虾夷扇贝育苗阶段幼体细菌性病原的确定

刘继晨; 金晓杰; 李明; 陈娟; 郭宗明; 马悦欣

doi:10.3969/j.issn.1001-909X.2018.01.009

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海洋学研究 ›› 2018, Vol. 36 ›› Issue (1) : 86-92. DOI: 10.3969/j.issn.1001-909X.2018.01.009

研究论文

虾夷扇贝育苗阶段幼体细菌性病原的确定

刘继晨^1,3, 金晓杰^*1, 李明², 陈娟¹, 郭宗明¹, 马悦欣³

作者信息 +

Identification of bacterial infections associated with larval development of Yesso scallop

LIU Ji-chen^1,3, JIN Xiao-jie^*1, LI Ming², CHEN Juan¹, GUO Zong-ming¹, MA Yue-xin³

Author information +

文章历史 +

摘要

本研究确定了獐子岛集团虾夷扇贝Patinopecten yessoensis育苗过程中幼体大规模死亡的病原菌。采用TCBS培养基从患病幼体中分离弧菌,其数量为1.12×10⁴ CFU/g,以V1菌株和V2菌株为优势菌,占总弧菌数的64.3%和32.1%。用血琼脂平板测定V1菌株和V2菌株的溶血性,2株菌分别呈β溶血和α溶血。用2株菌分别感染扇贝幼体,幼体的死亡率显著高于对照组(P<0.05),症状与自然状态下的发病症状一致,并且从感染死亡的幼体中分离到原感染菌株,确定V1菌株和V2菌株是造成扇贝幼体大规模死亡的病原菌。对2株病原菌进行致病基因分析,结果发现V1菌株含有胞外金属蛋白酶基因。经16S rRNA基因测序分析,V1菌株(GenBank登录号KR232924)和V2菌株(GenBank登录号KR232925)与Vibrio splendidus ctt 31/5 和 Vibrio tasmaniensis 007的相似度分别为99.9%和99.2%。用药敏纸片扩散法检测病原菌对常见抗生素的敏感性,结果表明利福平、氟苯尼考和磺胺异恶唑均能有效抑制病原菌的生长。

Abstract

The pathogens causing mortality in Yesso scallop (Patinopecten yessoensisis) larvae in the hatchery of Zhangzidao Group Co. Ltd. were identified. A high mortality of the Yesso scallop larvae occurred during the seedling process: the larvae lost their ability to swim, their cilia fell off and their valves closed, they then sank to the bottom of the nursery pond and died. Vibrio has been reported as the primary pathogen for massive mortalities of different bivalves both in hatchery and in the field worldwide. Total viable vibrios counts in moribund larvae were 1.12×10⁴ CFU/g on Tryptone Citrate Bile Sucrose agar plates. Two dominant strains, named V1 and V2, accounted for 64.3% and 32.1% of total vibrios counts respectively. Blood agar plates were used to measure hemolysis of isolated strains. Strain V1 exhibited beta hemolysis, while strain V2 exhibited alpha hemolysis. A challenge test showed that the signs of infected larvae were identical to those of natural moribund larvae, the cumulative mortality caused by strains V1 and V2 was significantly higher than that of the control (P<0.05) and bacteria were re-isolated from the dead larvae. Strains V1 (GenBank Accession No: KR232924) and V2 (GenBank Accession No: KF232925) shared 99.9% and 99.2% identity with Vibrio splendidus ctt 31/5 and Vibrio tasmaniensis 007, respectively, by 16S rDNA sequencing. The presence of genes coding for virulence factors was examined by PCR analysis, showing that strain V1 possessed the extracellular metalloprotease (vsm) gene. Antibiotic susceptibility assays showed that both strains were sensitive to the rifampicin, florfenicol and sulfafurazole.

导出引用

刘继晨, 金晓杰, 李明, 陈娟, 郭宗明, 马悦欣. 虾夷扇贝育苗阶段幼体细菌性病原的确定[J]. 海洋学研究. 2018, 36(1): 86-92 https://doi.org/10.3969/j.issn.1001-909X.2018.01.009

LIU Ji-chen, JIN Xiao-jie, LI Ming, CHEN Juan, GUO Zong-ming, MA Yue-xin. Identification of bacterial infections associated with larval development of Yesso scallop[J]. Journal of Marine Sciences. 2018, 36(1): 86-92 https://doi.org/10.3969/j.issn.1001-909X.2018.01.009

