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Isolation, identification and characteristics of pathogenic bacteria from Paralichthys olivaceus with ascites

  • Corresponding author: Lingyun QU, qly@fio.org.cn
  • Received Date: 2018-12-20
    Accepted Date: 2019-04-26
  • In order to determine the pathogen, which can cause the death of cultured Paralichthys olivaceus from ascites disease in Beidaihe area, Hebei Province, three dominant bacteria were isolated from P.olivaceus infected with ascites. The biological status of the isolates was determined by physiological and biochemical identification and 16S rRNA sequence alignment. The pathological characteristics of the isolates were further identified by virulence genes (toxR, vhhA, vhhB) and histopathological analysis. The results showed that the three isolates were Vibrio harveyi and the virulence genes of the three isolates were all positive. Pathological sections showed that the isolates could be used to treat multiple organs (intestine, kidney, spleen and liver) of P.olivaceus. The LD50 of strain BDHYPFS-Y1G to P.olivaceus was 5.88×106 CFU/g, which was lower than the toxicity of natural state. Drug susceptibility test indicated that all three isolates were highly sensitive to the nitrofurantoin. This study confirmed the pathogen of this ascites disease, and preliminarily studied the pathogenicity and drug sensitivity of the pathogen, which can provide scientific basis for the prevention and control of the disease in industrial culture of flounder.
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  • [1] 朱壮春, 史相国, 张淑杰, 等. 牙鲆腹水病病原研究[J]. 水产科学, 2006, 25(7): 325-329. doi: 10.3969/j.issn.1003-1111.2006.07.001Zhu Z C, Shi X G, Zhang S J, et al. The pathogenic bacteria of the ascites in Japanese flounder (Paralichthys olivaceus)[J]. Fisheries Science, 2006, 25(7): 325-329(in Chinese). doi: 10.3969/j.issn.1003-1111.2006.07.001
    [2] 李筠, 颜显辉, 陈吉祥, 等. 养殖大菱鲆腹水病病原的研究[J]. 中国海洋大学学报, 2006, 36(4): 649-654.Li Y, Yan X H, Chen J X, et al. Studies on the characteristics of pathogenic Edwardsiella tarda isolated from diseased Scophthalmus maximus[J]. Periodical of Ocean University of China, 2006, 36(4): 649-654(in Chinese).
    [3] 张伟妮, 周丽, 邢婧, 等. 养殖大菱鲆腹水症病原菌SR1的分离及鉴定[J]. 中国水产科学, 2006, 13(4): 603-609. doi: 10.3321/j.issn:1005-8737.2006.04.015Zhang W N, Zhou L, Xing J, et al. Isolation and identification of pathogen SR1 associated with swollen abdomen of cultured turbot (Scophthalmus maximus)[J]. Journal of Fishery Sciences of China, 2006, 13(4): 603-609(in Chinese). doi: 10.3321/j.issn:1005-8737.2006.04.015
    [4] 孙祎敏, 李楠, 宋杰, 等. 褐牙鲆腹水症病原菌的分离鉴定及其灭活疫苗的研制[J]. 水产科学, 2009, 28(11): 613-617. doi: 10.3969/j.issn.1003-1111.2009.11.001Sun Y M, Li N, Song J, et al. Isolation and identification of bacterial pathogen Aeromonas hydrophila and development of the inactivated vaccine in Japanese flounder Paralichthys olivaceus diseased with ascides[J]. Fisheries Science, 2009, 28(11): 613-617(in Chinese). doi: 10.3969/j.issn.1003-1111.2009.11.001
    [5] 胡璇, 孙敬锋, 陈成勋, 等. 养殖半滑舌鳎腹水病的病原分离鉴定及药物敏感性分析[J]. 天津农学院学报, 2014, 21(3): 12-16. doi: 10.3969/j.issn.1008-5394.2014.03.004Hu X, Sun J F, Chen C X, et al. Isolation and identification of pathogen of ascites disease from Cynoglossus semilaevis günther and its antibiotic sensitivity analysis[J]. Journal of Tianjin Agricultural University, 2014, 21(3): 12-16(in Chinese). doi: 10.3969/j.issn.1008-5394.2014.03.004
    [6] 王岚, 王印庚, 张正, 等. 养殖大菱鲆(Scophthalmus maximus)腹水病的病原多样性及其耐药性分析[J]. 渔业科学进展, 2017, 38(4): 17-24.Wang L, Wang Y G, Zhang Z, et al. Diversity and drug resistance of bacterial pathogens isolated from bacterial ascetic disease in cultured turbot Scophthalmus maximus[J]. Progress in Fishery Sciences, 2017, 38(4): 17-24(in Chinese).
    [7] 刘玉娟, 田新朋, 黄小芳, 等. 中国南海沉积环境可培养细菌多样性研究[J]. 微生物学通报, 2014, 41(4): 661-673.Liu Y J, Tian X P, Huang X F, et al. Diversity of cultivable bacteria isolated from marine sediment environments in South China Sea[J]. Microbiology China, 2014, 41(4): 661-673(in Chinese).
    [8] 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. doi: 10.1046/j.1365-2672.2003.02020.x
    [9] 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. doi: 10.2323/jgam.50.137
    [10] 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.Jiang K, Xu L W, Su Y L, et al. Isolation and identification of two pathogenetic Vibrio harveyi strains from pearl gentian[J]. Ecological Science, 2017, 36(6): 16-24(in Chinese).
