腹泻仔猪肠道菌群的ERIC-PCR指纹图谱分析
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摘要
【目的】分析腹泻仔猪周围环境菌群变化规律,为仔猪腹泻的诊断和治疗提供支持。【方法】采集健康对照和腹泻仔猪粪便、口腔以及产床和母猪乳头等样品,采用ERIC-PCR技术分析其菌群结构特征,并对粪便样品中的细菌进行分离和鉴定。【结果】对照组4类样品细菌ERIC-PCR指纹图谱十分相似,腹泻组各类样品之间电泳条带差异较大,出现了异常亮度的电泳条带,与对照组优势条带位置不同,其中口腔和母猪乳头部位电泳条带数显著低于对照组(P<0.05),菌群多样性指数降低,优势度指数升高。从粪便样品中分离出2株可疑菌株b1和b2,细菌染色和生化试验初步证明分离株b1和b2分别符合大肠杆菌和奇异变形杆菌的特征;利用ERIC-PCR技术分析分离株b1和b2的指纹图谱,发现分离优势细菌的ERIC-PCR指纹图谱包含于粪便样品的ERIC-PCR图谱中,证明样品的ERIC-PCR指纹图谱能够反映出优势菌群的特征;测序结果显示,分离株b1与大肠杆菌基因组同源性为99%,分离株b2与奇异变形杆菌基因组同源性为98%。【结论】腹泻仔猪周围环境菌群发生明显变化,ERIC-PCR技术可以快速、准确地监测和诊断菌群结构的变化,有助于细菌性仔猪腹泻的诊断和治疗。
【Objective】This study analyzed the changes of flora in the surrounding environment of diarrhea piglets to provide support for the diagnosis and treatment of diarrhea in piglets.【Method】Samples were collected from feces,mouth,obstetric table and sow nipple of health and diarrhea piglets.The bacterial community structure characteristics in the samples were analyzed by using ERIC-PCR technology,and the bacteria in fecal samples were isolated and identified.【Result】The ERIC-PCR fingerprints of the 4samples in control group were very similar,but the electrophoresis bands were different in diarrhea groups.Abnormal brightness electrophoretic bands appeared in diarrhea groups and the locations of bands were different from the control group.Especially,the mean numbers of bands in mouth and nipple samples were significantly lower than those of control group(P<0.05).In the diarrhea group,bacteria diversity index decreased,while the dominance index increased.Two suspicious strains b1 and b2were separated from fecal samples of diarrhea piglets.Bacterial staining and biochemical experiments showed that strains b1 and b2were identical with characteristics of Escherichia coli and Proteus.ERIC-PCR fingerprinting analysis showed that the ERIC-PCR fingerprints of the dominant bacteria were included in the ERIC-PCR map ofthe fecal samples,indicating that the ERIC-PCR fingerprint of the samples could reflect the characteristics of the dominant flora.The sequencing results showed that DNA sequences of bands in isolated b1 and b2were in 99% and 98% homology with Escherichia coli and Proteus,respectively.【Conclusion】The surrounding environmental flora of diarrhea piglets changed significantly.The ERIC-PCR technology can quickly and accurately monitor and diagnose the bacterial community structure characteristics,which is helpful for diagnosis and treatment of bacterial diarrhea in piglets.
【Objective】This study analyzed the changes of flora in the surrounding environment of diarrhea piglets to provide support for the diagnosis and treatment of diarrhea in piglets.【Method】Samples were collected from feces,mouth,obstetric table and sow nipple of health and diarrhea piglets.The bacterial community structure characteristics in the samples were analyzed by using ERIC-PCR technology,and the bacteria in fecal samples were isolated and identified.【Result】The ERIC-PCR fingerprints of the 4samples in control group were very similar,but the electrophoresis bands were different in diarrhea groups.Abnormal brightness electrophoretic bands appeared in diarrhea groups and the locations of bands were different from the control group.Especially,the mean numbers of bands in mouth and nipple samples were significantly lower than those of control group(P<0.05).In the diarrhea group,bacteria diversity index decreased,while the dominance index increased.Two suspicious strains b1 and b2were separated from fecal samples of diarrhea piglets.Bacterial staining and biochemical experiments showed that strains b1 and b2were identical with characteristics of Escherichia coli and Proteus.ERIC-PCR fingerprinting analysis showed that the ERIC-PCR fingerprints of the dominant bacteria were included in the ERIC-PCR map ofthe fecal samples,indicating that the ERIC-PCR fingerprint of the samples could reflect the characteristics of the dominant flora.The sequencing results showed that DNA sequences of bands in isolated b1 and b2were in 99% and 98% homology with Escherichia coli and Proteus,respectively.【Conclusion】The surrounding environmental flora of diarrhea piglets changed significantly.The ERIC-PCR technology can quickly and accurately monitor and diagnose the bacterial community structure characteristics,which is helpful for diagnosis and treatment of bacterial diarrhea in piglets.
