西藏牦牛源肠出血性大肠杆菌毒力基因检测与进化分析
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摘要
为调查西藏地区牦牛源肠出血性大肠杆菌的流行情况,利用PCR检测和生物进化分析方法,对西藏林芝、拉萨不同地、市149株牦牛源大肠杆菌的肠出血性大肠杆菌相关毒力基因进行了研究。结果表明:牦牛源肠出血性大肠杆菌6对毒力基因中,只检出ehxA基因,检出率为9.40%;对检测到的14株牦牛源肠出血性大肠杆菌进行克隆测序,经系统发育分析发现牦牛源肠出血性大肠杆菌菌株内同源性达到99.9%左右,与GenBank中所录其他菌株的同源性达到99%。因此,西藏牦牛源肠出血性大肠杆菌基因确实存在,其毒力基因主要为ehxA基因;西藏林芝、阿里、日喀则、昌都均有分布,拉萨、山南和那曲地区未检出,应引起重视。西藏地区要根据各地实际情况需要加强监测肠出血性大肠杆菌引起的腹泻,建立流行毒株预警机制并合理用药。
In order to investigate the epidemic situation of intestinal haemorrhagic Escherichia coli from Yak in Tibet area,the virulence genes of 149 of enterohemorrhagic E.coli strains from yak in Linzhi and Lhasa were studied by using PCR detection and phylogenetic analysis.The results showed:The ehxA gene was detected among the 6 pairs enterohemorrhagic E.coli virulence genes originated in yak and the detection rate was 9.40.%.Fourteen strains were then cloned and sequenced by phylogenetic analysis.It was found that enterohemorrhagic E.coli strains were of99.9% homology,and were 99.0% homology to other strains recorded in GenBank.These results indicated that the Tibetan exist enterohemorrhagic E.coli gene in yak,the main virulence gene was ehxA gene.Linzhi,Ali,Shigatse and Changdu in Tibet were the distribution areas.The enterohemorrhagic E.coli gene was not detected in Lhasa,Shannan and Nagqu.This study indicated that the survey for diarrhea caused by EHEC should be strengthened according to the actual situation.Early pandemic strain warning mechanism should then be established and the drug should be used rationally.
In order to investigate the epidemic situation of intestinal haemorrhagic Escherichia coli from Yak in Tibet area,the virulence genes of 149 of enterohemorrhagic E.coli strains from yak in Linzhi and Lhasa were studied by using PCR detection and phylogenetic analysis.The results showed:The ehxA gene was detected among the 6 pairs enterohemorrhagic E.coli virulence genes originated in yak and the detection rate was 9.40.%.Fourteen strains were then cloned and sequenced by phylogenetic analysis.It was found that enterohemorrhagic E.coli strains were of99.9% homology,and were 99.0% homology to other strains recorded in GenBank.These results indicated that the Tibetan exist enterohemorrhagic E.coli gene in yak,the main virulence gene was ehxA gene.Linzhi,Ali,Shigatse and Changdu in Tibet were the distribution areas.The enterohemorrhagic E.coli gene was not detected in Lhasa,Shannan and Nagqu.This study indicated that the survey for diarrhea caused by EHEC should be strengthened according to the actual situation.Early pandemic strain warning mechanism should then be established and the drug should be used rationally.
引文
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[12]王刚,罗润波,贡嘎,益西措姆,李家奎,索朗斯珠.西藏牦牛源大肠杆菌毒力基因检测与分型[J].中国兽医学报,2017,37(10):1913-1918Wang G,Luo R B,Gongga,Yixicuomu,Li J K,Suolangsizhu.Escherichia coli virulence gene detection and classification in Tibetan yak[J].Chinese Journal of Veterinary Science,2017,37(10):1913-1918(in Chinese)
[13] Angiuoli S V,Gussman A,Klimke W,Cochrane G,Field D,Garrity G,Kodira C D,Kyrpides N,Madupu R,Markowitz V,Tatusova T,Thomson N,White O.Toward an online repository of standard operating procedures(SOPs)for(meta)genomic annotation[J].Omics A Journal of Integrative Biology,2008,12(2):137
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[16]郭锐,谭臣,彭忠,华琳,曾东柱,吴斌.牛源肠出血性大肠埃希菌的分离鉴定及进化分析[J].动物医学进展,2015,36(8):11-17Guo R,Tan C,Peng Z,Hua L,Zeng D Z,Wu B.Isolation,identification and phylogenetic analysis of enterohemorrhagic Escherichia coli from cows[J].Progress in Veterinary Medicine,2015,36(8):11-17(in Chinese)