    [11] 薛淑霞, 冯守明, 孙金生. 海水工厂化养殖大菱鲆(Scophthalmus maximus)和褐牙鲆(Paralichthys olivaceus)腹水病病原菌的分离与鉴定[J]. 海洋与湖沼, 2006, 37(6): 548-554. doi: 10.3321/j.issn:0029-814X.2006.06.012Xue S X, Feng S M, Sun J S. Isolation and identification of pathogenic bacteria in swollen abdomen of cultured turbot (Scophthalmus maximus) and flounder (Paralichthys olivaceus)[J]. Oceanologia et Limnologia Sinica, 2006, 37(6): 548-554(in Chinese). doi: 10.3321/j.issn:0029-814X.2006.06.012
    [12] 王亚军, 魏文娟, 潘厚军, 等. 斑点叉尾鮰" 腹水症”病原的分离鉴定及体外芽孢杆菌拮抗试验[J]. 淡水渔业, 2018, 48(3): 72-77. doi: 10.3969/j.issn.1000-6907.2018.03.012Wang Y J, Wei W J, Pan H J, et al. Isolation and identification of the pathogen Aeromonas sobria from Ictalurus punctatus and the Bacillus antagonistic experiment in vitro[J]. Freshwater Fisheries, 2018, 48(3): 72-77(in Chinese). doi: 10.3969/j.issn.1000-6907.2018.03.012
    [13] Oakey H J, Owens L. A new bacteriophage, VHML, isolated from a toxin-producing strain of Vibrio harveyi in tropical Australia[J]. Journal of Applied Microbiology, 2000, 89(4): 702-709. doi: 10.1046/j.1365-2672.2000.01169.x
    [14] Munro J, Oakey J, Bromage E, et al. Experimental bacteriophage-mediated virulence in strains of Vibrio harveyi[J]. Diseases of Aquatic Organisms, 2003, 54(3): 187-194. doi: 10.3354/dao054187
    [15] 钟英斌, 张晓华, 陈吉祥, 等. 哈维氏弧菌溶血素基因vhhA在大肠杆菌中的表达及活性研究[J]. 中国海洋大学学报, 2007, 37(1): 97-102.Zhong Y B, Zhang X H, Chen J X, et al. Expression of Vibrio harveyi hemolysin gene vhhA in E.coli and its biological activities[J]. Periodical of Ocean University of China, 2007, 37(1): 97-102(in Chinese).
    [16] 许国晶, 绳秀珍, 战文斌. 温度对牙鲆皮肤黏液抗体产生的影响[J]. 中国水产科学, 2011, 18(6): 1377-1383.Xu G J, Sheng X Z, Zhan W B. Effect of temperature on production of skin mucus antibody in flounder (Paralichthys olivaceus)[J]. Journal of Fishery Sciences of China, 2011, 18(6): 1377-1383(in Chinese).
    [17] 钟硕良, 陈月忠, 周宸. 环境因子对发光细菌的生长及日本对虾仔虾感染死亡率的影响[J]. 中国水产科学, 2001, 8(1): 41-45. doi: 10.3321/j.issn:1005-8737.2001.01.010Zhong S L, Chen Y Z, Zhou C. Effects of environmental factors on growth of luminous bacteria (Vibrio harveyi) and mortality of infected post-larvae of Penaeus japonicus[J]. Journal of Fishery Sciences of China, 2001, 8(1): 41-45(in Chinese). doi: 10.3321/j.issn:1005-8737.2001.01.010
    [18] 陈建国, 陈超, 李炎璐, 等. 美洲黑石斑鱼(Centropristis striata)" 突眼症”的病原菌分离鉴定[J]. 渔业科学进展, 2017, 38(5): 32-40. doi: 10.11758/yykxjz.20160504001Chen J G, Chen C, Li Y L, et al. Isolation and classification of exophthalmic disease-inducing pathogenic bacterium from Centropristis striata[J]. Progress in Fishery Sciences, 2017, 38(5): 32-40(in Chinese). doi: 10.11758/yykxjz.20160504001
    [19] Austin B, Zhang X H. Vibrio harveyi: A significant pathogen of marine vertebrates and invertebrates[J]. Letters in Applied Microbiology, 2006, 43(2): 119-124. doi: 10.1111/lam.2006.43.issue-2
    [20] 王力, 张吉鹏, 刘美如, 等. 红鳍东方鲀源哈维弧菌毒力基因检测及分型研究[J]. 现代农业科技, 2018(2): 237-239, 242. doi: 10.3969/j.issn.1007-5739.2018.02.133Wang L, Zhang J P, Liu M R, et al. Virulence genes detection and BOX-PCR identification of Vibrio harveyi strains isolated from Takifugu rubripes[J]. Modern Agricultural Science and Technology, 2018(2): 237-239, 242(in Chinese). doi: 10.3969/j.issn.1007-5739.2018.02.133
    [21] Prayitno S B, Latchford J W. Experimental infections of crustaceans with luminous bacteria related to Photobacterium and Vibrio. Effect of salinity and pH on infectiosity[J]. Aquaculture, 1995, 132(1-2): 105-112. doi: 10.1016/0044-8486(94)00374-w
    [22] Tu Z G, Li H Y, Zhang X, et al. Complete genome sequence and comparative genomics of the golden pompano (Trachinotus ovatus) pathogen, Vibrio harveyi strain QT520[J]. PeerJ, 2017, 5: e4127. doi: 10.7717/peerj.4127
    [23] Qin Y X, Wang J, Su Y Q, et al. Studies on the pathogenic bacterium of ulcer disease in Epinephelus awoara[J]. Acta Oceanologica Sinica, 2006, 25(1): 154-159.
    [24] Becker P, Gillan D, Lanterbecq D, et al. The skin ulceration disease in cultivated juveniles of Holothuria scabra (Holothuroidea, Echinodermata)[J]. Aquaculture, 2004, 242(1-4): 13-30. doi: 10.1016/j.aquaculture.2003.11.018
    [25] Tendencia E A. The first report of Vibrio harveyi infection in the sea horse Hippocampus kuda Bleekers 1852 in the Philippines[J]. Aquaculture Research, 2004, 35(13): 1292-1294. doi: 10.1111/j.1365-2109.2004.01109.x
    [26] 张美霞, 耿慧君, 王丽丽, 等. 哈维弧菌对水产动物的致病性及其噬菌体防控研究进展[J]. 中国抗生素杂志, 2017, 42(9): 717-723. doi: 10.3969/j.issn.1001-8689.2017.09.001Zhang M X, Geng H J, Wang L L, et al. Research progress in using bacteriophages on the pathogenicity of Vibrio harveyi to aquatic animals and its biocontrol approach[J]. Chinese Journal of Antibiotics, 2017, 42(9): 717-723(in Chinese). doi: 10.3969/j.issn.1001-8689.2017.09.001
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Isolation, identification and characteristics of pathogenic bacteria from Paralichthys olivaceus with ascites

