一株牛蛙源金黄杆菌的分离鉴定及其致病特性
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摘要
【背景】美国牛蛙养殖过程中病害问题非常突出,尤其是细菌性病害,其病原种类多、病原菌复杂多样、蔓延速度快、发病死亡率高,一直是牛蛙养殖过程中防控的难点。【目的】确定从患病牛蛙体内分离到的一株细菌NW1203的分类地位和致病性。【方法】无菌操作从牛蛙体内取样划线分离细菌,通过形态观察、生理生化试验、16S rRNA基因序列比对进行种属鉴定,通过人工感染、溶血性试验和病理切片观察分析其致病特性。【结果】经形态和生理生化鉴定及16S rRNA基因序列比对,菌株NW1203为金黄杆菌属细菌,与Chryseobacterium sp. F30的相似性达100%,进化树也显示该菌与金黄杆菌属细菌聚类;溶血性试验表明,菌株NW1203对绵羊、小鼠和牛蛙的血细胞都呈完全溶血;人工感染试验及感染病蛙的组织切片观察显示,菌株NW1203对牛蛙具有较强致病性,可引起牛蛙肝、肾、脾等主要组织严重病变,LD50为4.753×103 CFU/g。【结论】明确了菌株NW1203为牛蛙新病原,为牛蛙疾病的防控提供了理论依据。
[Background] Diseases, especially bacterial diseases have been a big challenge in American bullfrog(Rana catesbiana) farming. Due to a high diversity of these pathogens, rapid spread of the disease as well as high mortality rate, the bacterial disease in the bullfrog is difficult to prevention and control.[Objective] In order to study the taxonomic status and pathogenicity, one bacterial strain NW1203 was isolated and identified from diseased R. catesbiana. [Methods] The strain NW1203 was isolated from R.catesbiana by streaking plate method under aseptic conditions, followed by morphological observation,physiological, biochemical tests and 16 S rRNA gene phylogenetic analyses to identify bacterial species. To investigate the pathogenicity of these bacteria, artificial infection followed by hemolytic test and histopathological examination were performed. [Results] Based on the characterization of physiological,biochemical examination and 16 S rRNA gene sequence alignment, the isolated bacteria NW1203 was identified as Chryseobacterium sp. with 100% sequences similarities to Chryseobacterium sp. F30.Furthermore, phylogenetic trees showed bacteria NW1203 was grouped with Chryseobacterium sp.Hemolytic tests showed the strain NW1203 exhibited strongly hemolytic to the erythrocyte of sheep, mice and bullfrog. Artificial infection test and tissue section observation of infected frogs indicated the strain NW1203 had strongly pathogenic to R. catesbiana with LD50 values of 4.753×103 CFU/g and could cause severe pathological changes in main tissues including liver, kidney and spleen. [Conclusion] This study indicates that the bacteria strain NW1203 was a new pathogen causing bacterial disease of R. catesbiana and provides a theoretical basis for the prevention and control of bullfrog diseases.
[Background] Diseases, especially bacterial diseases have been a big challenge in American bullfrog(Rana catesbiana) farming. Due to a high diversity of these pathogens, rapid spread of the disease as well as high mortality rate, the bacterial disease in the bullfrog is difficult to prevention and control.[Objective] In order to study the taxonomic status and pathogenicity, one bacterial strain NW1203 was isolated and identified from diseased R. catesbiana. [Methods] The strain NW1203 was isolated from R.catesbiana by streaking plate method under aseptic conditions, followed by morphological observation,physiological, biochemical tests and 16 S rRNA gene phylogenetic analyses to identify bacterial species. To investigate the pathogenicity of these bacteria, artificial infection followed by hemolytic test and histopathological examination were performed. [Results] Based on the characterization of physiological,biochemical examination and 16 S rRNA gene sequence alignment, the isolated bacteria NW1203 was identified as Chryseobacterium sp. with 100% sequences similarities to Chryseobacterium sp. F30.Furthermore, phylogenetic trees showed bacteria NW1203 was grouped with Chryseobacterium sp.Hemolytic tests showed the strain NW1203 exhibited strongly hemolytic to the erythrocyte of sheep, mice and bullfrog. Artificial infection test and tissue section observation of infected frogs indicated the strain NW1203 had strongly pathogenic to R. catesbiana with LD50 values of 4.753×103 CFU/g and could cause severe pathological changes in main tissues including liver, kidney and spleen. [Conclusion] This study indicates that the bacteria strain NW1203 was a new pathogen causing bacterial disease of R. catesbiana and provides a theoretical basis for the prevention and control of bullfrog diseases.
