一株感染深水网箱养殖许氏平鲉的病原菌分离与鉴定
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摘要
2016年7月,山东省长岛县深水网箱养殖许氏平鲉(Sebastes schlegeli)暴发严重皮肤溃疡症。作者对病鱼进行病原菌分离。通过形态学观察、常规生理生化试验、gyrB和16S rDNA基因克隆测序等方法对分离菌株进行鉴定。在病灶溃疡处分离到一株绝对优势菌BZ01,该菌株在TSB固体培养基上呈半透明菌落,在TCBS选择性培养基上菌落呈绿色。透射电镜观察为短棒状,具有单根极生鞭毛。人工回接感染证明,该菌株对许氏平鲉具有较强的致病力,可以引起皮肤溃疡等症状,且与自然感染症状一致,其LD_(50)为2.07×10~6 CFU/ml。通过gyrB和16S rDNA基因序列测定并构建系统发育树显示,菌株BZ01与弧菌属同源性最高,并在系统发育树中与轮虫弧菌(Vibrio rotiferianus)聚为一枝,结合形态及生理生化表型测定结果,将该菌株鉴定为轮虫弧菌(V.rotiferianus)。药敏试验结果显示,该菌株对四环素类、喹诺酮类、香豆素类、肽酰转移酶类高度敏感,而对大环内酯类、多肽类、磺胺类、β-内酰胺类、氨基糖苷类中度敏感或不敏感。
In July 2016, a severe disease outbreak occurred in offshore-cage cultured black rockfish(Sebastes schlegelii) in Changdao County, Shangdong Province, which resulted in significant mortality. The main symptom of diseased rockfish was skin ulcers on the body. In order to determine the cause of the disease, pathogenic bacteria were isolated from diseased fish and identified by various methods including morphological observation, traditional physiological and biochemical tests, and gyr B gene sequence alignment. A dominant bacterial strain was isolated from ulcerative skin and was marked as BZ01. The strain formed a subtransparent colony on TSB solid medium plate and a green colony on TCBS selective medium plate. Its shape was short and rod-like with a single polar flagellum when observed by transmission electron microscopy(TEM). An artificial infection experiment was carried out to verify its pathogenicity to black rockfish. The results demonstrated that this strain of BZ01 had strong pathogenicity to black rockfish and could cause death. The artificial infected black rockfishes showed the same symptoms as those of natural infection. The value of LD50 was calculated as 2.07×10~6 CFU/ml. Analysis of the gyrB gene sequence suggested that the strain of BZ01 was most closely related to Vibrio. In gyrB gene sequencing, it clustered into Vibrio rotiferianus in the phylogenetic tree with 100% confidence. Based on these results, this strain was identified as V. rotiferianus. At the same time, the drug sensitivity test showed that this strain of BZ01 was highly sensitive to tetracyclines(tetracycline, doxycycline), quinolones(norfloxacin, ofloxacin, fleroxacin, nalidixic acid), coumarin(streptonivicin), and peptidyl transferase(florfenicol), while medium sensitive or resistant to macrolides, peptides, sulfonamides, β-lactams, and aminoglycosides. This is the first report that V. rotiferianus could infect offshore-cage cultured rockfish in China, which would provide an effective reference for disease control in cultured S. schlegelii.