    Corresponding author: Lingyun QU, qly@fio.org.cn
  • 1. First Institute of Oceanography, MNR, Qingdao    266061, China
  • 2. Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao    266235, China
  • 3. Dalian Ocean University, Dalian    116023, China
  • 4. Beidaihe Central Experiment Station Chinese Academy of Fishery Sciences, Beidaihe    066100, China
  • 5. The Affiliated Hospital of Qingdao University, Qingdao    266032, China
  • 6. Qilu University of Technology, Jinan    250353, China

Abstract: In order to determine the pathogen, which can cause the death of cultured Paralichthys olivaceus from ascites disease in Beidaihe area, Hebei Province, three dominant bacteria were isolated from P.olivaceus infected with ascites. The biological status of the isolates was determined by physiological and biochemical identification and 16S rRNA sequence alignment. The pathological characteristics of the isolates were further identified by virulence genes (toxR, vhhA, vhhB) and histopathological analysis. The results showed that the three isolates were Vibrio harveyi and the virulence genes of the three isolates were all positive. Pathological sections showed that the isolates could be used to treat multiple organs (intestine, kidney, spleen and liver) of P.olivaceus. The LD50 of strain BDHYPFS-Y1G to P.olivaceus was 5.88×106 CFU/g, which was lower than the toxicity of natural state. Drug susceptibility test indicated that all three isolates were highly sensitive to the nitrofurantoin. This study confirmed the pathogen of this ascites disease, and preliminarily studied the pathogenicity and drug sensitivity of the pathogen, which can provide scientific basis for the prevention and control of the disease in industrial culture of flounder.

