Isolation, Identification and Characteristics of Pathogen from Sea Cucumber Apostichopus japonicus with Skin Ulceration Syndrome
LI Xiaoyu1,2,3, FENG Lijuan1, XU Le1, WANG Lili1,2,3, ZHANG Meixia1, LI Shuying3,4, LI Jibin3,4, XU Yongping1,2,3
1. School of Bioengineering, Dalian University of Technology, Dalian 116024, China; 2. Center for Food Safety of Animal Origin, Ministry of Education, Dalian 116600, China; 3. Professional Technology Innovation Center for Phage Application of Liaoning Province, Dalian 116600, China; 4. Dalian SEM Bio-Engineering Technology Co. Ltd., Dalian 116600, China
Abstract：In order to explore the death causes of sea cucumber Apostichopus japonicus with skin ulceration from a local farm in Dalian, a dominant bacterial strain AP-1 was isolated from the diseased sea cucumbers. The 16S rDNA sequence, morphological observation, physiological and biochemical characteristics results showed that it was Vibrio alginolyticus. The strain was artificially infected back to the healthy sea cucumber by the means of immersion infection, body wall wound immersion infection and intraperitoneal injection infection, respectively. The results showed that the immersion infection did not show pathological symptoms, whereas other two methods showed different degrees of dose-dependent lethality. The LC50 of the sea cucumber with the isolate was 5.23×106 cfu/mL in body wall wound immersion infection and 3.08×104 cfu/g in intraperitoneal injection infection. Virulence gene detection showed that the strain was positively amplified with the seven virulence genes including toxR, FlaA, Collagenase, fur, AspA, ompW and tlh. Drug resistance analysis showed that the strain was resistant to ampicillin, piperacillin, augmentin, cefazolin, cefotaxime and amikacin, and was intermediate to cefuroxime, cefoxitin, chloramphenicol, aztreonam and neomycin. However, it was sensitive to other 16 antibiotics. This finding will provide scientific basis and reference for sea cucumber industry in Dalian.
[1]PANGESTUTI R, ARIFIN Z. Medicinal and health benefit effects of functional sea cucumbers[J].Journal of Traditional and Complementary Medicine,2018,8(3):341-351. [2]王印庚,廖梅杰,李彬,等.海参“3.15”事件解读及产业可持续发展思考[J].科学养鱼,2020(9):4-6. [3]SWEET M J, BATEMAN K S. Diseases in marine invertebrates associated with mariculture and commercial fisheries[J].Journal of Sea Research,2015,104:16-32. [4]HAN Q X, KEESING J K, LIU D Y. A review of sea cucumber aquaculture, ranching, and stock enhancement in China[J].Reviews in Fisheries Science & Aquaculture,2016,24(4):326-341. [5]张春云,陈国福,徐仲,等.海参疾病学研究进展[J].水产科学,2011,30(10):644-648. [6]李强,孙康泰,张显昱.刺参“腐皮综合征”研究进展[J].中国农业科技导报,2013,15(6):40-45. [7]杨求华,葛辉,方旅平,等.池塘养殖刺参病原菌塔式弧菌的分离与鉴定[J].南方水产科学,2014,10(4):45-51. [8]魏霜.溶藻弧菌及其毒力相关基因的检测[D].广州:暨南大学,2013. [9]郭静.溶藻弧菌相关分离株分子鉴定和生理生态特性研究[D].宁波:宁波大学,2012. [10]Clinical and Laboratory Standards Institute. M45, Methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious bacteria[S]. Wayne: CLSI,2015. [11]杨昆明,张文润,马江霞,等.鲟源致病性鲁氏耶尔森菌的分离、鉴定及药敏研究[J].水产科学,2019,38(1):48-54. [12]GRIMES D J. The vibrios:scavengers, symbionts, and pathogens from the sea[J].Microbial Ecology,2020,80(3):501-506. [13]BUNPA S, CHAICHANA N, TENG J L L, et al. Outer membrane protein A (OmpA) is a potential virulence factor of Vibrio alginolyticus strains isolated from diseased fish[J].Journal of Fish Diseases,2020,43(2):275-284. [14]ZUO Y F, ZHAO L M, XU X J, et al. Mechanisms underlying the virulence regulation of new Vibrio alginolyticus ncRNA Vvrr1 with a comparative proteomic analysis[J].Emerging Microbes & Infections,2019,8(1):1604-1618. [15]李颖,关国华.微生物生理学[M].北京:科学出版社,2013. [16]赵小然,李艳,王莉芬,等.刺参体腔细胞表面抗原分类及体壁组织中免疫细胞的研究[J].大连海洋大学学报,2019,34(4):470-474. [17]LI H, QIAO G, LI Q, et al. Biological characteristics and pathogenicity of a highly pathogenic Shewanellamarisflavi infecting sea cucumber, Apostichopus japonicus[J].Journal of Fish Diseases,2010,33(11):865-877. [18]肖培华,唐永新,于乐河,等.刺参病害细菌感染试验[J].齐鲁渔业,2005,22(6):26-27. [19]杨嘉龙,周丽,绳秀珍,等.养殖刺参溃疡病病原菌RH2的鉴定及其生物学特性分析[J].水产学报,2007,31(4):504-511. [20]郑世忠,刘广东.大连海水养殖风险及其影响因素研究[J].渔业信息与战略,2019,34(3):194-198. [21]LI R J, DU Y P, MA H L, et al. Wound healing at the body wall of the sea cucumber Apostichopus japonicus by Chinese herbal treatment[J].Aquaculture Research,2021,52(2):693-701. [22]LYU T, SONG T X, LIU H J, et al. Isolation and characterization of a virulence related Vibrio alginolyticus strain Wz11 pathogenic to cuttlefish, Sepia pharaonis[J].Microbial Pathogenesis,2019,126:165-171. [23]张晶,陈佳璇,伍业健,等.广州地区水产品及外环境水体溶藻弧菌污染状况与毒力相关基因分析[J].中国卫生检验杂志,2016,26(4):539-541. [24]BRICKNELL I R, KING J A, BOWDEN T J, et al. Duration of protective antibodies, and the correlation with protection in Atlantic salmon (Salmosalar L.), following vaccination with an Aeromonas salmonicida vaccine containing iron-regulated outer membrane proteins and secretory polysaccharide[J].Fish & Shellfish Immunology,1999,9(2):139-151. [25]BORDAS M A, BALEBONA M C, ZORRILLA I, et al. Kinetics of adhesion of selected fish-pathogenic Vibrio strains of skin mucus of gilthead sea bream (Sparus aurata L.)[J].Applied and Environmental Microbiology,1996,62(10):3650-3654. [26]程海燕.溶藻弧菌毒力相关基因toxR、acfA的功能研究[D].湛江:广东海洋大学,2014. [27]杨霞,潘吉脉,张飘,等.鲤源杀鲑气单胞菌无色亚种的分离鉴定及毒力基因检测[J].扬州大学学报(农业与生命科学版),2021,42(1):24-29. [28]JIA A R, WOO N Y S, ZHANG X H. Expression, purification, and characterization of thermolabile hemolysin (TLH) from Vibrio alginolyticus[J].Diseases of Aquatic Organisms,2010,90(2):121-127. [29]李雪,张眉眉,张铭琰,等.辽宁省溶藻弧菌REP-PCR和PFGE分子分型、耐药谱的研究[J].中国微生态学杂志,2018,30(5):535-540. [30]封会茹,游京蓉,刘玉堂,等.溶藻弧菌引起暴发型食物中毒的病原学研究[J].中国食品卫生杂志,2003,15(4):331-334.
2022, Vol. 41 