引文
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[2]Yang CH.Wry head disease of bullfrog pathogen,pathology and sensitive drug research[D].Changsha:Master’s Thesis of Hunan Agricultural University,2010(in Chinese)杨春浩.牛蛙歪头病病原、病理及敏感药物研究[D].长沙:湖南农业大学硕士学位论文,2010
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[5]Yu Z,Xiao KY,Zhong L,et al.Pathogen identification and drug sensitivity of ascites disease in Rana catesbeiana[J].Freshwater Fisheries,2013,43(1):50-54(in Chinese)于喆,肖克宇,钟蕾,等.牛蛙腹水病病原鉴定和药敏试验[J].淡水渔业,2013,43(1):50-54
[6]Han YN,Yu YC,Wang XY,et al.Identification and sensitive drug test of pathogenic Proteus vulgaris from Rana catesbeiana[J].Microbiology China,2016,43(6):1280-1287(in Chinese)韩艳楠,余银春,王鑫毅,等.牛蛙致病变形菌的鉴定及其敏感药物筛选[J].微生物学通报,2016,43(6):1280-1287
[7]Hang XY,Zhou DR,Ye XP,et al.Isolation and identification on the pathogen of bullfrog Streptoccus disease[J].Acta Hydrobiologica Sinica,2012,36(2):361-364(in Chinese)杭小英,周冬仁,叶雪平,等.牛蛙无乳链球菌病病原的分离鉴定[J].水生生物学报,2012,36(2):361-364
[8]Yin LF,Chen GL.Diagnosis and prevention of common diseases of R.catesbiana[J].Scientific Fish Farming,2010(10):80(in Chinese)尹伦甫,陈公立.牛蛙常见疾病的诊断与防治[J].科学养鱼,2010(10):80
[9]Liu XL,Mei H,Bu LF,et al.Isolation and pathogenicity study of Elizabethkingia sp.from Channa argus[J].Journal of Ningbo University(Natural Science&Engineering Edition),2018,31(2):1-7(in Chinese)刘晓琳,梅寒,卜令飞,等.乌鳢伊丽莎白菌的分离鉴定及致病性研究[J].宁波大学学报:理工版,2018,31(2):1-7
[10]Kim KK,Kim MK,Lim JH,et al.Transfer of Chryseobacterium meningosepticum and Chryseobacterium miricola to Elizabethkingia gen.nov.as Elizabethkingia meningoseptica comb.nov.and Elizabethkingia miricola comb.nov.[J].International Journal of Systematic and Evolutionary Microbiology,2005,55(3):1287-1293
[11]Zhang Q,Liu G,Ma XL,et al.Acute toxicity test of Gamithromycin injection in mice[J].Modern Animal Husbandry Science&Technology,2017(3):4-5(in Chinese)张倩,刘刚,马晓玲,等.加米霉素注射液对小鼠的急性毒性试验[J].现代畜牧科技,2017(3):4-5
[12]Wang XY,Han YN,Jin S,et al.Identification and analysis on extracellular products characteristics of Chryseobacterium indologenes isolation from Channa argus[J].Acta Hydrobiologica Sinica,2016,40(3):641-646(in Chinese)王鑫毅,韩艳楠,金珊,等.乌鳢产吲哚金黄杆菌的鉴定及其胞外产物特性分析[J].水生生物学报,2016,40(3):641-646
[13]Zhao NX,Yuan GY.Medical Bacteria Name and Classification Identification[M].3rd ed.Jinan:Shandong University Press,2013:115-120(in Chinese)赵乃昕,苑广盈.医学细菌名称及分类鉴定[M].3版.济南:山东大学出版社,2013:115-120
[14]Hugo CJ,Segers P,Hoste B,et al.Chryseobacterium joostei sp.nov.,isolated from the dairy environment[J].International Journal of Systematic and Evolutionary Microbiology,2003,53(3):771-777
[15]Santos Y,Toranzo AE,Barja JL,et al.Virulence properties and enterotoxin production of Aeromonas strains isolated from fish[J].Infection and Immunity,1988,56(12):3285-3293