  • 腹水病是鲆鲽鱼类养殖过程中危害严重的疾病之一。国内报道的鲆鲽鱼类腹水病病原包括迟缓爱德华氏菌(Edwardsiella tarda)[1-2]、溶藻弧菌(Vibrio alginolyticus)[3]、嗜水气单胞菌(Aeromonas hydrophila)[4]、副溶血弧菌(Vibrio parahaemolyticus)[5]等。夏季是养殖鲆鲽鱼类腹水病频繁暴发的时期,患腹水病病鱼的主要特征为病鱼行动迟缓、摄食能力减弱、体表变黑、腹部膨胀,失去原有的体型比例;鱼体腹腔或肠道内有不同程度无色或淡黄色积液,肝脏由淡红色变成米黄色[6]。由于鲆鲽鱼类腹水病可由单个或多个病原菌共同引发,且传染性强、死亡率高,对不同地区病原菌的鉴定、腹水病的防控以及患病鱼的治疗造成了一定的困难。因此,加强对鲆鲽鱼类腹水病病原的分离和鉴定,详细了解鲆鲽鱼类腹水病病原对有效防控鲆鲽鱼类腹水病具有重要意义。

    2018年9月,河北北戴河地区养殖牙鲆发生腹水病,在半个月的病程中牙鲆累计死亡率超过60%。为查清病原,本实验室对患有腹水症的濒死牙鲆进行了病原菌分离纯化和验证,以期为牙鲆生产养殖过程中有效防治腹水病提供有效参考。

    • 患病牙鲆取自河北北戴河地区某牙鲆养殖厂,挑选具有典型腹水症状的牙鲆,体质量为(34±1) g,体长为(15±1) cm;健康牙鲆购自青岛胶南养殖场,体质量为(20±1) g,体长为(13±1) cm。

    • 无菌条件下,取具有典型腹水症状的濒死牙鲆的肝脏、肾脏、脾脏等病灶组织,研磨稀释后涂布于TCBS培养基和2216 E培养基,30 °C培养24 h后肉眼观察菌落形态、大小、颜色等,挑取不同形态的优势菌落进行3次以上的纯化培养,保存备用,菌株编号分别为BDHYPFS-Y1G、BDHYPFS-Y2P、BDHYPFS-Y2S。

    • 取健康和自然发病牙鲆的肝脏、脾脏、肾脏等组织,于Bouin氏液固定12~24 h后转入75%酒精保存。组织样品通过清洗、脱水、石蜡包埋、切片、H E染色、清洗及封片后,在倒置生物显微镜(尼康TE2000-U)下观察并拍照记录。

    • 将分离株BDHYPFS-Y1G、BDHYPFS-Y2P、BDHYPFS-Y2S分别接种于2216E固体培养基上,28 °C恒温培养24 h后,用无菌接种环挑取适量单菌落接种于细菌微量鉴定管中,用无菌封口膜封口,(36±1)°C恒温培养。

    • 挑取新鲜培养的单菌落于无菌水中,沸水浴99 °C 20 min,提取分离株的DNA。利用通用引物扩增16S rRNA基因全长,同时利用特异性引物检测其毒力基因(表1)。反应体系为60 μL:10 μmol/L的正/反向引物各3 μL,DNA模板3 μL,2×Taq mix 30 μL,无菌水补至60 μL。反应条件:95 °C预变性5 min,94 °C 变性 45 s,42~55 °C退火45 s(不同引物退火温度不同,表1),72 °C延伸 90 s,34个循环;72 °C终延伸10 min。PCR反应产物送至生工生物工程(上海)股份有限公司测序。将返回的测序菌种的 16S rRNA基因扩增序列通过EzBio Cloud数据库进行序列同源性分析,比对后将该菌种的16S rRNA基因扩增序列提交到GenBank进行序列号的注册,获取登录号。用Clustal W软件将NCBI数据库中获得的相似度较高的序列和其他种属的序列进行多序列匹配(multiple alignments),用MEGA 6.0采用邻接法(neighbor joining method)构建系统发育树, 并通过自举分析(bootstrap)进行置信度检测,自举数据集为1 000次。

