,22(3):275-278.[李军,冯娟,刘旭,等.香港地区养殖平鲷的病原菌(溶藻胶弧菌)研究[J].水产学报,1998,22(3):275-278.]
[18]Li Y Q,Jian J C,Wu Z H.Pathogen of Vibriosis of Lutjanus sanguineus[J].Journal of Zhanjiang Ocean University,2004,24(6):1-5.[李永芹,简纪常,吴灶和.红鳍笛鲷弧菌病病原的研究[J].湛江海洋大学学报,2004,24(6):1-5.]
[19]Biel F M,Allen F A,H?se C C.Autolysis in Vibrio tubiashii and Vibrio coralliilyticus[J].Canadian Journal of Microbiology,2014,60(2):57-63.
[20]Richards G P,Watson M A,Needleman D S,et al.Mortalities of Eastern and Pacific oyster larvae caused by the pathogens Vibrio coralliilyticus and Vibrio tubiashii[J].Applied&Environmental Microbiology,2015,81(1):292-297.
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[23]Liu Z Y,Wang Y G,Sun X Q.Correlation between bacteria associated with trash fish and diseases of cultured turbot(Scophthalmus maximus)[J].South China Fisheries Science,2009,5(5):44-51.[刘朝阳,王印庚,孙晓庆.小杂鱼携带细菌与大菱鲆疾病发生的相关性[J].南方水产科学,2009,5(5):44-51.]
[24]Pang H J,Liao M J,Li B,et al.Etiology and treatment of enteritis disease in cultured junviles of Apostichopus japonicus[J].Progress in Fishery Sciences,2017,38(3):188-197.[逄慧娟,廖梅杰,李彬,等.刺参(Apostichopus japonicus)保苗期“肠炎病”及其治疗方法[J].渔业科学进展,2017,38(3):188-197.]
[2]Hao Z L,Zhang X M,Zhang P D.Biological characteristics and propagation of Sepia esculenta[J].Chinese Journal of Ecology,2007,26(4):601-606.[郝振林,张秀梅,张沛东.金乌贼的生物学特性及增殖技术[J].生态学杂志,2007,26(4):601-606.]
[3]Zhao H J,Wei B F,Hu M,et al.Preliminary study on Sepia esculenta oosperm hatching and effects of different adhesion substrates[J].Transactions of Oceanology and Limnology,2004,26(3):64-68.[赵厚钧,魏邦福,胡明,等.金乌贼受精卵孵化及不同材料附着基附卵效果的初步研究[J].海洋湖沼通报,2004,26(3):64-68.]
[4]Zheng X D,Han S,Lin X Z,et al.Research progress in Cephalopod reproductive behaviors[J].Journal of Fishery Sciences of China,2009,16(3):459-465.[郑小东,韩松,林祥志,等.头足类繁殖行为学研究现状与展望[J].中国水产科学,2009,16(3):459-465.]
[5]Niu C,Yang C J,Huang Y X,et al.The efficacy of new spawning substrates for Sepia esculenta oosperm adhesion[J].Journal of Fishery Sciences of China,2017,24(6):1234-1244.[牛超,杨超杰,黄玉喜,等.金乌贼新型产卵附着基的实验研究[J].中国水产科学,2017,24(6):1234-1244.]
[6]Lei S H,Zhang X M,Zhang P D,et al.Early development of golden cuttlefish Sepia esculenta[J].Journal of Fishery Sciences of China,2014,21(1):37-43.[雷舒涵,张秀梅,张沛东,等.金乌贼的早期生长发育特征[J].中国水产科学,2014,21(1):37-43.]
[7]Brickle P,Olson P D,Littlewood D T J,et al.Parasites of Loligo gahi from waters off the Falkland Islands,with a phylogenetically based identification of their cestode larvae[J].Canadian Journal of Zoology,2001,79(12):2289-2296.
[8]Sangster C R,Smolowitz R M.Description of Vibrio alginolyticus infection in cultured Sepia officinalis,Sepia apama,and Sepia pharaonis[J].Biological Bulletin,2003,205(2):233-234.
[9]Mladineo I,Jozi?M.Aggregata infection in the common octopus,Octopus vulgaris(Linnaeus,1758),Cephalopoda:Octopodidae,reared in a flow-through system[J].Acta Adriatica,2005,46(2):193-199.
