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Isolation, identification and pathogenicity studies on visceral granulomatous disease in Acanthopagrus schlegelii

  • Corresponding author: Dong QIAN, qiandong@nbu.edu.cn
  • Received Date: 2018-10-13
    Accepted Date: 2018-12-24
  • A serious disease occurred in black seabream, Acanthopagrus schlegelii, cultivated in runway fish culture pond of mariculture farm in Taizhou, Zhejiang province, in April, 2017. The diseased fish showed sluggish swimming, anorexia and ulceration on surface. Moribund fish were dissected with symptom of massive ascites and swelling on liver, spleen and kidney, with white granulomatous nodules, especially in spleen. The bacteria were isolated with tryptone soybean agar, and all the fish with typical symptoms showed the pure culture of grayish white colony with Gram negative rods. The bacterial strain AS15, from the spleen of fish with most serious granulomatous, was choose for challenging test to healthy black seabream by injection intraperitoneally. Mortality was recorded with similar symptom with native infected seabream, with the median death dose of 6.5×104 CFU/ind. AS15 was identified as Edwardsiella tarda with API 20E biochemical bacteria identification system as well as 16S rDNA sequences blasted on NCBI, with 99% similarity to reference strains. With phylogenetic analysis for 16S rDNA, AS15 belonged to the same cluster with ETT883、LADL05-105, E. piscicida according to new classification for genus E.dsiella, and same cluster to LADL05-105 and NCIM2056, E. piscicida-like, the new recommended species of the genus, with 100% similarity on gyrB phylogenetic tree. The species-specific primers for genus E. dsiella were used for PCR test, and AS15 showed positive to EPL, the species primer for E. piscicida-like, and PCR negative to all other three primers of E. tarda, E. piscicida and E. ictalur. Ten pathogenic gene, including four fimbrial genes, and sodB, citC, esrB, mukF, katB, the related virulence gene were analyzed by PCR. The strain AS15 showed positive to all four fimbrial genes and other virulence gene except gene gadB. It could be considered that AS15, E. piscicida-like bacteria, the first pathogenic strain isolated and reported from marine cultivated seabream, was the pathogen of serious outbreak case of visceral granulomatous disease in black seabream in runway pond culture system.
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Isolation, identification and pathogenicity studies on visceral granulomatous disease in Acanthopagrus schlegelii

    Corresponding author: Dong QIAN, qiandong@nbu.edu.cn
  • 1. School of Marine Sciences, Ningbo University, Ningbo    315211, China
  • 2. Taizhou Extention Station for Fishery Technique, Taizhou    318000, China
  • 3. Zhejiang Hongye Seafood Company, Taizhou    318000, China

Abstract: A serious disease occurred in black seabream, Acanthopagrus schlegelii, cultivated in runway fish culture pond of mariculture farm in Taizhou, Zhejiang province, in April, 2017. The diseased fish showed sluggish swimming, anorexia and ulceration on surface. Moribund fish were dissected with symptom of massive ascites and swelling on liver, spleen and kidney, with white granulomatous nodules, especially in spleen. The bacteria were isolated with tryptone soybean agar, and all the fish with typical symptoms showed the pure culture of grayish white colony with Gram negative rods. The bacterial strain AS15, from the spleen of fish with most serious granulomatous, was choose for challenging test to healthy black seabream by injection intraperitoneally. Mortality was recorded with similar symptom with native infected seabream, with the median death dose of 6.5×104 CFU/ind. AS15 was identified as Edwardsiella tarda with API 20E biochemical bacteria identification system as well as 16S rDNA sequences blasted on NCBI, with 99% similarity to reference strains. With phylogenetic analysis for 16S rDNA, AS15 belonged to the same cluster with ETT883、LADL05-105, E. piscicida according to new classification for genus E.dsiella, and same cluster to LADL05-105 and NCIM2056, E. piscicida-like, the new recommended species of the genus, with 100% similarity on gyrB phylogenetic tree. The species-specific primers for genus E. dsiella were used for PCR test, and AS15 showed positive to EPL, the species primer for E. piscicida-like, and PCR negative to all other three primers of E. tarda, E. piscicida and E. ictalur. Ten pathogenic gene, including four fimbrial genes, and sodB, citC, esrB, mukF, katB, the related virulence gene were analyzed by PCR. The strain AS15 showed positive to all four fimbrial genes and other virulence gene except gene gadB. It could be considered that AS15, E. piscicida-like bacteria, the first pathogenic strain isolated and reported from marine cultivated seabream, was the pathogen of serious outbreak case of visceral granulomatous disease in black seabream in runway pond culture system.