      基因
      gene
      片段大/bp
      fragment size
      PCR引物序列(5′-3′)
      primer sequence of PCR
      退火温度/°C
      annealing temperature
      参考文献
      references
      16S rRNA 1 500 27-F:AGAGTTTGATCCTGGCTCAG
      1492rrrr:GGTTACCTTGTTACGACTT
      55 Liu等[7]
      toxR 390 ToxR-F:TTCTGAAGCAGCACTCAC
      ToxR-R:TCGACTGGTGAAGACTCA
      42 Conejero等[8]
      vhhA 1300 vhhA-F:ATCATGAATAAAACTATTACGTTACT
      vhhA-R:GAAAGGATGGTTTGACAAT
      53 Conejero等[9]
      vhhB 216 VhhB-F:TCAGTGCCTCTCAAGTAAGA
      VhhB-R:GCTTGATAACACTTTGCGGT
      53 Conejero等[9]

      Table 1.  Primer sequence of ascites pathogen

    • 按照蒋魁[10]等人的方法,取健康牙鲆幼鱼180尾,暂养一周后进行人工回感试验。将纯化的菌株BDHYPFS-Y1G用无菌生理盐水制成菌悬液,将菌液浓度调整成108 CFU/mL,十倍梯度稀释至104 CFU/mL,共5个实验组,每组3个平行,每个平行10尾鱼,每尾鱼腹腔注射0.3 mL菌悬液,对照组注射同等体积的生理盐水。在水温(19±1) °C下连续观察7 d并记录鱼的发病特征、死亡情况及濒死症状。解剖观察濒死鱼体内的器官病变情况,取病变组织及腹水进行病原菌的分离鉴定。采用寇氏法计算菌株BDHYPFS-Y1G的半致死量:

      Xg为死亡率为 100%组的对数剂量, d对数组距, ∑P为各组死亡率之和。

    • 采用药敏纸片扩散法对3株优势菌进行药敏试验。取100 μL新鲜菌液涂布于2216E琼脂培养基上,用无菌镊子将药敏纸片(购于杭州微生物试剂有限公司)贴于培养基表面,30 °C培养24 h后,根据杭州微生物试剂有限公司提供的《药敏试验纸片法的抑菌范围解释标准》,确定病原菌株对抗生素的耐药性测定抑菌圈直径。

    2.   结果
    • 患病牙鲆表现为食欲减退或丧失,体色发黑,游动失去平衡,游动力减弱,腹部肿大,(图Ⅰ-1)严重时肠道挤出肛门外,半个月内累计死亡率高达60%以上。濒死病鱼解剖后肠道底部肿大(图Ⅰ-2),部分鱼体肠道肿胀透明,腹腔内充满淡黄色腹水(图Ⅰ-3),肠道内少量白便,肝脏呈乳白色(图Ⅰ-4),部分鱼体肝脏有出血点。

      Figure 图版I.  Clinical symptoms of naturally infected P. olivaceus

    • 与对照组相比,患病牙鲆肠道上皮组织中的杯状细胞数量增多,固有层毛细血管网萎缩,黏膜层和黏膜固有层组织分离(图Ⅱ-2)。患病牙鲆肝脏内呈明显脂肪变性和空泡变性,细胞核被挤压至一侧,胞质间出现大小不等的空泡,细胞界限不明显,胞内物质凝缩,染色变深嗜碱性,肝组织出现血红细胞侵润(图Ⅱ-4)。患病牙鲆脾脏中的组织结构松散,红髓、白髓结构不清晰,椭圆体萎缩,结构变形出现大小不等的缝隙(图Ⅱ-6)。患病牙鲆肾脏的造血组织出现局灶性大面积坏死;肾小球上皮细胞萎缩溶解,基膜解体破碎,肾间质结构松散(图Ⅱ-8)。

      Figure 图版II.  Histopathological figure of diseased P. olivaceus

    • 通过分离纯化方法分别在病鱼的肾脏、脾脏、肝脏中获得3株优势菌,其菌落特征较为一致,在2216E培养基上均为灰白色、不透明、平滑、边缘无透明圈 (图1-1);在TCBS培养基上为黄色、不透明、平滑、边缘无透明圈(图1-2)。

