马链球菌马亚种新疆分离株的分离及耐药性和进化分析
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摘要
为了解新疆马链球菌马亚种流行株的分子特性和遗传变异情况,对2017年采集的病马淋巴组织进行病原分离培养、生化鉴定和药敏试验,并对该分离菌株的SeM基因进行了PCR扩增和测序分析。同时,将该分离株与本实验室2013—2017年分离的10个分离株的SeM基因与国外分离株构建系统进化树。结果表明,成功分离鉴定了马链球菌马亚种ZZM17-1株,该菌株对阿莫西林、环丙沙星、克林霉素、多西环素、庆大霉素、左氧氟沙星、四环素、磺胺甲恶唑、利福平、诺氟沙星、土霉素、头孢噻呋、磺胺嘧啶钠和恩诺沙星等14种药物敏感,对青霉素和氨苄西林表现明显的耐药。基于SeM的系统进化树表明新疆地区马链球菌马亚种分离株分别属于Ⅰ群和Ⅳ群。该结果丰富了国内马链球菌马亚种的基因数据,为本地区开展马腺疫的防治提供了理论依据。
To understand the molecular characteristic and genetic evolution of the epidemic strain of Streptococcus equi subsp.equi in Xinjiang, the lymph nodes of horse infected with suspected strangles in 2017 were collected for bacterium culture,biochemical identification and microbial susceptibility test,then the SeM gene was amplified by PCR and sequenced.The SeM genes of 10 isolates in our laboratory from 2013—2017 and those from other countries were used to construct phylogenetic trees.The results showed that S.equi subsp.equi strain ZZM17-1 was successfully isolated and this isolate was sensitive to fourteen microbiotics including amoxicillin,ciprofloxacin,clindamycin,doxycycline,gentamicin,levofloxacin,tetracycline,sulfamethoxazole,rifampicin,norfloxacin,oxytetracycline,ceftiofur,sulfadiazine sodium and enrofloxacin,but was apparently resistant to penicillin and ampicillin.Phylogenetic analysis based on Se M revealed that Xinjiang isolates belong to 2 different gene groups,Ⅰand Ⅳ group.This result provides theoretical basis for the prevention and treatment of strangles in this area.
To understand the molecular characteristic and genetic evolution of the epidemic strain of Streptococcus equi subsp.equi in Xinjiang, the lymph nodes of horse infected with suspected strangles in 2017 were collected for bacterium culture,biochemical identification and microbial susceptibility test,then the SeM gene was amplified by PCR and sequenced.The SeM genes of 10 isolates in our laboratory from 2013—2017 and those from other countries were used to construct phylogenetic trees.The results showed that S.equi subsp.equi strain ZZM17-1 was successfully isolated and this isolate was sensitive to fourteen microbiotics including amoxicillin,ciprofloxacin,clindamycin,doxycycline,gentamicin,levofloxacin,tetracycline,sulfamethoxazole,rifampicin,norfloxacin,oxytetracycline,ceftiofur,sulfadiazine sodium and enrofloxacin,but was apparently resistant to penicillin and ampicillin.Phylogenetic analysis based on Se M revealed that Xinjiang isolates belong to 2 different gene groups,Ⅰand Ⅳ group.This result provides theoretical basis for the prevention and treatment of strangles in this area.
引文
[1]陈新华,康立超,黄新,等.马腺疫链球菌的分离与鉴定[J].中国兽医杂志,2013,49(1):38-39.CHEN Xin-hua,KANG Li-chao,HUANG Xin,et al.Isolation and identification of Streptococcus equi[J].Chinese Journal of Veterinary Medicine,2013,49(1):38-39.(in Chinese)
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[7]BOSCHWITZ J S,TIMONEY J F.Characterization of the antiphagocytic activity of equine fibrinogen for Streptococcus equi subsp.equi[J].Microbial Pathogenesis,1994,17(2):121-129.
[8]KELLY C,BUGG M,ROBINSON C,et al.Sequence variation of the SeM gene of Streptococcus equi allows discrimination of the source of strangles outbreaks[J].Journal of Clinical Microbiology,2006,44(2):480-486.