中图分类号： S917.1

参考文献

[1] LI Wen-ji, TAN Ke-fei. The inspiration on massive mortality of scallop Patinopecten yessoensis resolved by Japan[J]. Fisheries Science, 2009, 28(10): 609-612.
李文姬, 谭克非. 日本解决虾夷扇贝大规模死亡的启示[J]. 水产科学, 2009, 28(10): 609-612.
[2] TORKILDSEN L, LAMBERT C, NYLUND A, et al. Bacteria associated with early life stages of great scallop, Pecten maximus: impact on larval survival[J]. Aquaculture International, 2005, 13(6): 575-592.
[3] NICOLAS J L, CORRE S, GAUTHIER G, et al. Bacterial problems associated with scallop Pecten maximus larval culture[J]. Diseases of Aquatic Organisms, 1996, 27(1): 67-76.
[4] ROJAS R, MIRANDA C D, OPAZO R, et al. Characterization and pathogenicity of Vibrio splendidus strains associated with massive mortalities of commercial hatchery-reared larvae of scallop Argopecten purpuratus(Lamarck, 1819)[J]. Journal of Invertebrate Pathology, 2015, 124(1): 61-69.
[5] PAILLARD C, LE ROUX F, BORREGO J J. Bacterial disease in marine bivalves, a review of recent studies: Trends and evolution[J]. Aquatic Living Resources, 2004, 17(4): 477-498.
[6] LIU Rui, QIU Li-mei, YU Zuo-an, et al. Identification and characterisation of pathogenic Vibrio splendidus from Yesso scallop (Patinopecten yessoensis) cultured in a low temperature environment[J]. Journal of Invertebrate Pathology, 2013, 114(2): 144-150.
[7] TENG Wei-ming, LI Wen-ji, ZHANG Ming, et al. Isolation, identification and pathogenicity of Vibrio chagasii from Patinopecten yessoensis[J]. Journal of Fisheries of China, 2012, 36(6): 937-943.
滕炜鸣, 李文姬, 张明, 等. 虾夷扇贝脓胞病病原的分离、鉴定与致病性[J]. 水产学报, 2012, 36(6): 937-943.
[8] SANDLUND N, TORKILDSEN L, MAGNESEN T, et al. Immunohistochemistry of great scallop Pecten maximus larvae experimentally challenged with pathogenic bacteria[J]. Diseases of Aquatic Organisms, 2006, 69(2-3): 163-173.
[9] GUISANDE J A, LAGO E P, PRADO S, et al. Genotypic diversity of culturable Vibrio species associated with the culture of oysters and clams in Galicia and screening of their pathogenic potential[J]. Journal of Shellfish Research, 2008, 27(4): 801-809.
[10] KESARCODI-WATSON A, KASPAR H, LATEGAN M J, et al. Two pathogens of Greenshell^TM mussel larvae, Perna canaliculus: Vibrio splendidus and a V. coralliilyticus/neptunius-like isolate[J]. Journal of Fish Diseases, 2009, 32(6): 499-507.
[11] 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 and Environmental Microbiology, 2015, 81(1): 292-297.
[12] BEAZ-HIDALGO R, DIÉGUEZ A L, CLEENWERCK I, et al. Vibrio celticus sp. nov., a new Vibrio species belonging to the Splendidus clade with pathogenic potential for clams[J]. Systematic and Applied Microbiology, 2010, 33(6): 311-315.
[13] GENARD B, MINER P, NICOLAS J L, et al. Integrative study of physiological changes associated with bacterial infection in Pacific Oyster larvae[J]. PLoS One, 2013, 8(5): e64534.
[14] PRADO S, DUBERT J, ROMALDE J L, et al. Vibrio ostreicida sp. nov., a new pathogen of bivalve larvae[J]. International Journal of Systematic and Evolutionary, 2014, 64(5): 1 641-1 646.
[15] KONEMAN E W, ALLEN S D, JANDA W M, et al. Diagnóstico Microbiológico[M]. Mexico City, Mexico: Médica Panamericana Press, 2001: 1-158.
[16] SUGUMAR G, NAKAI T, HIRATA Y, et al. Vibrio splendidus biovar II as the causative agent of bacillary necrosis of Japanese oyster Crassostrea gigas larvae[J]. Diseases of Aquatic Organisms, 1998, 33(2): 111-118.
[17] SUN Xue-ying, LIU Ji-chen, LI Ming, et al. Characterization of culturable bacterial diversity in the water of Yesso scallop Patinopecten yessoensis larva rearing tanks[J]. Journal of Dalian Ocean University, 2017, 32(3): 323-328.
孙雪莹, 刘继晨, 李明, 等. 虾夷扇贝苗种培育水中可培养细菌群落分析[J]. 大连海洋大学学报, 2017, 32(3): 323-328.