  • 黑棘鲷(Acanthopagrus schlegelii)属鲈形目(Perciformes)、鲷科(Sparidae)、棘鲷属(Acanthopagrus),是一种广泛分布于我国东南沿海及中国台湾、日本、东南亚等国家和地区的名贵经济鱼类。黑鲷生长迅速、肉质鲜嫩、营养价值高,对温度和盐度的适应范围广、食谱杂、抗病力较强,不作长距离洄游,是人工养殖和沿海增殖放流的优良品种,拥有广阔的市场前景[1]

    随着黑鲷市场需求的增加,人工养殖规模和增长速度不断扩大。我国2003—2011年鲷养殖总产量由4.23万t增加到5.63万t,年均增长3.69%;2011—2016年鲷养殖年均增长5.12%,其中2014—2016年期间年均增长率为12.08%。浙江省鲷养殖规模为全国第四,是重要黑鲷养殖省[2]。随着黑棘鲷养殖规模扩大和养殖密度提高,各种疾病相继出现,已报道了杀鱼假交替单胞菌(Pseudoalteromonas piscicida)引起的黑鲷尾鳍溃烂病[3],拟态弧菌(Vibrio mimicus)引起的黑鲷幼鱼腹水病[4],恶臭假单胞菌(Pseudomonas putida)引起的黑鲷肠炎[5]和迟缓爱德华菌(Edwardsiella tarda)引起的黑鲷感染[6]等。

    2017年4月,浙江台州某海水养殖公司跑道式黑鲷养殖池发生严重疾病,累计死亡4 000尾以上,死亡率80%以上,造成极大经济损失。患病黑鲷出现剧烈游动、活力下降、食欲减退、体表溃疡等症状,病鱼剖检可见肝脏、脾脏、肾脏出现明显的白色结节,个别患病黑鲷脾脏、肾脏布满白色结节,同时伴随大量腹水。本实验对患病黑鲷进行了细菌分离,从典型内脏结节病濒死黑鲷的肝脏、脾脏、肾脏等组织分离到15株细菌(SA-1~SA-15),并对有较强致病力的AS15菌株进行了形态、生理生化性状、16S rDNAgyrB序列鉴定、系统发育树分析、致病性和致病基因研究。本文报道了这一研究结果。

    • 患病濒死黑鲷采自浙江台州某海水养殖公司,体质量(43±2) g;攻毒用健康黑鲷为浙江宏野海产品有限公司提供,购自福建省漳州市沼安县,体质量(25±2) g,于曝气人工海水中暂养7 d,每日虹吸换水30%以上,暂养期间水温维持在(24±1) °C,并保持充氧;用于种特异性引物鉴别的菌株AS15、AS12分离自不同患病黑鲷,其他参考菌株:迟缓爱德华菌59-T-S2、59-T-S3、1501-T及杀香鱼假单胞菌YC-17-02为本实验室分离保存。

    • 细菌鉴定条API 20E试剂购自生物梅里埃中国有限公司;胰酪大豆胨培养液(TSB)和胰酪大豆胨琼脂(TSA)购自北京陆桥技术股份有限公司;革兰染色液购自杭州滨和微生物试剂有限公司;基因组DNA提取试剂盒购自北京全式金生物技术有限公司;Taq DNA聚合酶、2×TaqMix购自北京康为世纪生物科技有限公司;引物合成和序列测定均委托上海华大基因科技有限公司。

    • 患病黑鲷自2017年4月初发病,于4月中旬在现场捞取反应迟缓、体表溃疡的患病黑鲷,用75%酒精棉球消毒鱼体表面,选择肝脏、脾脏、肾脏肿大和典型白色结节的病鱼,无菌取肝脏、脾脏、肾脏于TSA平板上划线分离。

    • TSA平板分离菌28 °C培养48 h后,肝脏、脾脏、肾脏均分离到直径为0.5~1 mm的灰白色菌落纯培养,5尾典型患病黑鲷共挑取15个单菌落,于TSA平板上划线纯化,从5尾病鱼肝脏、脾脏、肾脏共分离到15株菌株(SA1~SA15),用20%甘油TSB菌悬液保存于−80 °C备用。