[9]IJAZ M,VELINENI S,TIMONEY J F.Selective pressure for allelic diversity in SeM of Streptococcus equi does not affect immunoreactive proteins SzPSe or Se18.9[J].Infection,Genetics and Evolution,2011,11(5):1159-1163.
[10]TIMONEY J F,MUKHTAR M M.The protective M proteins of the equine group C streptococci[J].Veterinary Microbiology,1993,37(3-4):389-395.
[11]TIMONEY J F,EGGERS D.Serum bactericidal responses to Streptococcus equi of horses following infectionorvaccination[J].EquineVeterinaryJournal,1985,17(4):306-310.
[12]LINDAHL S,SODERLUND R,FROSTH S,et al.Tracing outbreaks of Streptococcus equi infection(strangles)in horses using sequence variation in the SeM gene and pulsed-field gel electrophoresis[J].Veterinary Microbiology,2011,153(1-2):144-149.
[13]LIBARDONI F,VIELMO A,FARIAS L,et al.Diversity of SeM in Streptococcus equi subsp.equi isolated from strangles outbreaks[J].Veterinary Microbiology,2013,162(2):663-669.
[14]康立超,陈新华,钟发刚,等.马腺疫链球菌的分离鉴定与疫苗的研制[J].黑龙江畜牧兽医,2013(5):109-111.KANG Li-chao,CHEN Xin-hua,ZHONG Fa-gang,et al.Isolation,identification and vaccine development of Streptococcus equi[J].Heilongjiang Animal Science and Veterinary Medicine,2013(5):109-111.(in Chinese)
[15]FLOCK M,JACOBSSON K,FRYKBERG L,et al.Recombinant Streptococcus equi proteins protect mice in challenge experiments and induce immune response in horse[J].Infection and Immunity,2004,72(6):3228-3236.
[16]刘云涛,王彩蝶,夏利宁,等.马链球菌马亚种新疆株SeM基因的克隆及序列差异分析[J].西北农业学报,2015,24(3):14-19.LIU Yun-tao,WANG Cai-die,XIA Li-ning,et al.Cloning and sequence difference analysis of the SeM gene of Streptococcus equi subsp.equi isolated in Xinjiang[J].Acta Agriculturae Borealioccid-entalis Sinica,2015,24(3):14-19.(in Chinese)
[17]SHEORAN A S,SPONSELLER B T,HOLMES M A,et al.Serum and mucosal antibody isotype responses to M-like protein(SeM)of Streptococcus equi in convalescent and vaccinated horses[J].Veterinary Immunology&Immunopathology,1997,59(3):239-251.
[18]TIMONEY J F,ARTIUSHIN S C,BOSCHWITZ J S.Comparison of the sequences and functions of Streptococcus equi M-like proteins SeM and SzPSe[J].Infection&Immunity,1997,65(9):3600-3605.
[2]周红,李月,李成贵,等.马链球菌兽疫亚种串联表达蛋白对小鼠免疫效果的评价[J].中国兽医科学,2015,45(11):1136-1141.ZHOU Hong,LI Yue,LI Cheng-gui,et al.Immunity effect assessment in mice of a tandem fusion protein composed of M-like protein,fibronectin-binding protein and IgG-binding protein of Streptococcus equi ssp.zooepidemicus[J].Chinese Veterinary Science,2015,45(11):1136-1141.(in Chinese)
[3]王淑杰,姜成钢,武嘉男,等.致病性马兽疫链球菌的分离鉴定、生物学特性及其M-like基因的序列分析[J].中国预防兽医学报,2012,34(11):878-881.WANG Shu-jie,JIANG Cheng-gang,WU Jia-nan,et al.Isolation and identification of a Streptococcus equi subsp.zooepidemicus and the M-like gene analysis[J].Chinese Journal of Preventive Veterinary Medicine,2012,34(11):878-881.(in Chinese)
[4]BOSCHWITZ J S,TIMONEY J F.Inhibition of C3 deposition on Streptococcus equi subsp.equi by M protein:a mechanism for survival in equine blood[J].Infection and Immunity,1994,62(8):3515-3520.