引文
[1]张欣,胡兴权,王伟立.仔猪腹泻病的诊断与防治[J].畜禽业,2012(7):70-72.Zhang X,Hu X Q,Wang W L.Diagnosis and prevention of diarrhea in piglets[J].Livestock and Poultry Industry,2012(7):70-72.
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[4]赵桂英,段纲,杨亮宇,等.仔猪消化道菌群变化与仔猪腹泻的关系[J].家畜生态,2004,25(4):44-47.Zhao G Y,Duan G,Yang L Y,et al.Relationship between microflora changes in digestive tract of piglets and diarrhea in piglets[J].Livestock Ecology,2004,25(4):44-47.
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[7]罗永久,钟志军,彭广能,等.不同食性动物肠道菌群的ERICPCR指纹图谱分析[J].中国兽医学报,2014,34(11):1764-1769.Luo Y J,Zhong Z J,Peng G N,et al.ERIC-PCR fingerprinting analysis of intestinal flora in different feeding animals[J].Chinese Journal of Veterinary Science,2014,34(11):1764-1769.
[8] Shannon C E,Weaver W.The mathematical theory of communication[M].Urbana:University of Illinois Press,1998.
[9] Hunter P R,Gaston M A.Numerical index of the discriminatory ability of typing systems:an application of Simpson’s index of diversity[J].Journal of Clinical Microbiology,1988,26(11):2465-2466.
[10]陈祥,高崧,王雷,等.华东地区致初生仔猪腹泻大肠杆菌的O血清型和毒力因子[J].微生物学报,2004,44(1):96-100.Chen X,Gao S,Wang L,et al.Serogroups and virulence factors of Escherichia coli associated with diarrhea in piglets in eastern China[J].Acta Microbiologica Sinica,2004,44(1):96-100.
[11]倪莉,徐丽敏,马宁,等.山羊奇异变形杆菌毒力因子zapA基因的克隆及蛋白结构预测[J].西南师范大学学报(自然科学版),2012,37(6):46-50.Ni L,Xu L M,Ma N,et al.Cloning and prediction of protein structure of virulence factors zapAgene of Proteus mirabilis from goat[J].Journal of Southwest China Normal University(Natural Science Editon),2012,37(6):46-50.
[12]李云飞,秦翠丽,王忠泽,等.兔粪中微生物区系ERIC-PCR DNA指纹图谱的建立及分析[J].畜牧兽医学报,2016,47(4):716-722.Li Y F,Qin C L,Wang Z Z,et al.Establishment and analysis of ERIC-PCR DNA fingerprinting of microbial flora in rabbit feces[J].Acta Veterinaria et Zootechnica Sinica,2016,47(4):716-722.
[13]潘玉善.禽源大肠杆菌、奇异变形杆菌多重耐药的分子特征及CTX-M基因传播扩散机制[D].郑州:河南农业大学,2012.Pan Y S.Molecular characterization and mechanism of dissemination of CTX-M-type extended-spectrum-β-lactamase genes in multidrug-resistant isolates of Escherichia coli and Proteus mirabilis from poultry[D].Zhengzhou:Henan Agricultural University,2012.
[14]田存锋,吴同山,王敬军,等.仔猪腹泻的病因分析及防控措施探索[J].广东畜牧兽医科技,2015(3):48-50.Tian C F,Wu T S,Wang J J,et al.Pathogeny analysis and prevention and control measures of piglet diarrhea[J].Guangdong Journal of Animal and Veterinary Science,2015(3):48-50.
[15] Ruiz V L A,Bersano J G,Carvalho A F,et al.Case-control study of pathogens involved in piglet diarrhea[J].BMC Research Notes,2016,9(1):22.