      选取脾脏白色结节最典型的5号患病黑鲷脾脏分离菌株AS15于胰酪大豆肉汤(TSB) 28 °C振荡培养24 h;7 000 r/min离心10 min,pH 7.4灭菌磷酸盐缓冲液(PBS)洗涤3次,制成菌悬液,以8×108、8×107、8×106、8×105、8×104 CFU/mL菌悬液按0.1 mL/尾腹腔注射健康黑鲷,空白对照注射等体积灭菌PBS缓冲溶液。水簇箱(24±2) °C充气养殖,每日记录发病和死亡情况,分离濒死或死亡黑鲷细菌。LD50采用karber法计算[7]

    • 挑取28 °C培养18~24 h纯化AS15单菌落,制备菌涂片,革兰染色,观察菌体形态;同时取AS15单菌落,制成菌悬液,接种至API 20E细菌鉴定条中,恒温培养24~48 h,按鉴定操作说明显色并记录结果,按梅里埃细菌鉴定软件及《伯杰氏系统细菌手册》第2版进行鉴定。

    • 挑取纯化AS15单菌落,双蒸水混匀,100 °C煮沸10 min,冰浴5 min,12 000 r/min离心2 min,取上清液作为模板进行PCR扩增;16S rDNA扩增引物:27F 5′- AGAGTTTGATCCTGGCTC AG-3',1492R 5'- GGTTACCTTGTTACGACTT-3'[8];50 μLPCR反应体系:27F、1492R正反引物(10 μmol/L) 各1 μL,模板2 μL,2×mix 25 μL,双蒸水补足体积,PCR扩增条件:预变性94 °C 5 min;变性94 °C 30 s,退火54 °C 30 s,延伸72 °C 90 s,35个循环;72 °C终延伸10 min[9]gyrB PCR引物:gyrB-F 5'-GGATAACGCGATTGACGAAG-3',gyrB-R 5'-GCCGTGARCAAARTCRAA-3'[10],50 μL PCR反应体系:gyrB-F、gyrB-R正反引物(10 μmol/L) 各1 μL,模板2 μL,2×mix 25 μL,双蒸水补足体积,扩增条件:预变性94 °C 5 min;变性95 °C 30 s,退火55 °C 30 s,延伸72 °C 2 min,45个循环;72 °C终延伸7 min[10]。1%琼脂糖凝胶电泳检测PCR产物,PCR产物测序后BLAST分析。

    • 将16S rDNAgyrB序列通过NCBI数据库BLAST比对分析,采用MEGA7.0软件基于邻接法(Neighbor-Joining, NJ),对AS15 16S rDNA基因和gyrB基因同源性较高的迟缓爱德华菌(Edwardsiella tarda)、杀鱼爱德华菌(Edwardsiella piscicida)、类杀鱼爱德华菌(Edwardsiella Piscicida-like)、鲇鱼爱德华菌(Edwardsiella ictaluri)、保科爱德华菌(Edwardsiella hoshinae)等菌株构建系统进化树[11]

    • 参考文献合成爱德华菌属内种特异性引物,对用于AS15的种鉴别。各引物序列:迟缓爱德华菌(ET)、鲇鱼爱德华菌(ESC)、杀鱼爱德华菌(EP)[10]、类杀鱼爱德华菌(EPL)[10](表1);鉴别用菌株参见“实验鱼及菌株”部分;PCR条件:预变性95 °C 5 min;变性95 °C 15 s,退火58 °C 15 s,延伸72 °C 15 s,35个循环;72 °C终延伸5 min[10]

      引物
      primer
      引物序列
      primer sequence
      参考文献
      references
      ET-F CAGTGATAAAAAGGGGTGGA [10]
      ET-R CTACACAGCAACGACAACG
      ESC-F ACTTATCGCCCTCGCAAC [10]
      ESC-R GCCTCTGATAAGTGGTTTCG
      EP-F CTTTGATCATGGTTGCGGAA [10]
      EP-R CGGCGTTTTCTTTTCTCG
      EPL-F TTTGATCGGGTACGCTGT [10]
      EPL-R AATTGCTCTATACGCACGC