      Figure 1.  Colony morphologies of BDHYPFS-Y1G on different plates

    • 利用细菌微量鉴定管对分离株BDHYPFS-Y1G、BDHYPFS-Y2P、BDHYPFS-Y2S进行鉴定,结果显示,3株分离株生理生化鉴定结果相同,均可发酵肌醇、山梨糖醇、鼠李糖、蔗糖、蜜二糖、苦杏仁苷、甘露醇、鼠李糖、蔗糖、甘露醇、山梨醇、密二糖、苦杏仁苷、棉子糖、水杨苷、麦芽糖;可以进行赖氨酸脱羧、鸟氨酸脱羧、色氨酸脱羧酶、尿素酶、VP、明胶液化、葡萄糖产气;在1%、3%、6%、10%Nacl胨水中生长;精氨酸双水解酶、阿拉伯糖发酵、H2S产气等11个项目均为阴性(表2)。

      试验项目
      test item
      结果 result试验项目
      test item
      结果 result
      123123
      阿拉伯糖 arabinose 鼠李糖 rhamnose + + +
      H2S - - - 蔗糖 sucrose + + +
      明胶 gelatin + + + 甘露醇 mannitol + + +
      赖氨酸脱羧酶 lysine decarboxylase + + + 山梨醇 sorbitol + + +
      鸟氨酸脱羧酶 ornithine decarboxylase + + + 密二糖 melibiose + + +
      精氨酸双水解酶 arginine dihydrolase - - - 苦杏仁苷 amygdalin + + +
      色氨酸脱羧酶 tryptophane decarboxylase - - - 肌醇发酵 inositol fermentation - - -
      ONPG - - - 棉子糖 raffinose + + +
      葡萄糖产气 glucose gas production + + + 七叶苷 raffinose - - -
      V-P + + + 水杨苷 saligenin + + +
      尿素酶 urea enzymes + + + 木糖 xylose - - -
      西蒙氏橼酸盐 simone citrate - - - 无盐胨水 no salt peptone water - - -
      脂酶 lipoidase - - - 1%NaCl + + +
      麦芽糖 maltose + + + 3%NaCl + + +
      吲哚试验 indole test - - - 6%NaCl + + +
      10%NaCl + + +
      注:+. 表示阳性,-. 表示阴性;1. 菌株BDHYPFS-Y2S,2. 菌株BDHYPFS-Y2P,3. 菌株BDHYPFS-Y1G
      Notes:+. positive, -. negative; 1. bacteria of BDHYPFS-Y2S, 2. bacteria of BDHYPFS-Y2P, 3. bacteria of BDHYPFS-Y1G

      Table 2.  Physiological and biochemical characteristics of pathogenic bacteria

    • 对菌株BDHYPFS-Y2S、BDHYPFS-Y2P、BDHYPFS-Y1G进行PCR扩增,得到的16S rRNA基因片段序列结果输入到EzBio Cloud数据库中进行序列比对,发现菌株BDHYPFS-Y2S、BDHYPFS-Y2P、BDHYPFS-Y1G均为哈氏弧菌,并与哈氏弧菌参考菌株同源性分别为99.25%、99.72%、99.11%。

      选择GenBank中已报道的同源性较高的哈氏弧菌以及其他的种属的细菌的16S rRNA基因序列进行同源性比对。利用MEGA 6.0软件采用邻接法构建系统发育树,结果显示,菌株BDHYPFS-Y2S、BDHYPFS-Y2P、BDHYPFS-Y1G均与哈氏弧菌V. harveyi一致性最高(图2)。

      Figure 2.  Phylogenetic tree based on 16S rRNA gene sequence

      毒力基因相关特异性PCR结果显示,菌株BDHYPFS-Y2S、BDHYPFS-Y2P、BDHYPFS-Y1G扩增toxR基因、vhhB基因、vhhA基因结果均为阳性(图3)。

      Figure 3.  Electropherogram figure of virulence gene

    • 人工回感试验发现,菌株BDHYPFS-Y1G能引发牙鲆出现腹水症状,并导致死亡;注射BDHYPFS-Y1G后24 h内开始出现腹水症状,感染期间对照组未见死亡情况;BDHYPFS-Y1G感染的实验组死亡鱼体出现的腹水症状,与养殖场中自然发病的牙鲆症状相同。同时,从感染的病鱼中再次分离到优势菌,生理生化鉴定结果与菌株BDHYPFS-Y1G一致,且16S rRNA序列比对与菌株BDHYPFS-Y1G一致性为100%。试验表明,菌株BDHYPFS-Y1G为牙鲆腹水病病原。