In July 2016, a severe disease outbreak occurred in offshore-cage cultured black rockfish(Sebastes schlegelii) in Changdao County, Shangdong Province, which resulted in significant mortality. The main symptom of diseased rockfish was skin ulcers on the body. In order to determine the cause of the disease, pathogenic bacteria were isolated from diseased fish and identified by various methods including morphological observation, traditional physiological and biochemical tests, and gyr B gene sequence alignment. A dominant bacterial strain was isolated from ulcerative skin and was marked as BZ01. The strain formed a subtransparent colony on TSB solid medium plate and a green colony on TCBS selective medium plate. Its shape was short and rod-like with a single polar flagellum when observed by transmission electron microscopy(TEM). An artificial infection experiment was carried out to verify its pathogenicity to black rockfish. The results demonstrated that this strain of BZ01 had strong pathogenicity to black rockfish and could cause death. The artificial infected black rockfishes showed the same symptoms as those of natural infection. The value of LD50 was calculated as 2.07×10~6 CFU/ml. Analysis of the gyrB gene sequence suggested that the strain of BZ01 was most closely related to Vibrio. In gyrB gene sequencing, it clustered into Vibrio rotiferianus in the phylogenetic tree with 100% confidence. Based on these results, this strain was identified as V. rotiferianus. At the same time, the drug sensitivity test showed that this strain of BZ01 was highly sensitive to tetracyclines(tetracycline, doxycycline), quinolones(norfloxacin, ofloxacin, fleroxacin, nalidixic acid), coumarin(streptonivicin), and peptidyl transferase(florfenicol), while medium sensitive or resistant to macrolides, peptides, sulfonamides, β-lactams, and aminoglycosides. This is the first report that V. rotiferianus could infect offshore-cage cultured rockfish in China, which would provide an effective reference for disease control in cultured S. schlegelii.
引文
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Chen ZQ,Yao ZX,Lin M,et al.Characterization of pathogenic bacteria Vibrio rotiferianus isolated from Cynoglossus semilaevis Günther.Biotechnology Bulletin,2012(6):147-153[陈政强,姚志贤,林茂,等.半滑舌鳎病原菌轮虫弧菌(Vibrio rotiferianus)的分离与鉴定.生物技术通报,2012(6):147-153]
Chowdhury PR,Boucher Y,Hassan KA,et al.Genome Sequence of Vibrio rotiferianus Strain DAT722.Journal of Bacteriology,2011,193(13):3381-3382
Gomez-Gil B,Thompson FL,Thompson CC,et al.Vibrio rotiferianus sp.nov,isolated from cultures of the rotifer Brachionus plicatilis.International Journal of Systematic and Evolutionary Microbiology,2003,53(1):239-243
Gu ZH,Fu ZR,Qian H,et al.Artificial breeding and related factors in Schlegel's rockfish Sebastes schlegeli in Tianjin.Fisheries Science,2015,34(8):519-522[顾中华,傅志茹,钱红,等.天津地区许氏平鲉人工育苗研究.水产科学,2015,34(8):519-522]
Han HJ,Kim DY,Kim WS,et al.Atypical Aeromonas salmonicida infection in the black rockfish,Sebastes schlegeli Hilgendorf,in Korea.Journal of Fish Diseases,2011,34(1):47-55