[18] GUAN Feng, GAO Wei, CHEN Ji-gang, et al. Virulence characteristics and antimicrobial susceptibility of three pathogenic bacteria isolated from the channels and ports in Ningbo and Zhoushan[J]. Journal of Marine Sciences, 2015, 33(4):77-82.
管峰, 高威, 陈吉刚, 等. 宁波-舟山港口航道3种致病性细菌毒力特征与耐药性[J]. 海洋学研究, 2015, 33(4):77-82.
[19] YE Ying-wu, WANG Yu-san, SHEN Zi-yu. National guide to clinical laboratory procedures[M]. second editon. Nanjing: Southeast University Press, 1997.
叶应妩, 王毓三, 申子瑜. 全国临床检验操作规程 [M].第二版.南京: 东南大学出版社, 1997.
[20] SCHULZE A D, ALABI A O, TATTERSALL-SHELDRAKE A R, et al. Bacterial diversity in a marine hatchery: Balance between pathogenic and potentially probiotic bacterial strains[J]. Aquaculture, 2006, 256(1-4): 50-73.
[21] RIQUELME C, TORANZO A E, BARJA J L, et al. Association of Aeromonas hydrophila and Vibrio alginolyticus with larval mortalities of scallop (Argopecten purpuratus)[J]. Journal of Invertebrate Pathology, 1996, 67(1): 213-218.
[22] LACOSTE A, NORMANNO G, ADDANTE N, et al. A Vibrio splendidus strain is associated with summer mortality of juvenile oysters Crassostrea gigas in the Bay of Morlaix (North Brittan y, France)[J]. Diseases of Aquatic Organisms, 2001, 46(2): 139-145.
[23] GARNIER M, LABREUCHE Y, GARCIA C, et al. Evidence for the involvement of pathogenic bacteria in summer mortalities of the Pacific oyster Crassostrea gigas[J]. Microbial Ecology, 2007,53(2): 187-196.
[24] WAECHTER M, LE ROUX F, NICOLAS J L, et al. Characterization of pathogenic bacteria of the cupped oyster Crassostrea gigas[J]. Comptes Rendus Biologies, 2002, 325(3): 231-238.
[25] GÓMEZ-LEÓN J, VILLAMIL L, LEMOS M L, et al. Isolation of Vibrio alginolyticus and Vibrio splendidus from aquacultured carpet shell clam (Ruditapes decussatus) larvae associated with mass mortalities[J]. Applied and Environmental Microbiology, 2005, 71(1): 98-104.
[26] BINESSE J, DELSERT C, SAULNIER D, et al. Metalloprotease vsm is the major determinant of toxicity for extracellular products of Vibrio splendidus[J]. Applied and Environmental Microbiology, 2008, 74(23): 7 108-7 117.
[27] LE ROUX F, BINESSE J, SAULNIER D, et al. Construction of a Vibrio splendidus mutant lacking the metalloprotease gene vsm by use of a novel counter selectable suicide vector[J]. Applied and Environmental Microbiology, 2007, 73(3): 777-784.
[28] LAGO E P, NIETO T P, SEGUIN R F. Fast detection of Vibrio species potentially pathogenic for mollusc[J]. Veterinary Microbiology, 2009, 139(3-4): 339-346.
[29] DENG Huan, HE Chong-bo, ZHOU Zun-chun, et al. Isolation and pathogenicity of pathogens from skin ulceration disease and viscera ejection syndrome of the sea cucumber Apostichopus japonicas[J]. Aquaculture, 2009,287(1-2): 18-27.
[30] VANHOVE A S, DUPERTHUY M, CHARRIÈRE G M, et al. Outer membrane vesicles are vehicles for the delivery of Vibrio tasmaniensis virulence factors to oyster immune cells[J]. Environmental Microbiology, 2015, 17(4): 1 152-1 165.
[31] MIRANDA C D, ROJAS R, ABARCA A, et al. Effect of florfenicol and oxytetracycline treatments on the intensive larval culture of the Chilean scallop Argopecten purpuratus (Lamarck, 1819)[J]. Aquaculture Research, 2014, 45(1): 16-30.
[32] DUBERT J, OSORIO C R, PRADO S, et al. Persistence of Antibiotic Resistant Vibrio spp. in Shellfish Hatchery Environment[J]. Microbial Ecology, 2015, 72(4) :1-10.
[33] RIQUELME C, JORQUERA M, ROJAS A, et al. Addition of inhibitor-producing bacteria to mass cultures of Argopecten purpuratus larvae (Lamarck, 1819)[J]. Aquaculture, 2001, 192(2-4): 111-119.
[34] KESARCODI-WATSON A, MINER P, NICOLAS J L, et al. Pathogenic threats and probiotic use in larviculture of the scallop, Pecten maximus[J]. Aquaculture Research, 2016, 47(4): 1 221-1 230.