Isolation and Identification of Pathogenetic Vibrio harveyi from Ark Shell Tegillarca granosa
TENG Shuangshuang1, ZHANG Jiongming1, ZHANG Libing2, XIE Shangshu3, WANG Yaohua1, XIAO Guoqiang1,4, YANG Qianyuan1,4, FANG Jun1
1.Wenzhou Key Laboratory of Marine Genetics and Breeding, Zhejiang Key Laboratory of Exploitation and Preservation of Coastal Bio-Resource, Zhejiang Mariculture Research Institute, Wenzhou 325005, China; 2.Yueqing Agricultural and Rural Bureau, Wenzhou 325699, China; 3.Ruian Agricultural and Rural Bureau, Wenzhou 325200, China; 4.College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 316022, China
Abstract：In order to study the species and related characteristics of the pathogenic strain J14 which was isolated from the sick ark shell Tegillarca granosa in Zhejiang province, the infection test, morphological observation, physiological and biochemical test, and 16S rRNA gene sequence analysis were performed. The results showed that the J14 strain was strong pathogenicty as the LC50 of J14 to ark shell was 7.74×106 cells/mL. The strain of J14 was gram negative, and exhibited round, bulged and yellow in thiosulfate-citrate-bile-salts-sucrose agar. Biochemical characteristics showed that the strain belonged to the Vibrio. The sequence analysis of 16S rRNA gene revealed that the strain shared high homology with V. harveyi using BLAST from GenBank database and the strain clustered with V. harveyi into one branch in the phylogenetic tree analysis. According to the morphological and physiological-biochemical characteristics, as well as the results of 16S rRNA gene sequence analysis, the strain was identified as V. harveyi. The results of virulence gene test showed that the strain carried seven typical virulence genes, including luxR, toxR, vhhA, vhhB, toxS, chiA and serine protease genes. The susceptibility test showed that the strain was not sensitive to penicillin, ampicillin, amoxicillin and bacitracin, moderately sensitive to erythromycin, polymyxin B, and sensitive to 26 different antibiotics such as gentamicin, kanamycin, and cotrimoxazole. The findings provide a reference with the identification of V. harveyi and the prevention and control of bacterial disease in ark shell.
[1]WANG H, LIU J W, WANG Y, et al. Vibrio marisflavi sp. nov. , isolated from seawater[J].International Journal of Systematic and Evolutionary Microbiology,2011,61(3):568-573. [2]徐先栋.华南海水养殖地区哈氏弧菌分子流行病学特征及其毒力菌株基因组分析[D].海口:海南大学,2017:1. [3]陈盛峰,陈佳,禤金彩,等.北海近岸养殖海域3种弧菌的分离鉴定及耐药性分析[J].动物医学进展,2016,37(6):29-34. [4]孟俞辰,牛雪可,邱文,等.中国东海海水中弧菌的分离鉴定及耐药性分析[J].贵州医科大学学报,2018,43(4):378-382. [5]张晓华,林禾雨,孙浩.弧菌科分类学研究进展[J].中国海洋大学学报(自然科学版),2018,48(8):43-56. [6]王瑞旋,耿玉静,冯娟,等.杂色鲍哈维弧菌耐药质粒的鉴定和分析[J].南方水产科学,2012,8(2):1-6. [7]曾德乾,冯娟,徐力文,等.海水养殖鱼哈维弧菌分离株的耐药谱型分析[J].中国水产科学,2015,22(1):129-138. [8]王保坤,余俊红,李筠,等.花鲈弧菌病病原菌(哈维氏弧菌)的分离与鉴定[J].中国水产科学,2002,9(1):52-55. [9]LI M F, WANG C L, SUN L. A pathogenic Vibrio harveyi lineage causes recurrent disease outbreaks in cultured Japanese flounder (Paralichthys olivaceus) and induces apoptosis in host cells[J].Aquaculture,2011,319(1/2):30-36. [10]TALPUR A D. Mentha piperita (Peppermint) as feed additive enhanced growth performance, survival, immune response and disease resistance of Asian seabass, Lates calcarifer (Bloch) against Vibrio harveyi infection[J].Aquaculture,2014,420/421:71-78. [11]HASHEM M, EL-BARBARY M. Vibrio harveyi infection in Arabian surgeon fish (Acanthurus sohal) of Red Sea at Hurghada, Egypt[J].The Egyptian Journal of Aquatic Research,2013,39(3):199-203. [12]MOHI M M, KURATANI M, MIYAZAKI T, et al. Histopathological studies on Vibrio harveyi-infected tiger puffer, Takifugu rubripes (Temminck et Schlegel), cultured in Japan[J].Journal of Fish Diseases,2010,33(10):833-840. [13]华昕彤,刘鹰,陈冰,等.