      根据半致死量实验结果,采用寇氏法计算LD50=5.88×106 CFU/g(表3)。

      分组
      group
      菌液浓度/(CFU/mL)
      concentration of bacterium
      剂量/m
      Ldosage
      数量/尾
      quantity
      累计死亡/尾
      cumulative death
      死亡率/%
      death rate
      1 108 0.3 30 30 100
      2 107 0.3 30 24 80
      3 106 0.3 30 9 30
      4 105 0.3 30 2 6
      5 104 0.3 30 0 0
      对照组
      control
      0.9% NaCl 0.3 30 0 0

      Table 3.  Infection of flounder challenged by BDHYPFS-Y1G

    • 菌株BDHYPFS-Y2S、BDHYPFS-Y2P、BDHYPFS-Y1G对17种抗菌药物的药敏试验结果显示,BDHYPFS-Y2S、BDHYPFS-Y2P、BDHYPFS-Y1G均对呋喃妥因抗菌药物敏感;对左氧氟沙星、诺氟沙星抗菌药物中度敏感;对庆大霉素、磺胺异恶唑、四环素、7种抗菌药物耐药(表4)。

      药物
      antibiotics
      药量/(μg/片)
      concentration
      RIS抑菌圈直径/mm diameter of inhibited zone
      BDHYPFS-Y2SBDHYPFS-Y1GBDHYPFS-Y2P
      呋喃妥因 nitrofurantoin 300 ≤14 15-16 ≥17 22(S) 18(S) 18(S)
      萘啶酸 nalidixic acid 30 ≤13 14-18 ≥19 20(S) 19(S) 13(R)
      痢特灵 furazolidone 300 ≤14 15-16 ≥17 18(S) 16(I) 17(S)
      氧氟沙星 ofloxacin 5 ≤12 13-15 ≥16 18(S) 13(I) 14(I)
      头孢他啶 fortum 30 ≤14 15-17 ≥18 11(R) 16(I) 14(I)
      头孢噻肟 cefotaxime 30 ≤14 15-22 ≥23 15(I) 22(S) 15(I)
      左氧氟沙 levofloxacin 5 ≤13 14-16 ≥17 14(I) 14(I) 14(I)
      诺氟沙星 norfloxacin 10 ≤12 13-16 ≥17 14(I) 12(I) 14(I)
      奈替米星 netilmicin 30 ≤12 13-14 ≥15 14(I) 14(I) 18(S)
      氟苯尼考 florfenicol 30 ≤12 13-17 ≥18 9(R) 11(R) 11(R)
      新霉素 neomycin 30 ≤12 13-16 ≥17 0(R) 9(R) 0(R)
      美满霉素 Minocycline 30 ≤14 15-18 ≥19 0(R) 0(R) 0(R)
      庆大霉素 gentamycin 10 ≤12 13-14 ≥15 0(R) 11(R) 7(R)
      四环素 tetracycline 30 ≤14 15-18 ≥19 0(R) 0(R) 0(R)
      磺胺异恶 sulfisoxazole 300 / / / 0(R) 0(R) 0(R)
      青霉素 penicillin 10 / / / 0(R) 0(R) 0(R)
      氨苄西林 ampicillin 10 / / / 0(R) 0(R) 0(R)
      注:S. 高度敏感;I. 中度敏感;R.低敏感度或不敏感
      Notes: S. highly susceptible;I. intermediately susceptible;R. denoted low or no sensitivity