      Table 1.  Specific primer sequences

    • 对分离菌株AS15侵袭相关的菌毛基因fimAfimBfimCfimD和毒力相关sodBcitCesrBgodBmukFkatB进行分析,各致病基因引物见表2

      目的基因
      target gene
      引物序列
      primer sequence
      参考文献
      references
      fimA fimA-F:CGGTAAAGTTGAGTTTACGGGTG [12]
      fimA-R:TGTAACCGTGTTGGCGTAAG
      fimB fimB-F:CTATATGGTGCAGACCTG [12]
      fimB-R:GCTGAAGGAGACTGTATTG
      fimC fimC-F:AACACCGGTATCAGCGGAAC [12]
      fimC-R:GTTGAATCGGTATGGCGTC
      fimD fimD-F:GGTAACCTGATTTGGCGTTC [12]
      fimD-R:GGATCACCTGGATCTTATCC
      sodB sodB-F:ATGTCATTCGAATTACCTGC [13]
      sodB-R:TCGATGTAATAAGCGTGTTCCCA
      citC citC-F:TTTCCGTTTGTGAATCAGGTC [14]
      citC-R:AATGTTTCGGCATAGCGTTG
      esrB esrB-F:GATCATGCCTTGCTAGCC [15]
      esrB-R:TCGGCGACCAGCTTGAGA
      godB godB-F:ATTCCCGCTTTGGTTCAGA [16]
      godB-R:GAGGAGCCGATAGTATTGGTA
      mukF mukF-F:TTTGGACGGTGAAATGAGC [16]
      mukF-R:CGTTGCGGTGCCAGTGAA
      katB katB-F:GATGCGATCAAGTTCCCGGA [17]
      katB-R:ACCTGGATGTACAGATCCCAT

      Table 2.  PCR primers for pathogenic gene of AS15

    2.   结果
    • 2017年4月初,跑道式养殖池患病黑鲷出现剧烈游动,随后活力下降、食欲减退,鱼体表面出现溃疡,并陆续死亡。黑鲷剖检可见肝脏、脾脏、肾脏有白色结节,严重者肝脏充血并肿大,脾脏表面及内部布满白色肉芽肿性结节,且伴随大量腹水;解剖镜下可见白色结节紧密排列分布脾脏内外(图版)。

      Figure 图版.  Symptoms of naturally infected black seabream, A. schlegelii

    • 分离菌株AS15腹腔注射感染健康黑鲷,注射2 d后黑鲷呈食欲减退、反应迟钝,濒死黑鲷沿水簇箱快速转圈、头朝上漂浮水中,陆续死亡。最高浓度组注射第2天开始死亡,感染2~6 d死亡黑鲷脾脏肿大、内脏无明显结节症状;第7天后死亡黑鲷肉眼可观察到脾脏出现与自然发病鲷相同的白色结节(图1),第8天后发病和死亡鱼脾脏可见明显白色结节,第11天后不再出现死亡,对照组未出现死亡。攻毒发病所分离菌株菌体形态、菌毛基因等与AS15相同,表明AS15可引起健康黑鲷发病并出现自然发病相同症状。用寇氏法计算出AS15对黑鲷的半数致死浓度为6.5×104 CFU/尾(表3)。

      Figure 1.  Challenge the symptoms of infection, A. schlegelii

      注射浓度/(CFU/mL)
      injection concentration
      死亡鱼数/(尾/d) mortality number 累计死亡数/尾
      total mortality
      死亡率/%
      mortality rate
      1 2 3 4 5 6 7 8 9 10 11 12 13 14
      8×108 0 2 3 3 2 2 1 0 0 0 0 0 0 0 13 100
      8×107 0 1 2 3 2 2 1 1 0 0 0 0 0 0 12 92
      8×106 0 0 2 1 1 2 2 1 1 0 0 0 0 0 10 77
      8×105 0 0 0 1 1 2 2 0 1 0 0 0 0 0 7 54
      8×104 0 0 0 0 0 0 1 2 1 0 1 0 0 0 5 38
      对照 control 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
      注:注射剂量0.1 mL/尾
      Notes: the injection dose was 0.1 mL/tailil