[17] Kehl K S,Havens P,Behnke C E,Acheson D W.Evaluation of the premier EHEC assay for detection of Shiga toxinproducing Escherichia coli[J].Journal of Clinical Microbiology,1997,35(8):2051
[18] Kendall M M,Gruber C C,Parker C T,Sperandio V.Ethanolamine controlsexpressionofgenesencoding components involved in interkingdom signaling and virulence in enterohemorrhagic Escherichia coli O157∶H7[J].Mbio,2012,3(3):6-6
[19] Luzader D H,Clark D E,Gonyar L A,Kendall M M.EutR is a direct regulator of genes that contribute to metabolism and virulence in enterohemorrhagic Escherichia coli O157∶H7[J].Journal of Bacteriology,2013,195(21):4947-4953
[20]斯木吉德,敖日格乐,王纯洁,吴美云.6种血清型牛源致病性大肠杆菌的体外抑菌效果研究[J].黑龙江畜牧兽医,2009(9):60-70Simujide,Aorigele,Wang C J,Wu M Y.Bacteriostatic effect of 6serotype bovine pathogenic Escherichia coli in vitro[J].Heilongjiang Animal Science and Veterinary Medicine,2009(9):60-70(in Chinese)
[21]陈明勇,张冰,陈德威,曾群辉.牦牛致病性大肠杆菌生物学特性研究[J].中国农业大学学报,2001,6(3):119-122Chen M Y,Zhang B,Chen D W,Zeng Q H.Study on biological characteristics of pathogenic Escherichia coli in yak[J].Journal of China Agricultural University,2001,6(3):119-122(in Chinese)
[22]李玉,敖日格乐,王纯洁,斯木吉德,白同日格.临床型乳房炎奶牛致病性大肠杆菌血清型研究[J].中国奶牛,2010(12):40-41Li Y,Aorigele,Wang C J,Simujide,Baitongrige.Serotype of pathogenic Escherichia coli in dairy cows with clinical mastitis[J].China Dairy Cattle,2010(12):40-41(in Chinese)
[23]张天华,敖日格乐,王纯洁,斯木吉德,满达.奶牛粪样中致病性大肠埃希菌的分离与鉴定[J].动物医学进展,2012,33(5):122-124Zhang T H,Aorigele,Wang C J,Simujide,Manda.Isolation and identification of pathogenic Escherichia coli from feces of dairy cows[J].Progress in Veterinary Medicine,2012,33(5):122-124(in Chinese)
[24] Kaplan B S,Cleary T G,Obrig T G.Recent advances in understanding the pathogenesis of the hemolytic uremic syndromes[J].Pediatric Nephrology,1990,4(3):276-283
[2]张秀丽,廖兴广,张丁,张聪恪,王想霞,夏胜利.河南省首次检出O157∶H7大肠埃希氏菌[J].中国人兽共患病杂志,2000,16(3):102-103Zhang X L,Liao X G,Zhang D,Zhang C K,Wang X X,Xia S L.Escherichia coli O157∶H7isolated from row pork in Henan Province[J].Chinese Journal of Zoonoses,2000,16(3):102-103(in Chinese)
[3] Bettelheim K A.Enterohaemorrhagic Escherichia coli:A new problem,an old group of organisms[J].Australian Veterinary Journal,2010,73(1):20-26
[4] Hofmann S L.Southwestern internal medicine conference:Shiga-like toxins in hemolytic-uremic syndrome and thrombotic thrombocytopenic purpura[J].American Journal of the Medical Sciences,1993,306(6):398-406
[5] Holmes A D.Shiga and shiga-like toxins[J].Microbiological Reviews,1987,51(2):206-220
[6] Schmidt H,Plaschke B,Franke S,Russman H,Schwarzkopf A,Heesemann J,Karch H.Differentiation in virulence patterns of Escherichia coli possessing eae genes[J].Medical Microbiology and Immunology,1994,183(1):23-31
[7] Armstrong G L,Hollingsworth J,Morris J G.Emerging foodborne pathogens:Escherichia coli O157∶H7as a model of entry of a new pathogen into the food supply of the developed world[J].Epidemiologic Reviews,1996,18(1):29-51
[8]白向宁,赵爱兰,夏胜利,熊衍文,徐建国.非O157志贺毒素大肠杆菌分离株的多位点序列分型研究[J].中国人兽共患病学报,2012,28(6):544-548,560Bai X N,Zhao A L,Xia S L,Xiong Y W,Xu J G.Multilocus sequence typing of non-O157Shiga toxin-producing Escherichia coli isolates[J].Chinese Journal of Zoonoses,2012,28(6):544-548,560(in Chinese)
[9]陈祥,崔一晨,高崧,焦新安.肠出血性大肠杆菌传播模式[J].中国人兽共患病学报,2008,24(6):574-577Chen X,Cui Y C,Gao S,Jiao X A.Transmission mode of enterohemorrhagic Escherichia coli[J].Chinese Journal of Zoonoses,2008,24(6):574-577(in Chinese)