[16]Hantsis-Zacharov E,Shakéd T,Senderovich Y,et al.Chryseobacterium oranimense sp.nov.,a psychrotolerant,proteolytic and lipolytic bacterium isolated from raw cow’s milk[J].International Journal of Systematic and Evolutionary Microbiology,2008,58(11):2635-2639
[17]Weon HY,Kim BY,Yoo SH,et al.Chryseobacterium soli sp.nov.and Chryseobacterium jejuense sp.nov.,isolated from soil samples from Jeju,Korea[J].International Journal of Systematic and Evolutionary Microbiology,2008,58(2):470-473
[18]Cho SH,Lee KS,Shin DS,et al.Four new species of Chryseobacterium from the rhizosphere of coastal sand dune plants,Chryseobacterium elymi sp.nov.,Chryseobacterium hagamense sp.nov.,Chryseobacterium lathyri sp.nov.and Chryseobacterium rhizosphaerae sp.nov.[J].Systematic and Applied Microbiology,2010,33(3):122-127
[19]Holmes B,Steigerwalt AG,Nicholson AC.DNA-DNAhybridization study of strains of Chryseobacterium,Elizabethkingia and Empedobacter and of other usually indole-producing non-fermenters of CDC groups IIc,IIe,IIh and IIi,mostly from human clinical sources,and proposals of Chryseobacterium bernardetii sp.nov.,Chryseobacterium carnis sp.nov.,Chryseobacterium lactis sp.nov.,Chryseobacterium nakagawai sp.nov.and Chryseobacterium taklimakanense comb.nov.[J].International Journal of Systematic and Evolutionary Microbiology,2013,63(12):4639-4662
[20]Lo CI,Sankar SA,Mediannikov O,et al.High-quality genome sequence and description of Chryseobacterium senegalense sp.nov.[J].New Microbes&New Infections,2016,10:93-100
[21]Loch TP,Faisal M.Chryseobacterium aahli sp.nov.,isolated from lake trout(Salvelinus namaycush)and brown trout(Salmo trutta),and emended descriptions of Chryseobacterium ginsenosidimutans and Chryseobacterium gregarium[J].International Journal of Systematic and Evolutionary Microbiology,2014,64(5):1573-1579
[22]Fang H,Chen CZ,Zhang XJ.Aquacultural Animal Pathogenic Bacteriology[M].Beijing:China Agriculture Press,2010:499-512(in Chinese)房海,陈翠真,张晓君.水产养殖动物病原细菌学[M].北京:中国农业出版社,2010:499-512
[23]Dong L.Identification and aethionlogical of pathogentic bactetia of cultured Turbot(Scophthalmus maximus)[D].Qingdao:Master’s Thesis of Ocean University of China,2009(in Chinese)董丽.养殖大菱鲆几种重要细菌性疾病病原菌的鉴定及其病原学初步研究[D].青岛:中国海洋大学硕士学位论文,2009
[24]Shahi N,Sharma P,Pandey J,et al.Characterization and pathogenicity study of Chryseobacterium scophthalmum recovered from gill lesions of diseased golden mahseer,Tor putitora(Hamilton,1822)in India[J].Aquaculture,2018,485:81-92
[25]Yang YB,Ai XH,Song Y,et al.Isolation,identification and antibiotic sensitivity of Chryseobacterium indologenes from Trionyx sinensis[J].Acta Hydrobiologica Sinica,2018,42(4):786-793(in Chinese)杨移斌,艾晓辉,宋怿,等.中华鳖源致病性产吲哚金黄杆菌分离、鉴定及药敏特性分析[J].水生生物学报,2018,42(4):786-793