      Table 3.  Results of artificial infection experiment

    • AS15 TSA培养基28 °C培养24~48 h可形成圆形、微隆起、光滑、湿润、半透明、直径0.5~1 mm灰白色菌落(图2-a);菌体为短杆状菌体,无荚膜,不形成芽孢,革兰染色呈阴性(图2-b)。

      Figure 2.  The morphology and bacteria of AS 15

    • 采用API 20E生化鉴定条对AS15进行鉴定,分离菌株氧化酶为阴性,葡萄糖发酵,赖氨酸、鸟氨酸、苯丙氨酸、H2S、枸橼酸、明胶液化、葡萄糖产酸、硝酸盐还原、甲基红等呈阳性,尿素酶呈弱阳性,半乳糖苷、精氨酸、吲哚、VP反应、甘露醇、肌醇、山梨醇、鼠李醇、蔗糖、蜜二糖、苦杏仁苷、阿拉伯糖等呈阴性(表4),与类杀鱼爱德华菌LADL05-105和迟缓爱德华菌典型菌ATCC15947相似度为86.2%,与杀鱼爱德华菌ETT883的相似度为82.8%。

          生化项目
        biochemical items
      实验菌株 experimental strains     生化项目
        biochemical items
      实验菌株 experimental strains
      AS15 LADL05-105[10] ET883[10] ATCC 15947[4] AS15 LADL05-105[10] ET883[10] ATCC 15947[4]
      革兰染色
      Gram stain
      Gram - - - - 甲基红
      Methlred
      MR + + + +
      氧化酶
      Oxidase
      OX - - - - 葡萄糖
      Glucose
      GLU + + + +
      葡萄糖发酵
      Glucose
      GLU O/F F F F F 甘露醇
      Mannitol
      MAN - - - -
      运动性
      Moveability
      MOV + + + + 肌醇
      Inositol
      INO - - - -
      半乳糖苷
      Galactoside
      ONPG - + + - 山梨醇
      Sorbitol
      SOR - - - -
      精氨酸
      Arginine
      ADH - - - - 鼠李醇
      Rhamnol
      RHA - - - -
      赖氨酸
      Lysine
      LDC + + + + 蔗糖
      Sucrase
      SAC - + + +
      鸟氨酸
      Ornithine
      ODC + + + + 蜜二糖
      Melibiose
      MEL - - - -
      苯丙氨酸
      Phenylalanine
      TDA - - - - 苦杏仁苷
      Amygdalin
      AMY - - - -
      枸橼酸
      Citric acid
      CIT + + - - 阿拉伯糖
      Arabinose
      ARA - - - -
      硫化氢
      Hydrothion
      H2S + + + + 硝酸盐还原
      Nitrate reduction
      NO2 + + + +
      尿素
      Carbamide
      URE ± - - - 亚硝酸还原
      Nitrite reduction
      N2 - - - -
      吲哚
      Indole
      IND + + + + 麦康凯琼脂
      MacConkey Agar
      McC + + + +
      丙酮酸盐
      Pyruvate
      VP - - - - SS琼脂
      SS Agar
      SS-gar + + + +
      明胶
      Gelatin
      GLN + - - -
      注:+. 阳性,. −.阴性;下同
      Notes: +. positive, −. negative; the same below

      Table 4.  Biochemical identification of strain AS15

    • 测定了AS15 16S rDNA全序列,经NCBI数据库BLAST比对,序列相似度为99%,排前5位菌株分别为迟缓爱德华菌Ya-1(KF032624)和N1(KJ725306)、鲇爱德华氏菌GYQ(KF952599)、类杀鱼爱德华氏菌LADL05-105(CP011516)和EA181011(CP011364)。

    • 为确定AS15的分类地位,测定了16S rDNAgyrB基因全序列,分别得到1 438 bp和1 792 bp的序列。系统进化树分析结果显示,AS15的16S rDNA序列在系统进化树上与杀鱼爱德华菌ETT883[11] (KC202809)和类杀鱼爱德华菌LADL05-105[9](JX866968)聚为一簇,同源性为99%;gyrB基因序列与类杀鱼爱德华菌NCIM2056[10](JN700741)和LADL05-105 (JX867004)同源性达100%,与杀鱼爱德华菌ETT883(JN700725)同源性达97%(图34)。

