工厂化淡水养殖凡纳滨对虾甲壳溃疡症病因与防治研究
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摘要
为探明工厂化淡水养殖凡纳滨对虾甲壳溃疡症的病因和防控方法,进行了病原分离鉴定、毒力基因检测、攻毒试验和外用内服药敏试验。从患虾肝胰腺和发病池水中经TCBS琼脂分离纯化菌株,经培养特性、形态学、生理生化和16S rRNA基因序列分析,分别鉴定得凡隆气单胞菌F-L株和点状气单胞菌F-W株。进一步从凡隆气单胞菌F-L株中检出气溶素、细胞毒性肠毒素、酯酶、鞭毛、核酶、丝氨酸蛋白酶、热不稳定性肠毒素和弹性蛋白酶8种毒力基因,点状气单胞菌F-W株则检出气溶素、热不稳定性肠毒素、酯酶、鞭毛、弹性蛋白酶和溶血素6种毒力基因。两株菌浸泡攻毒都能引起凡纳滨对虾死亡,半数致死浓度分别为10~(7. 68)和10~(7. 8)cfu/m L,可初步判定气单胞菌为本病病原。药敏试验和防治试验结果显示,恩诺沙星内服、菌毒克外用消毒以及促进创伤愈合、调控虾池水质和虾肠肝健康有利于本病防控。此研究能为防控气单胞菌引起的凡纳滨对虾甲壳溃疡提供借鉴。
In order to determine the pathogen of the Carapace ulcer of Litopenaeus vannamei in factory freshwater aquaculture and provide the methods of prevention and treatment,analysis of isolation and identification of pathogens,virulence genes,toxicity test,the influence of phase related antibiotics and topical disinfectants on the aquatic animal were couducted. Samples were collected and isolated from the hepatopancreas of diseased shrimps and attacked water,and cultured with TCBS medium,screening for dominant strains of each group,respectively called F-L and F-W. Each group was identified based on the culture characteristics and forms,sequences of16 S rRNA gene and biochemical identification. The result of the F-L was Aeromonas veronii,and of the F-W was Aeromonas punctata.In virulence genes test,F-L strains detected 8 genes,respectively were aer A,Act,Lip,Fla,Exu,Ser,Alt and ahy B,and F-W strains had aer A,Alt,Lip,Fla,ahy B and hly A. In toxicity test,both strains of bacteria could cause the death of Litopenaeus vannamei,and median lethal dose was 10~(7. 68) and 10~(7. 8) cfu/m L,respectively. The study would provide a basis of the effective prevention and control of Carapace ulcer of Litopenaeus vannamei caused by Aeromonas.
In order to determine the pathogen of the Carapace ulcer of Litopenaeus vannamei in factory freshwater aquaculture and provide the methods of prevention and treatment,analysis of isolation and identification of pathogens,virulence genes,toxicity test,the influence of phase related antibiotics and topical disinfectants on the aquatic animal were couducted. Samples were collected and isolated from the hepatopancreas of diseased shrimps and attacked water,and cultured with TCBS medium,screening for dominant strains of each group,respectively called F-L and F-W. Each group was identified based on the culture characteristics and forms,sequences of16 S rRNA gene and biochemical identification. The result of the F-L was Aeromonas veronii,and of the F-W was Aeromonas punctata.In virulence genes test,F-L strains detected 8 genes,respectively were aer A,Act,Lip,Fla,Exu,Ser,Alt and ahy B,and F-W strains had aer A,Alt,Lip,Fla,ahy B and hly A. In toxicity test,both strains of bacteria could cause the death of Litopenaeus vannamei,and median lethal dose was 10~(7. 68) and 10~(7. 8) cfu/m L,respectively. The study would provide a basis of the effective prevention and control of Carapace ulcer of Litopenaeus vannamei caused by Aeromonas.
引文
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[17]张冬星,康元环,田佳鑫,等.鲫鱼源致病性维氏气单胞菌的分离鉴定及药敏试验[J].中国兽医科学,2017(3):366-374.
[18]YANG Q,ZHAO M,WANG K Y,et al. Multidrug-resistant Aeromonas veronii recovered from channel catfish(Ictalurus punctatus)in China:prevalence and mechanisms of fluoroquinolone resistance[J]. Microbial Drug Resistance,2016,23(4):473-479.
[19]彭开松,佘锐萍,吴襟.两种寡聚糖对罗非鱼生长及抗凡隆气单胞菌感染的影响[J].中国兽医科学,2011(1):41-47.
[20]HAO K,LIU J Y,LING F,et al. Effects of dietary administration of Shewanella haliotis,D4,Bacillus cereus,D7 and Aeromonas bivalvium,D15,single or combined,on the growth,innate immunity and disease resistance of shrimp,Litopenaeus vannamei[J]. Aquaculture,2014,428-429(3):141-149.