[26]Boran H,Terzi E,Altinok I,et al.Bacterial diseases of cultured Mediterranean horse mackerel(Trachurus mediterraneus)in sea cages[J].Aquaculture,2013,396-399:8-13
[27]Bernardet JF,Vancanneyt M,Matte-Tailliez O,et al.Polyphasic study of Chryseobacterium strains isolated from diseased aquatic animals[J].Systematic and Applied Microbiology,2005,28(7):640-660
[28]Didinen BI,Onuk EE,?ztürk T,et al.First report of Chryseobacterium sp.from Koi(Cyprinus carpio)in Turkey[J].The Israeli Journal of Aquaculture,2016,68:1341-1348
[29]Vandamme P,Bernardet JF,Segers P,et al.NOTES:New perspectives in the classification of the Flavobacteria:Description of Chryseobacterium gen.nov.,Bergeyella gen.nov.,and Empedobacter nom.rev.[J].International Journal of Systematic Bacteriology,1994,44(4):827-831
[30]Li M,Song TT,Zheng RQ,et al.Isolation,identification and drug sensitivity test of the pathogen of Quasipaa spinosa[J].Journal of Anhui Agricultural Sciences,2016,44(3):64-66,98(in Chinese)李明,宋婷婷,郑荣泉,等.棘胸蛙歪头病病原分离·鉴定与药敏试验[J].安徽农业科学,2016,44(3):64-66,98
[2]Yang CH.Wry head disease of bullfrog pathogen,pathology and sensitive drug research[D].Changsha:Master’s Thesis of Hunan Agricultural University,2010(in Chinese)杨春浩.牛蛙歪头病病原、病理及敏感药物研究[D].长沙:湖南农业大学硕士学位论文,2010
[3]Liu XD,Yang JX,Gong H,et al.Characterization of an iridovirus isolate from Rana catesbiana[J].Chinese Journal of Animal Infectious Diseases,2012,20(1):16-21(in Chinese)刘晓东,杨金先,龚晖,等.一株牛蛙源虹彩病毒的分离及鉴定[J].中国动物传染病学报,2012,20(1):16-21
[4]Yang DW,Lv JY,Wu JY,et al.Preliminary study on common diseases and control of Rana catesbeiana[J].Fisheries Science and Technology,2000(3):27-30(in Chinese)杨大伟,吕军仪,吴金英,等.牛蛙(Rana catesbeiana)常见病害及防治的初步研究[J].水产科技,2000(3):27-30
[5]Yu Z,Xiao KY,Zhong L,et al.Pathogen identification and drug sensitivity of ascites disease in Rana catesbeiana[J].Freshwater Fisheries,2013,43(1):50-54(in Chinese)于喆,肖克宇,钟蕾,等.牛蛙腹水病病原鉴定和药敏试验[J].淡水渔业,2013,43(1):50-54
[6]Han YN,Yu YC,Wang XY,et al.Identification and sensitive drug test of pathogenic Proteus vulgaris from Rana catesbeiana[J].Microbiology China,2016,43(6):1280-1287(in Chinese)韩艳楠,余银春,王鑫毅,等.牛蛙致病变形菌的鉴定及其敏感药物筛选[J].微生物学通报,2016,43(6):1280-1287
[7]Hang XY,Zhou DR,Ye XP,et al.Isolation and identification on the pathogen of bullfrog Streptoccus disease[J].Acta Hydrobiologica Sinica,2012,36(2):361-364(in Chinese)杭小英,周冬仁,叶雪平,等.牛蛙无乳链球菌病病原的分离鉴定[J].水生生物学报,2012,36(2):361-364
[8]Yin LF,Chen GL.Diagnosis and prevention of common diseases of R.catesbiana[J].Scientific Fish Farming,2010(10):80(in Chinese)尹伦甫,陈公立.牛蛙常见疾病的诊断与防治[J].科学养鱼,2010(10):80