      Figure 3.  Phylogenetic tree and analysis for 16S rDNA sequence of AS15

      Figure 4.  Phylogenetic tree and analysis for gyrB sequence of AS15

      根据生化性状、6S rDNAgyrB基因全序列分子系统进化树分析结果,AS15可鉴定初步为类杀鱼爱德华菌成员[18]

    • 采用迟缓爱德华菌、鲇鱼爱德华菌、杀鱼爱德华菌和类杀鱼爱德华菌种特异性引物,对本次发病黑鲷分离菌株、人工感染再分离株及实验室保存迟缓爱德华菌分离菌株进行种特异性PCR鉴定(表5),实验室保存的迟缓爱德华菌株可用ET特异引物扩增出大小为114 bp特异性条带;发病黑鲷分离株AS12、AS15以及人工感染后再次分离的菌株AS15-2可用类杀鱼爱德华菌种特异引物扩增出128 bp特异性条带,用迟缓爱德华菌、鲇鱼爱德华菌和杀鱼爱德华菌的种特异引物未扩增出相应条带;对照菌株大黄鱼内脏白色结节病病原杀香鱼假单胞菌YC-17-02采用4对爱德华菌种特异性引物均未扩增出相应条带。结果提示,本次分离的菌株AS15为类杀鱼爱德华菌(图5)。

      编号
      no.
      菌株名
      name
      分离时间
      time
      分离地区
      location
      分离宿主
      host
         种特异性引物 species-specific PCR
         ET    EP    ESC    EPL
      1 59-T-S2 2015年9月 余姚 黄颡鱼 + - - -
      2 59-T-S3 2013年9月 余姚 黄颡鱼 + - - -
      3 1501-T 2015年4月 新昌 鲟鱼 + - - -
      4 YC-17-02 2017年5月 椒江 大黄鱼 - - - -
      5 AS12 2017年4月 临海 发病黑鲷4 - - - +
      6 AS15 2017年4月 临海 发病黑鲷5 - - - +
      7 AS15-2 2018年6月 宁波 攻毒发病黑鲷 - - - +

      Table 5.  Edwardsiella sp isolates from different fish identififed by spices-specific primers

      Figure 5.  Identification of Edwardsiella isolates by species-specific primers

    • 对分离株AS15的致病基因进行了检测,菌毛基因检测结果显示,AS15的重要菌毛基因fimAfimBfimCfimD等4个与爱德华菌侵袭和致病相关的菌毛基因均为阳性(图6-a),对4个菌毛基因的PCR产物进行测序在NCBI数据库序列比对,结果发现,fimAfimBfimC基因与杀鱼爱德华菌LADL05-105(CP011516.1)和EA181011(CP011364.1)相似性高达98%,fimD基因与爱德华菌S11-285(CP016044.1)相似性为99%,表明AS15有较强的侵袭力;对sodBcitCesrBmukFkatBgadB等6个致病基因进行了PCR检测,结果显示,AS15对sodBcitCesrBmukFkatB均为阳性,NCBI数据库中比对发现sodB基因与杀鱼爱德华菌WFE 1(MG225525)相似性高达97%,citC基因与杀鱼爱德华菌ETW41 (CP019440)相似性99%,esrBkatBmukF基因与杀鱼爱德华菌LADL05-105(CP011516)相似性分别高达98%、99%和96%。

      Figure 6.  Main pathogenic gene detection of AS15 with PCR

    3.   讨论
    • 爱德华菌是多种鱼类、爬行类和其他经济动物的病原,也可引起人类疾病,是人鱼共患病的潜在病原。爱德华菌可感染石斑鱼(Epinephelus sp.)、罗非鱼(Oreochromis spp.)、尖吻鲈(Lates calcarifer)、加州鲈(Micropterus salmoides)、牙鲆(Paralichthys olivaceus)、大比目鱼(Pleuronectiformes)等海水鱼类[18-24]及斑点叉尾鮰(Ietalurus punetaus)、大马哈鱼(Oncorhynchus keta)、溪红点鲑(Salvelinus leucomaenis)、养殖鲤科鱼等[25-32]淡水鱼类,还可感染海参和龟鳖类[10],造成极大经济损失。本实验对引起海水跑道式养殖黑鲷暴发疾病的病原菌进行了研究,将从典型症状的患病黑鲷分离株AS15进行人工感染健康黑鲷后可引发内脏白色结节,半数致死浓度为6.5×104 CFU/尾,表现出极强毒力;该菌株经形态、生理生化鉴定、16S rDNAgyrB基因系统进化树分析,其16S rDNA与杀鱼爱德华菌代表株ETT883等聚为一簇,gyrB进化树显示与类杀鱼爱德华菌NCIM2056等聚为一簇;通过爱德华菌属种特异性引物鉴定,AS15可用类杀鱼爱德华菌EPL扩增出128 bp特异性片段,序列分析证实为类杀鱼爱德华菌片段,表明该分离株为类爱德华菌成员。分离菌株AS15具有fimAfimBfimCfimD等菌毛基因和sodBcitCesrBmukFkatB等毒力基因,致病力强,是引起本次黑鲷疾病暴发的病原。