红鳍东方鲀感染哈氏弧菌前后组织中socs3基因的表达差异分析[J].大连海洋大学学报,2019,34(1):33-40. [14]SUDHEESH P S, XU H S. Pathogenicity of Vibrio parahaemolyticus in tiger prawn Penaeus monodon Fabricius:possible role of extracellular proteases[J].Aquaculture,2001,196(1/2):37-46. [15]SERASPE E B, GABOTERO S, DELA PEA M R, et al. Evaluation of dietary freeze-dried Chaetoceros calcitrans supplementation to control Vibrio harveyi infection on Penaeus monodon juvenile[J].Aquaculture,2014,432:212-216. [16]HUANG H H, LIU X L, XIANG J H, et al. Immune response of Litopenaeus vannamei after infection with Vibrio harveyi[J].Aquaculture,2013,406/407:115-120. [17]朱贝贝,方皓,李煜,等.文蛤病原菌哈氏弧菌的分离鉴定与药敏试验[J].山东畜牧兽医,2017,38(9):4-5. [18]杨星星,管嘉兴,房文红,等.乐清湾泥蚶、缢蛏体内及养殖水环境细菌菌群的初步调查[J].海洋渔业,2003,25(2):73-76. [19]朱凝瑜,孔蕾,郑天伦.浙江省海产品中副溶血性弧菌污染情况分析[J].浙江农业科学,2013,54(8):1013-1015. [20]王娟娟,包永波,王素芳,等.泥蚶血红蛋白异源四聚体酶解多肽抗菌活性及其机理研究[J].海洋学报,2015,37(12):106-115. [21]周德庆,徐德强,胡宝龙.微生物学实验教程[M].3版.北京:高等教育出版社,2013:95-98. [22]LANE D J. 16S/23S rRNA sequencing[G]//STACKEBRANDT E, GOODFELLOW M. Nucleic acid techniques in bacterial systematics. New York:John Wiley & Sons,1991:115-175. [23]张晓君,毕可然,阎斌伦,等.中国对虾糠虾幼体病原哈氏弧菌的鉴定及毒力基因检测[J].渔业科学进展,2014,35(3):105-111. [24]CONEJERO M J U, HEDREYDA C T. Isolation of partial toxR gene of Vibrio harveyi and design of toxR-targeted PCR primers for species detection[J].Journal of Applied Microbiology,2003,95(3):602-611. [25]CONEJERO M J U, HEDREYDA C T. PCR detection of hemolysin (vhh) gene in Vibrio harveyi[J].The Journal of General and Applied Microbiology,2004,50(3):137-142. [26]RUWANDEEPIKA H A D, DEFOIRDT T, BHOW-MICK P P, et al. Presence of typical and atypical virulence genes in Vibrio isolates belonging to the Harveyi clade[J].Journal of Applied Microbiology,2010,109(3):888-899. [27]Clinical and Laboratory Standards Institute. M45, Methods for antimicrobial dilution and disksusceptibility testing of infrequently isolatedor fastidious bacteria[S].3rd ed. Wayne: CLSI,2016:34-35. [28]杭州微生物试剂有限公司.杭州微生物药敏试验纸片抑菌范围解释标准[EB/OL].(2018-03-09)[2020-07-08]. http://www.hangwei-media.com/support/t_doc/2018-03-09/6.html. [29]HOLT J G, KRIEG N R, SNEATH P H, et al. Bergey′s manual of determinative bacteriology[M].9th ed. London:Williams & Wikins Press,1994:190-194,259-271. [30]张晓华,钟英斌,陈吉祥.哈维氏弧菌的生物学特性、流行病学及检测技术[J].中国海洋大学学报(自然科学版),2007,37(5):740-748. [31]陈献稿,吴淑勤,石存斌,等.斜带石斑鱼病原菌(哈维氏弧菌)的分离与鉴定[J].中国水产科学,2004,11(4):313-317. [32]谢珍玉,胡超群.弧菌毒力基因水平转移与进化的研究进展[J].热带海洋学报,2005,24(3):86-95. [33]XU X D, LIU K F, WANG S F, et al. Identification of pathogenicity, investigation of virulent gene distribution and development of a virulent strain-specific detection PCR method for Vibrio harveyi isolated from Hainan Province and Guangdong Province, China[J].Aquaculture,2017,468:226-234. [34]NAKAMURA Y, ITOH T, MATSUDA H, et al. Biased biological functions of horizontally transferred genes in prokaryotic genomes[J].Nature Genetics,2004,36(7):760-766. [35]OCHMAN H, LAWRENCE J G, GROISMAN E A. Lateral gene transfer and the nature of bacterial innovation[J].Nature,2000,405(6784):299-304. [36]蒋魁,徐力文,苏友禄,等.两株珍珠龙趸病原性哈维弧菌(Vibrio harveyi)的分离与鉴定[J].生态科学,2017,36(6):16-24. [37]蒋魁,徐力文,苏友禄,等.2012年—2014年南海海水养殖鱼类病原菌哈维弧菌分离株的耐药性分析[J].南方水产科学,2016,12(6):99-107. [38]王春艳,阎斌伦.连云港市赣榆县水产养殖环境中哈氏弧菌耐药性研究[J].淮海工学院学报(自然科学版),2013,22(1):88-91. [39]RAISSY M, MOUMENI M, ANSARI M, et al. Antibiotic resistance pattern of some Vibrio strains isolated from seafood [J]. Iranian Journal of Fisheries Sciences,2012,11(3):618-626. [40]吴越,郑伊诺,陆荣茂,等.温州地区水产品中哈维氏弧菌的耐药分析[J].水产科学,2020,39(6):837-843. [41]水产养殖用药明白纸[R].北京:中华人民共和国农业农村部渔业渔政管理局,2019.
2022, Vol. 41 