[9]Liu XL,Mei H,Bu LF,et al.Isolation and pathogenicity study of Elizabethkingia sp.from Channa argus[J].Journal of Ningbo University(Natural Science&Engineering Edition),2018,31(2):1-7(in Chinese)刘晓琳,梅寒,卜令飞,等.乌鳢伊丽莎白菌的分离鉴定及致病性研究[J].宁波大学学报:理工版,2018,31(2):1-7
[10]Kim KK,Kim MK,Lim JH,et al.Transfer of Chryseobacterium meningosepticum and Chryseobacterium miricola to Elizabethkingia gen.nov.as Elizabethkingia meningoseptica comb.nov.and Elizabethkingia miricola comb.nov.[J].International Journal of Systematic and Evolutionary Microbiology,2005,55(3):1287-1293
[11]Zhang Q,Liu G,Ma XL,et al.Acute toxicity test of Gamithromycin injection in mice[J].Modern Animal Husbandry Science&Technology,2017(3):4-5(in Chinese)张倩,刘刚,马晓玲,等.加米霉素注射液对小鼠的急性毒性试验[J].现代畜牧科技,2017(3):4-5
[12]Wang XY,Han YN,Jin S,et al.Identification and analysis on extracellular products characteristics of Chryseobacterium indologenes isolation from Channa argus[J].Acta Hydrobiologica Sinica,2016,40(3):641-646(in Chinese)王鑫毅,韩艳楠,金珊,等.乌鳢产吲哚金黄杆菌的鉴定及其胞外产物特性分析[J].水生生物学报,2016,40(3):641-646
[13]Zhao NX,Yuan GY.Medical Bacteria Name and Classification Identification[M].3rd ed.Jinan:Shandong University Press,2013:115-120(in Chinese)赵乃昕,苑广盈.医学细菌名称及分类鉴定[M].3版.济南:山东大学出版社,2013:115-120
[14]Hugo CJ,Segers P,Hoste B,et al.Chryseobacterium joostei sp.nov.,isolated from the dairy environment[J].International Journal of Systematic and Evolutionary Microbiology,2003,53(3):771-777
[15]Santos Y,Toranzo AE,Barja JL,et al.Virulence properties and enterotoxin production of Aeromonas strains isolated from fish[J].Infection and Immunity,1988,56(12):3285-3293
[16]Hantsis-Zacharov E,Shakéd T,Senderovich Y,et al.Chryseobacterium oranimense sp.nov.,a psychrotolerant,proteolytic and lipolytic bacterium isolated from raw cow’s milk[J].International Journal of Systematic and Evolutionary Microbiology,2008,58(11):2635-2639
[17]Weon HY,Kim BY,Yoo SH,et al.Chryseobacterium soli sp.nov.and Chryseobacterium jejuense sp.nov.,isolated from soil samples from Jeju,Korea[J].International Journal of Systematic and Evolutionary Microbiology,2008,58(2):470-473
[18]Cho SH,Lee KS,Shin DS,et al.Four new species of Chryseobacterium from the rhizosphere of coastal sand dune plants,Chryseobacterium elymi sp.nov.,Chryseobacterium hagamense sp.nov.,Chryseobacterium lathyri sp.nov.and Chryseobacterium rhizosphaerae sp.nov.[J].Systematic and Applied Microbiology,2010,33(3):122-127
[19]Holmes B,Steigerwalt AG,Nicholson AC.DNA-DNAhybridization study of strains of Chryseobacterium,Elizabethkingia and Empedobacter and of other usually indole-producing non-fermenters of CDC groups IIc,IIe,IIh and IIi,mostly from human clinical sources,and proposals of Chryseobacterium bernardetii sp.nov.,Chryseobacterium carnis sp.nov.,Chryseobacterium lactis sp.nov.,Chryseobacterium nakagawai sp.nov.and Chryseobacterium taklimakanense comb.nov.[J].International Journal of Systematic and Evolutionary Microbiology,2013,63(12):4639-4662