[5]ARTIUSHIN S C,TIMONEY J F,SHEORAN A S,et al.Characterization and immunogenicity of pyrogenic mitogens SePE-H and SePE-I of Streptococcus equi[J].Microbial Pathogenesis,2002,32(2):71-85.
[6]MEEHAN M,MULDOWNEY D A,WATKINS N J,et al.Localization and characterization of the ligand binding domain of the fibrinogen binding protein(FgBP)of Streptococcus equi subsp.equi[J].Microbiology,2000,146(5):1187-1194.
[7]BOSCHWITZ J S,TIMONEY J F.Characterization of the antiphagocytic activity of equine fibrinogen for Streptococcus equi subsp.equi[J].Microbial Pathogenesis,1994,17(2):121-129.
[8]KELLY C,BUGG M,ROBINSON C,et al.Sequence variation of the SeM gene of Streptococcus equi allows discrimination of the source of strangles outbreaks[J].Journal of Clinical Microbiology,2006,44(2):480-486.
[9]IJAZ M,VELINENI S,TIMONEY J F.Selective pressure for allelic diversity in SeM of Streptococcus equi does not affect immunoreactive proteins SzPSe or Se18.9[J].Infection,Genetics and Evolution,2011,11(5):1159-1163.
[10]TIMONEY J F,MUKHTAR M M.The protective M proteins of the equine group C streptococci[J].Veterinary Microbiology,1993,37(3-4):389-395.
[11]TIMONEY J F,EGGERS D.Serum bactericidal responses to Streptococcus equi of horses following infectionorvaccination[J].EquineVeterinaryJournal,1985,17(4):306-310.
[12]LINDAHL S,SODERLUND R,FROSTH S,et al.Tracing outbreaks of Streptococcus equi infection(strangles)in horses using sequence variation in the SeM gene and pulsed-field gel electrophoresis[J].Veterinary Microbiology,2011,153(1-2):144-149.
[13]LIBARDONI F,VIELMO A,FARIAS L,et al.Diversity of SeM in Streptococcus equi subsp.equi isolated from strangles outbreaks[J].Veterinary Microbiology,2013,162(2):663-669.
[14]康立超,陈新华,钟发刚,等.马腺疫链球菌的分离鉴定与疫苗的研制[J].黑龙江畜牧兽医,2013(5):109-111.KANG Li-chao,CHEN Xin-hua,ZHONG Fa-gang,et al.Isolation,identification and vaccine development of Streptococcus equi[J].Heilongjiang Animal Science and Veterinary Medicine,2013(5):109-111.(in Chinese)
[15]FLOCK M,JACOBSSON K,FRYKBERG L,et al.Recombinant Streptococcus equi proteins protect mice in challenge experiments and induce immune response in horse[J].Infection and Immunity,2004,72(6):3228-3236.
[16]刘云涛,王彩蝶,夏利宁,等.马链球菌马亚种新疆株SeM基因的克隆及序列差异分析[J].西北农业学报,2015,24(3):14-19.LIU Yun-tao,WANG Cai-die,XIA Li-ning,et al.Cloning and sequence difference analysis of the SeM gene of Streptococcus equi subsp.equi isolated in Xinjiang[J].Acta Agriculturae Borealioccid-entalis Sinica,2015,24(3):14-19.(in Chinese)
[17]SHEORAN A S,SPONSELLER B T,HOLMES M A,et al.Serum and mucosal antibody isotype responses to M-like protein(SeM)of Streptococcus equi in convalescent and vaccinated horses[J].Veterinary Immunology&Immunopathology,1997,59(3):239-251.
[18]TIMONEY J F,ARTIUSHIN S C,BOSCHWITZ J S.Comparison of the sequences and functions of Streptococcus equi M-like proteins SeM and SzPSe[J].Infection&Immunity,1997,65(9):3600-3605.