[10]Furuya H.Two new dicyemids from Sepia longipes(Moll Usca:Cephalopoda:Decapoda)[J].Journal of Parasitology,2009,95(3):681-689.
[11]Furuya H.A new dicyemid from Sepiella japonica(Mollusca:Cephalopoda:Decapoda)[J].Journal of Parasitology,2008,94(1):223.
[12]Furuya H.Redescription of Dicyemennea nouveli(Phylum:Dicyemida)from Enteroctopus dofleini(Mollusca:Cephalopoda:Octopoda)[J].Journal of Parasitology,2008,94(5):1064-1070.
[13]Yang M C.Veterinary Statistics[M].Beijing:China Prospect Press,1990:232.[杨茂成.兽医统计学[M].北京:中国展望出版社,1990:232.]
[14]Holt J G,Krieg N R,Pha S,et al.Bergey’s Manual of Determinative Bacteriology[M].9th Edition.Philadelphia:Williams&Wilkins Company,1994:482-484.
[15]Dong X Z,Cai M Y.Common Bacterial System Identification Manual[M].Beijing:Science Press,2002:107-116.[东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2002:107-116.]
[16]Hao J G,Wang Y G,Liao M J,et al.The effects of three types of feed supplements on the growth of Apstichopus japonicus cultured in cages[J].Progress in Fishery Sciences,2015,36(5):102-110.[郝继光,王印庚,廖梅杰,等.饲料中添加3种不同投入品对筏式浅海网箱刺参(Apstichopus japonicus)养殖生长的影响[J].渔业科学进展,2015,36(5):102-110.]
[17]Li J,Feng J,Liu X,et al.Vibro alginolyticus,a pathogen of Sparus sarba cultured in Hongkong[J].Journal of Fisheries of China,1998,22(3):275-278.[李军,冯娟,刘旭,等.香港地区养殖平鲷的病原菌(溶藻胶弧菌)研究[J].水产学报,1998,22(3):275-278.]
[18]Li Y Q,Jian J C,Wu Z H.Pathogen of Vibriosis of Lutjanus sanguineus[J].Journal of Zhanjiang Ocean University,2004,24(6):1-5.[李永芹,简纪常,吴灶和.红鳍笛鲷弧菌病病原的研究[J].湛江海洋大学学报,2004,24(6):1-5.]
[19]Biel F M,Allen F A,H?se C C.Autolysis in Vibrio tubiashii and Vibrio coralliilyticus[J].Canadian Journal of Microbiology,2014,60(2):57-63.
[20]Richards G P,Watson M A,Needleman D S,et al.Mortalities of Eastern and Pacific oyster larvae caused by the pathogens Vibrio coralliilyticus and Vibrio tubiashii[J].Applied&Environmental Microbiology,2015,81(1):292-297.
[21]Elston R A,Hasegawa H,Humphrey K L,et al.Re-emergence of Vibrio tubiashii in bivalve shellfish aquaculture:severity,environmental drivers,geographic extent and management[J].Diseases of Aquatic Organisms,2008,82(2):119.
[22]Hasegawa H,Lind E J,Boin M A,et al.The extracellular metalloprotease of Vibrio tubiashii is a major virulence factor for Pacific oyster(Crassostrea gigas)larvae[J].Applied and Environmental Microbiology,2008,74(13):4101-4110.
[23]Liu Z Y,Wang Y G,Sun X Q.Correlation between bacteria associated with trash fish and diseases of cultured turbot(Scophthalmus maximus)[J].South China Fisheries Science,2009,5(5):44-51.[刘朝阳,王印庚,孙晓庆.小杂鱼携带细菌与大菱鲆疾病发生的相关性[J].南方水产科学,2009,5(5):44-51.]
[24]Pang H J,Liao M J,Li B,et al.Etiology and treatment of enteritis disease in cultured junviles of Apostichopus japonicus[J].Progress in Fishery Sciences,2017,38(3):188-197.[逄慧娟,廖梅杰,李彬,等.刺参(Apostichopus japonicus)保苗期“肠炎病”及其治疗方法[J].渔业科学进展,2017,38(3):188-197.]