[20]Lo CI,Sankar SA,Mediannikov O,et al.High-quality genome sequence and description of Chryseobacterium senegalense sp.nov.[J].New Microbes&New Infections,2016,10:93-100
[21]Loch TP,Faisal M.Chryseobacterium aahli sp.nov.,isolated from lake trout(Salvelinus namaycush)and brown trout(Salmo trutta),and emended descriptions of Chryseobacterium ginsenosidimutans and Chryseobacterium gregarium[J].International Journal of Systematic and Evolutionary Microbiology,2014,64(5):1573-1579
[22]Fang H,Chen CZ,Zhang XJ.Aquacultural Animal Pathogenic Bacteriology[M].Beijing:China Agriculture Press,2010:499-512(in Chinese)房海,陈翠真,张晓君.水产养殖动物病原细菌学[M].北京:中国农业出版社,2010:499-512
[23]Dong L.Identification and aethionlogical of pathogentic bactetia of cultured Turbot(Scophthalmus maximus)[D].Qingdao:Master’s Thesis of Ocean University of China,2009(in Chinese)董丽.养殖大菱鲆几种重要细菌性疾病病原菌的鉴定及其病原学初步研究[D].青岛:中国海洋大学硕士学位论文,2009
[24]Shahi N,Sharma P,Pandey J,et al.Characterization and pathogenicity study of Chryseobacterium scophthalmum recovered from gill lesions of diseased golden mahseer,Tor putitora(Hamilton,1822)in India[J].Aquaculture,2018,485:81-92
[25]Yang YB,Ai XH,Song Y,et al.Isolation,identification and antibiotic sensitivity of Chryseobacterium indologenes from Trionyx sinensis[J].Acta Hydrobiologica Sinica,2018,42(4):786-793(in Chinese)杨移斌,艾晓辉,宋怿,等.中华鳖源致病性产吲哚金黄杆菌分离、鉴定及药敏特性分析[J].水生生物学报,2018,42(4):786-793
[26]Boran H,Terzi E,Altinok I,et al.Bacterial diseases of cultured Mediterranean horse mackerel(Trachurus mediterraneus)in sea cages[J].Aquaculture,2013,396-399:8-13
[27]Bernardet JF,Vancanneyt M,Matte-Tailliez O,et al.Polyphasic study of Chryseobacterium strains isolated from diseased aquatic animals[J].Systematic and Applied Microbiology,2005,28(7):640-660
[28]Didinen BI,Onuk EE,?ztürk T,et al.First report of Chryseobacterium sp.from Koi(Cyprinus carpio)in Turkey[J].The Israeli Journal of Aquaculture,2016,68:1341-1348
[29]Vandamme P,Bernardet JF,Segers P,et al.NOTES:New perspectives in the classification of the Flavobacteria:Description of Chryseobacterium gen.nov.,Bergeyella gen.nov.,and Empedobacter nom.rev.[J].International Journal of Systematic Bacteriology,1994,44(4):827-831
[30]Li M,Song TT,Zheng RQ,et al.Isolation,identification and drug sensitivity test of the pathogen of Quasipaa spinosa[J].Journal of Anhui Agricultural Sciences,2016,44(3):64-66,98(in Chinese)李明,宋婷婷,郑荣泉,等.棘胸蛙歪头病病原分离·鉴定与药敏试验[J].安徽农业科学,2016,44(3):64-66,98
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