Jin CY.Identification and characterization of pathogenic bacteria causing vibriosis in cultured American white shrimp(Penaeus vannamei).Marine Environmental Science,2013,32(5):647-651[金春英.南美白对虾弧菌病病原的分离和鉴定.海洋环境科学,2013,32(5):647-651]
Kang S,Shin G,Shin Y,et al.Experimental evaluation of pathogenicity of Lactococcus Garvieae in black rockfish(Sebastes Schlegeli).Journal of Veterinary Science,2004,5(4):387-390
Labbate M,Boucher Y,Chowdhury PR,et al.Integration of a laterally acquired gene into a cell network important for growth in a strain of Vibrio rotiferianus.BMC Microbiology,2011,11:253
Li CH,Chen J,Shi YH,et al.Characterization of Listonella anguillarum as the aetiological agent of vibriosis occurred in cultured ayu(Plecoglossus altivelis)in Ninghai country,China.Acta Microbiologica Sinica,2009,49(7):931-937[李长红,陈炯,史雨红,等.宁海地区香鱼弧菌病病原菌鉴定.微生物学报,2009,49(7):931-937]
Masahiro S,Shizuo A,Masanori K.A streptococcal disease of cultured jacopever Sebastes schlegeli.Aquaculture Science,1986,34(3):171-177
Meng P,Zhan WB,Sheng XZ.Identification and phylogenetic analysis of pathogen HV0811 isolated from the diseased Sebastodes fuscescens.Marine Sciences,2010,34(5):48-54[孟鹏,战文斌,绳秀珍.从患病黑鲪分离病原菌HV0811的鉴定及其系统发育分析[J].海洋科学,2010,34(5):48-54]
Sheng XZ,Meng P,Xu XL,et al.Identification and lipopolysaccharide antigenicity analysis of Mycobacterium marinum isolated from Sebastes schlegeli.Journal of Fisheries of China,2011,35(10):1524-1531[绳秀珍,孟鹏,徐晓丽,等.从许氏平鲉体表分离海分枝杆菌的鉴定及其脂多糖的抗原性分析.水产学报,2011,35(10):1524-1531]
Wang GS,Wang YG,Zhang Z,et al.Isolation,identification and pathogenicity of Listonella anguillarum from diseased cultured Sebastes schlegelii.Journal of Fisheries of China,2012,36(8):1290-1296[王庚申,王印庚,张正,等.引起养殖许氏平鲉死亡的鳗利斯顿氏菌的分离鉴定及致病性.水产学报,2012,36(8):1290-1296]
Wang XR,Chen SQ,Mo ZL,et al.Isolation and identification of Aeromonas salmonicida associated with furunculosis in cultured sablefish(Anoplopoma fimbria).Progress in Fishery Science,2017,38(5):25-31[王晓冉,陈四清,莫照兰,等.养殖裸盖鱼(Anoplopoma fimbria)疥疮病病原菌的分离与鉴定.渔业科学进展,2017,38(5):25-31]
Won KM,Park SI.Pathogenicity of Vibrio harveyi to cultured marine fishes in Korea.Aquaculture,2008,285(1):8-13
Yang QH,Zhen LY,Huang ZC,et al.Identification and characterization of pathogen Vibrio rotiferianus,a pathogen isolated from Hippocampus erectus with tail-rot disease.Journal of Fishery Sciences of China,2017,24(5):1131-1140[杨求华,郑乐云,黄种持,等.线纹海马尾部溃烂症病原轮虫弧菌的分离鉴定及特性分析.中国水产科学,2017,24(5):1131-1140]
Chen JG,Chen C,Zhang TT,et al.Isolation and classification of exophthalmic disease-inducing pathogenic bacterium from Ctropristis striata.Progress in Fishery Science,2017,38(5):32-40[陈建国,超陈,李炎路,等.美洲黑石斑鱼(Centropristis striata)“突眼症”的病原茵分离鉴定.渔业科学进展,2017,38(5):32-40]
Chen ZQ,Yao ZX,Lin M,et al.Characterization of pathogenic bacteria Vibrio rotiferianus isolated from Cynoglossus semilaevis Günther.Biotechnology Bulletin,2012(6):147-153[陈政强,姚志贤,林茂,等.半滑舌鳎病原菌轮虫弧菌(Vibrio rotiferianus)的分离与鉴定.生物技术通报,2012(6):147-153]
Chowdhury PR,Boucher Y,Hassan KA,et al.Genome Sequence of Vibrio rotiferianus Strain DAT722.Journal of Bacteriology,2011,193(13):3381-3382