      Table 4.  Drug sensitive test results of BDHYPFS-Y2S、BDHYPFS-Y2P、BDHYPFS-Y1G

    3.   讨论
    • 腹水症是一种综合性病症,从临床症状上看,腹水病共同性特征为腹腔有积液,不同的致病菌可导致不同的患病症状。迟缓爱德华氏菌感染大菱鲆时,其症状为体表有出血点、肝脏、肾脏、脾脏、胆囊肿大[11];溶藻弧菌感染牙鲆的主要症状为体表腐烂[11];温和气单胞菌感染的腹水病症状为眼球突出、鳍基部出血、内脏出血[12];本研究患病牙鲆腹水特征与其他腹水病报道一致,腹腔内黄色积液增多,腹腔压力增大,导致消化道末端挤出肛外。患病牙鲆肝脏呈乳白色,呈现贫血症状。本实验分别从肾脏、脾脏、肝脏中分离出3株优势菌BDHYPFS-Y2S、BDHYPFS-Y1G、BDHYPFS-Y2P,经生理生化及分子生物学方法鉴定3株分离株均为哈氏弧菌。通过预实验,选取3株中毒性最强的菌株BDHYPFS-Y1G对健康牙鲆进行人工感染实验,能够复制牙鲆自然发病症状,且从人工感染的患腹水病牙鲆的病灶组织中再次得到分离株,人工感染分离株与自然分离株BDHYPFS-Y1G相比,生理生化特征一致,16S rRNA序列一致性为100%,证实哈氏弧菌为患腹水病牙鲆的病原菌。由于目前国内哈氏弧菌引起牙鲆腹水症爆发的报道相对较少,本研究可以提供填补哈维弧菌致牙鲆患腹水病例证以供后续腹水病防控研究。

      溶血活性通常为寄生型细菌及病毒所共有特征,通过溶解红细胞、分解血红蛋白来获取本身所需铁离子,有研究表明噬菌体如VHML等可能通过基因的水平转移导致哈氏弧菌产生溶血活性[13-14]。本研究观察自然发病状态下的牙鲆的肠、肝脏、脾脏、肾脏均有不同程度的损伤,呈全身性感染。多器官共同病理特征组织泛白,细胞呈现空泡变性、破裂变形,血红细胞侵润、溶解。本研究分离的3株哈氏弧菌溶血素基因vhhAvhhB检测均呈阳性,通过病理特征推测为哈氏弧菌的溶血素基因vhhAvhhB表达胞外溶血素,溶血素侵染宿主血细胞膜并引起细胞破裂溶解[15],呈现本研究观察到的病理特征。

      影响哈氏弧菌感染鱼类的外界环境因素有温度、pH、盐度等,其中温度是影响鱼类的生长率、存活率和抗病能力最重要的环境因素之一,且作用于病原菌在鱼体内的增殖。本研究回感实验表明,实验水温为(19±1) °C时,哈氏弧菌分离株BDHYPFS-Y1G的半数致死量LD50为5.88×106 CFU/g,低于自然状态下的毒性。许国晶等[16]研究发现水温在9~21 °C之间,牙鲆的黏液抗体水平随水温的升高而升高,达到峰值的时间随温度升高而加快,且在最适生活温度(21 °C)时,抗体应答强度最大;对比腹水病爆发现场水温(26±1) °C,本研究实验水温为(19±1) °C,牙鲆的黏液抗体水平接近峰值、免疫力强;钟硕良等[17]研究表明,水温在24~30 °C范围内,水体中哈氏弧菌的生长及对日本对虾(Penaeus japonicus)仔虾的感染死亡率随水温上升而递增。陈建国等[18]人发现,环境温度低于15 °C时,哈氏弧菌增殖遭到抑制,而在28~37 °C时,其生长速度加快,表明哈维弧菌在腹水病爆发现场水温(26±1) °C下比人工感染时毒力更强。病原菌引发养殖动物疾病的状况是一个宿主、病原菌、环境因素相互作用的结果。病原菌毒力会随温度变化而发生变化,在实际养殖生产过程中的病害防控中,需要密切关注环境温度等因素对疫情防控的作用。

      哈氏弧菌作为海洋鱼类和无脊椎动物致病菌[19],其感染范围十分广泛,引起的病症也有多种。其可使红鳍东方鲀(Takifugu rubripes)、虾(Artemia nauplii)、金鲳鱼(Trachinotus ovatus)、石斑鱼(Epinephelus awoara)、海参(Holothuria scabra)、海马(Hippocampus kuda等)[20-25]多种水产动物产生发光弧菌病、急性败血症、突眼和慢性皮肤溃疡等[26]。目前,抗生素治疗仍是治疗水生动物细菌性疾病的常用方法之一,本实验的药敏试验结果表明菌株BDHYPFS-Y2S、BDHYPFS-Y2P、BDHYPFS-Y1G均对呋喃妥因抗菌药物高度敏感;对左氧氟沙星、诺氟沙星抗菌药物中度敏感;然而对庆大霉素、磺胺异恶唑、四环素等7种抗菌药物耐药,呈现多重耐药现象,说明引发此次牙鲆腹水病的病原菌具有较高的耐药性,可能与长期使用抗生素相关,这也为以后加强牙鲆养殖过程中的管理提供了参考。

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