[21]WU Y S,LEE M C,HUANG C T,et al. Effects of traditional medical herbs“minor bupleurum decoction”on the non-specific immune responses of white shrimp(Litopenaeus vannamei)[J]. Fish&Shellfish Immunology,2017,64:218-225.
[2]农业部渔政渔业管理局.中国渔业统计年鉴[M].北京:中国农业出版社,2017:22-24.
[3]彭开松,佘锐萍,杨玉荣,等.温室池水环境与罗非鱼运动性气单胞菌败血症爆发的关系[J].农业工程学报,2006,22(10):189-192.
[4]CAO H,JIAN A,HE S,et al. Aeromonas schubertii:a potential pathogen for freshwater cultured whiteleg shrimp,Penaeus vannamei[J]. The Israeli Journal of Aquaculture-Bamidgeh,2015,67(1104):1-6.
[5]彭开松,佘锐萍,杨玉荣,等.鸡β-防御素13对尼罗罗非鱼生长和疾病抗性的影响[J].中国兽医科学,2009,39(5):432-436.
[6]JANDA J M,ABBOTT S L. The genus Aeromonas:taxonomy,pathogenicity,and infection[J]. Clinical Microbiology Reviews,2010,23(1):35-73.
[7]潘晓艺,沈锦玉,李建应,等.青虾“软壳综合症”病原及其特性[J].微生物学通报,2009,36(10):1571-1576.
[8]曹海鹏,温乐夫,杨移斌,等.克氏原螯虾源致病性豚鼠气单胞菌的分离及其生物学特性[J].水生生物学报,2014,38(6):1047-1053.
[9]朱若林,马腾,陈露,等.鳙鱼维氏气单胞菌的分离鉴定及其毒力基因检测[J].安徽农业大学学报,2017,44(2):229-233.
[10]姜英辉,房保海,祝素珍,等. SNT 3229-2012,食品消毒剂和防腐剂杀菌效果评价方法[S].北京:中华人民共和国国家质量监督检验检疫总局,2012:2-17.
[11]刘玉庆. EUCAST欧盟药敏试验标准[M].北京:中国质检出版社,2016:3-20.
[12]李永仁,王金雨,陶秉春.南美白对虾褐斑病综合防治的几点经验[J].中国水产,2010(7):65-66.
[13]董靖,丁浩,胥宁,等.杂交鲶维氏气单胞菌鉴定及联合药敏试验研究[J].中国预防兽医学报,2017(11):897-901.
[14]LYU A,SONG Y,HU X,et al. Aeromonas veronii,associated with skin ulcerative syndrome,isolated from the goldfish(Carassius auratus)in China[J]. Israeli Journal of Aquaculture-Bamidgeh,2016,68(1320):1-10.
[15]YANG Y,LI J,LIU X,et al. Improving extracellular production of Serratia marcescens lytic polysaccharide monooxygenase CBP21 and Aeromonas veronii B565 chitinase Chi92 in Escherichia coli and their synergism[J]. Amb Express,2017,7(170):1-9.
[16]ZHANG D,XU D H,QIU J,et al. Chitin degradation and utilization by virulent Aeromonas hydrophila strain ML10-51K.[J]. Archives of Microbiology,2017,199(4):1-7.
[17]张冬星,康元环,田佳鑫,等.鲫鱼源致病性维氏气单胞菌的分离鉴定及药敏试验[J].中国兽医科学,2017(3):366-374.
[18]YANG Q,ZHAO M,WANG K Y,et al. Multidrug-resistant Aeromonas veronii recovered from channel catfish(Ictalurus punctatus)in China:prevalence and mechanisms of fluoroquinolone resistance[J]. Microbial Drug Resistance,2016,23(4):473-479.
[19]彭开松,佘锐萍,吴襟.两种寡聚糖对罗非鱼生长及抗凡隆气单胞菌感染的影响[J].中国兽医科学,2011(1):41-47.
[20]HAO K,LIU J Y,LING F,et al. Effects of dietary administration of Shewanella haliotis,D4,Bacillus cereus,D7 and Aeromonas bivalvium,D15,single or combined,on the growth,innate immunity and disease resistance of shrimp,Litopenaeus vannamei[J]. Aquaculture,2014,428-429(3):141-149.
[21]WU Y S,LEE M C,HUANG C T,et al. Effects of traditional medical herbs“minor bupleurum decoction”on the non-specific immune responses of white shrimp(Litopenaeus vannamei)[J]. Fish&Shellfish Immunology,2017,64:218-225.