Gomez-Gil B,Thompson FL,Thompson CC,et al.Vibrio rotiferianus sp.nov,isolated from cultures of the rotifer Brachionus plicatilis.International Journal of Systematic and Evolutionary Microbiology,2003,53(1):239-243
Gu ZH,Fu ZR,Qian H,et al.Artificial breeding and related factors in Schlegel's rockfish Sebastes schlegeli in Tianjin.Fisheries Science,2015,34(8):519-522[顾中华,傅志茹,钱红,等.天津地区许氏平鲉人工育苗研究.水产科学,2015,34(8):519-522]
Han HJ,Kim DY,Kim WS,et al.Atypical Aeromonas salmonicida infection in the black rockfish,Sebastes schlegeli Hilgendorf,in Korea.Journal of Fish Diseases,2011,34(1):47-55
Jin CY.Identification and characterization of pathogenic bacteria causing vibriosis in cultured American white shrimp(Penaeus vannamei).Marine Environmental Science,2013,32(5):647-651[金春英.南美白对虾弧菌病病原的分离和鉴定.海洋环境科学,2013,32(5):647-651]
Kang S,Shin G,Shin Y,et al.Experimental evaluation of pathogenicity of Lactococcus Garvieae in black rockfish(Sebastes Schlegeli).Journal of Veterinary Science,2004,5(4):387-390
Labbate M,Boucher Y,Chowdhury PR,et al.Integration of a laterally acquired gene into a cell network important for growth in a strain of Vibrio rotiferianus.BMC Microbiology,2011,11:253
Li CH,Chen J,Shi YH,et al.Characterization of Listonella anguillarum as the aetiological agent of vibriosis occurred in cultured ayu(Plecoglossus altivelis)in Ninghai country,China.Acta Microbiologica Sinica,2009,49(7):931-937[李长红,陈炯,史雨红,等.宁海地区香鱼弧菌病病原菌鉴定.微生物学报,2009,49(7):931-937]
Masahiro S,Shizuo A,Masanori K.A streptococcal disease of cultured jacopever Sebastes schlegeli.Aquaculture Science,1986,34(3):171-177
Meng P,Zhan WB,Sheng XZ.Identification and phylogenetic analysis of pathogen HV0811 isolated from the diseased Sebastodes fuscescens.Marine Sciences,2010,34(5):48-54[孟鹏,战文斌,绳秀珍.从患病黑鲪分离病原菌HV0811的鉴定及其系统发育分析[J].海洋科学,2010,34(5):48-54]
Sheng XZ,Meng P,Xu XL,et al.Identification and lipopolysaccharide antigenicity analysis of Mycobacterium marinum isolated from Sebastes schlegeli.Journal of Fisheries of China,2011,35(10):1524-1531[绳秀珍,孟鹏,徐晓丽,等.从许氏平鲉体表分离海分枝杆菌的鉴定及其脂多糖的抗原性分析.水产学报,2011,35(10):1524-1531]
Wang GS,Wang YG,Zhang Z,et al.Isolation,identification and pathogenicity of Listonella anguillarum from diseased cultured Sebastes schlegelii.Journal of Fisheries of China,2012,36(8):1290-1296[王庚申,王印庚,张正,等.引起养殖许氏平鲉死亡的鳗利斯顿氏菌的分离鉴定及致病性.水产学报,2012,36(8):1290-1296]
Wang XR,Chen SQ,Mo ZL,et al.Isolation and identification of Aeromonas salmonicida associated with furunculosis in cultured sablefish(Anoplopoma fimbria).Progress in Fishery Science,2017,38(5):25-31[王晓冉,陈四清,莫照兰,等.养殖裸盖鱼(Anoplopoma fimbria)疥疮病病原菌的分离与鉴定.渔业科学进展,2017,38(5):25-31]
Won KM,Park SI.Pathogenicity of Vibrio harveyi to cultured marine fishes in Korea.Aquaculture,2008,285(1):8-13
Yang QH,Zhen LY,Huang ZC,et al.Identification and characterization of pathogen Vibrio rotiferianus,a pathogen isolated from Hippocampus erectus with tail-rot disease.Journal of Fishery Sciences of China,2017,24(5):1131-1140[杨求华,郑乐云,黄种持,等.线纹海马尾部溃烂症病原轮虫弧菌的分离鉴定及特性分析.中国水产科学,2017,24(5):1131-1140]
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