[10] Bolger A M,Lohse M,Usadel B.Trimmomatic:A flexible trimmer for Illumina sequence data[J].Bioinformatics,2014,30(15):2114-2120
[11] Zerbino D R,Birney V E:Algorithms for de novo short read assembly using de Bruijn graphs[J].Genome Research,2008,18(5):821-829
[12]王刚,罗润波,贡嘎,益西措姆,李家奎,索朗斯珠.西藏牦牛源大肠杆菌毒力基因检测与分型[J].中国兽医学报,2017,37(10):1913-1918Wang G,Luo R B,Gongga,Yixicuomu,Li J K,Suolangsizhu.Escherichia coli virulence gene detection and classification in Tibetan yak[J].Chinese Journal of Veterinary Science,2017,37(10):1913-1918(in Chinese)
[13] Angiuoli S V,Gussman A,Klimke W,Cochrane G,Field D,Garrity G,Kodira C D,Kyrpides N,Madupu R,Markowitz V,Tatusova T,Thomson N,White O.Toward an online repository of standard operating procedures(SOPs)for(meta)genomic annotation[J].Omics A Journal of Integrative Biology,2008,12(2):137
[14] Kaper J B,Nataro J P,Mobley H L.Pathogenic Escherichia coli[J].International Journal of Medical Microbiology,2005,295(6-7):355-356
[15] Fagan P K,Hornitzky M A,Bettelheim K A,Djordjevic S P.Detection of Shiga-like toxin(stx1 and stx2),intimin(eaeA),and enterohemorrhagic Escherichia coli(EHEC)hemolysin(EHEC hlyA)genes in animal feces by multiplex PCR[J].Applied and Environmental Microbiology,1999,65(2):868-872
[16]郭锐,谭臣,彭忠,华琳,曾东柱,吴斌.牛源肠出血性大肠埃希菌的分离鉴定及进化分析[J].动物医学进展,2015,36(8):11-17Guo R,Tan C,Peng Z,Hua L,Zeng D Z,Wu B.Isolation,identification and phylogenetic analysis of enterohemorrhagic Escherichia coli from cows[J].Progress in Veterinary Medicine,2015,36(8):11-17(in Chinese)
[17] Kehl K S,Havens P,Behnke C E,Acheson D W.Evaluation of the premier EHEC assay for detection of Shiga toxinproducing Escherichia coli[J].Journal of Clinical Microbiology,1997,35(8):2051
[18] Kendall M M,Gruber C C,Parker C T,Sperandio V.Ethanolamine controlsexpressionofgenesencoding components involved in interkingdom signaling and virulence in enterohemorrhagic Escherichia coli O157∶H7[J].Mbio,2012,3(3):6-6
[19] Luzader D H,Clark D E,Gonyar L A,Kendall M M.EutR is a direct regulator of genes that contribute to metabolism and virulence in enterohemorrhagic Escherichia coli O157∶H7[J].Journal of Bacteriology,2013,195(21):4947-4953
[20]斯木吉德,敖日格乐,王纯洁,吴美云.6种血清型牛源致病性大肠杆菌的体外抑菌效果研究[J].黑龙江畜牧兽医,2009(9):60-70Simujide,Aorigele,Wang C J,Wu M Y.Bacteriostatic effect of 6serotype bovine pathogenic Escherichia coli in vitro[J].Heilongjiang Animal Science and Veterinary Medicine,2009(9):60-70(in Chinese)
[21]陈明勇,张冰,陈德威,曾群辉.牦牛致病性大肠杆菌生物学特性研究[J].中国农业大学学报,2001,6(3):119-122Chen M Y,Zhang B,Chen D W,Zeng Q H.Study on biological characteristics of pathogenic Escherichia coli in yak[J].Journal of China Agricultural University,2001,6(3):119-122(in Chinese)
[22]李玉,敖日格乐,王纯洁,斯木吉德,白同日格.临床型乳房炎奶牛致病性大肠杆菌血清型研究[J].中国奶牛,2010(12):40-41Li Y,Aorigele,Wang C J,Simujide,Baitongrige.Serotype of pathogenic Escherichia coli in dairy cows with clinical mastitis[J].China Dairy Cattle,2010(12):40-41(in Chinese)
[23]张天华,敖日格乐,王纯洁,斯木吉德,满达.奶牛粪样中致病性大肠埃希菌的分离与鉴定[J].动物医学进展,2012,33(5):122-124Zhang T H,Aorigele,Wang C J,Simujide,Manda.Isolation and identification of pathogenic Escherichia coli from feces of dairy cows[J].Progress in Veterinary Medicine,2012,33(5):122-124(in Chinese)
[24] Kaplan B S,Cleary T G,Obrig T G.Recent advances in understanding the pathogenesis of the hemolytic uremic syndromes[J].Pediatric Nephrology,1990,4(3):276-283
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