      传统上爱德华菌属分为迟缓爱德华氏菌,鲇爱德华菌、保科爱德华菌[33]3个种,以迟缓爱德华菌分布和感染宿主最为广泛[34],又分为两个生物群,其中1型为典型的迟缓爱德华菌,表现为对阿拉伯糖、蔗糖、海藻糖和甘露醇等多种糖类不利用。2012年根据迟缓爱德华菌株毒力的不同,又提出EdwG I型和EdwG II型两个不同基因型[35],EdwG I型含有Ⅲ型分泌系统(T3SS)和VI型分泌系统(T6SS),对鱼类具有更强致病性,该型迟缓爱德华菌现命名为杀鱼爱德华菌,以分离自欧洲鳗的ETT883为代表菌株[11],原迟缓爱德华菌中无T3SS或T6SS菌株仍称为迟缓爱德华菌,以ATCCT15947为代表菌株(分离自病人)[36]。随后又将杀鱼爱德华菌分为杀鱼爱德华菌和类杀鱼爱德华菌2个种[18],两者序列存在差异,生化性状上,现报道的迟缓爱德华菌和杀鱼爱德华菌,枸橼酸多为阴性,而类杀鱼爱德华菌枸橼酸反应多为阳性,两者还可通过特异的种鉴定性引物加以区分。本文报道的黑鲷AS15菌株,可引起黑鲷内脏结节症状,与引起大口黑鲈鱼心、肝、肾肉芽肿性结节的杀鱼爱德华菌相似[18],生化性状与迟缓爱德华菌典型菌和类杀鱼爱德华菌相似度为86.2%,与杀鱼爱德华菌相似度为82.8%;16S rDNA进化树分析表明,AS15与杀鱼爱德华菌ETT883和类杀鱼爱德华菌LADL05-105聚为一簇;gyrB进化树与类杀鱼爱德华菌NCIM2056(JN700741)和LADL05-105(JX867004)同源性为100%,与杀鱼爱德华菌ETT883(JN700725)同源性达97%;爱德华菌属种特异性引物PCR分析表明,AS15仅对类杀鱼爱德华菌(EPL)有扩增带。综上分析,确定AS15属类杀鱼爱德华菌。这是我国首次从海水鲷科鱼类中检出致病性类杀鱼爱德华菌。

      对于爱德华菌的致病因子已开展大量研究,发现了皮肤毒素[37]、溶血素[38]、保护细菌在巨噬细胞增殖的抗调理因子[39]、血凝素[33]、细胞侵袭因子[40]等多种毒力因子,这些因子包括逃避机体免疫、增强细菌入侵能力的侵袭因子,也包括可在体内破坏机体组织和细胞的溶血系等因子[41]。菌毛基因与迟缓爱德华菌的致病性存在很强的相关性,Sakai等[12]研究发现17株致病性菌株中可检测到4种菌毛基因,而14株非致病菌株13株未检出任何菌毛基因[12]。本文对黑鲷分离株AS15的4种菌毛基因进行了检测,表明4种菌毛基因均为阳性,此外还检测了抵抗因子(sodB)、柠檬酸裂解酶(citC)、毒力启动因子(esrB)、杀伤因子(mukF)、过氧化氢酶(katB)和谷氨酸脱梭酶同工酶(gadB)等6个致病基因,结果显示,AS15的sodBcitCesrBmukFkatB等5个毒力基因均为阳性,其中包括T3SS分泌系统调节基因esrB[15]。这些分析,为AS15致病机理的深入研究奠定了基础,同时为该菌毒力的判断提供了依据。

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