[16] Khosravi A D,Hoveizavi H,Mohammadian A,et al.Genotyping of multidrug-resistant strains of Pseudomonas aeruginosa isolated from burn and wound infections by ERIC-PCR[J].Acta Cirurgica Brasileira,2016,31(3):206-211.
[17] Madhusudana Rao B,Surendran P K.Genetic heterogeneity of non-O1and non-O139 Vibrio cholerae isolates from shrimp aquaculture system:a comparison of RS-,REP-and ERICPCR fingerprinting approaches[J].Letters in Applied Microbiology,2010,51(1):65-74.
[18]陈玲,张菊梅,杨小鹃,等.南方食品中沙门氏菌污染调查及分型[J].微生物学报,2013,53(12):1326-1333.Chen L,Zhang J M,Yang X J,et al.Investigation and typing of Salmonellacontamination in food in South China[J].Acta Microbiologica Sinica,2013,53(12):1326-1333.
[19] Chen L,Li Y,Yu Y.Soil bacterial and fungal community successions under the stress of chlorpyrifos application and molecular characterization of chlorpyrifos-degrading isolates using ERIC-PCR[J].Journal of Zhejiang University(Science B),2014,15(4):322.
[20] Tungao W,Nimnoi P,Lumyong S,et al.Determination of bacterial diversity in bulk soil and rhizospheres using molecular fingerprintings and phenotypic features[J].World Applied Sciences Journal,2012,19(11):1613-1620.
[21] Pieper R,Janczyk P,Urubschurov V,et al.Effect of a single oral administration of Lactobacillus plantarum DSMZ 8862/8866before and at the time point of weaning on intestinal microbial communities in piglets[J].International Journal of Food Microbiology,2009,130(3):227-232.
[22] Bhandari S K,Xu B,Nyachoti C M,et al.Evaluation of alternatives to antibiotics using an Escherichia coli K88model of piglet diarrhea:effects on gut microbial ecology[J].Journal of Animal Science,2008,86(4):836-847.
[23] Deng H,Zhuang Y,Cai Y,et al.Isolation and molecular biological analysis of pig Proteus mirabilis[J].Chinese Journal of Veterinary Medicine,2015,3:6.
[24] Chen X,Wang Y W,Wang Y,et al.Isolation,identification and phylogenetic analysis of Proteus mirabilis from goats with diarrhea[J].Chin Anim Husb Vet Med,2014,41(8):215-218.
[25]张博.城市污水中肠杆菌的抗生素耐药性和多种耐药基因的检测[D].哈尔滨:东北农业大学,2013.Zhang B.Detection of antibiotic resistance and multidrug resistance genes of Enterobacteriaceae isolated from municipal wastewater[D].Harbin:Northeast Agricultural University,2013.
[2]孙涛,李东春,朱雅宁.提高新生仔猪成活率的有效对策[J].中国畜禽种业,2009(1):39-40.Sun T,Li D C,Zhu Y N.Effective measures to improve the survival rate of new born piglets[J].The Chinese Livestock and Poultry Breeding,2009(1):39-40.
[3]李志亚.猪传染性胃肠炎与猪流行性腹泻的区别[J].现代农业科技,2010(1):371.Li Z Y.The difference between transmissible gastroenteritis and porcine epidemic diarrhea[J].Modern Agricultural Science and Technology,2010(1):371.
[4]赵桂英,段纲,杨亮宇,等.仔猪消化道菌群变化与仔猪腹泻的关系[J].家畜生态,2004,25(4):44-47.Zhao G Y,Duan G,Yang L Y,et al.Relationship between microflora changes in digestive tract of piglets and diarrhea in piglets[J].Livestock Ecology,2004,25(4):44-47.
[5] Katara J,Deshmukh R,Singh N K,et al.Molecular typing of native Bacillus thuringiensis isolates from diverse habitats in India using REP-PCR and ERIC-PCR analysis[J].The Journal of General and Applied Microbiology,2012,58(2):83-94.
[6] Yuan W,Chai T J,Miao Z M.ERIC-PCR identification of the spread of airborne Escherichia coli in pig houses[J].Science of the Total Environment,2010,408(6):1446-1450.
[7]罗永久,钟志军,彭广能,等.不同食性动物肠道菌群的ERICPCR指纹图谱分析[J].中国兽医学报,2014,34(11):1764-1769.Luo Y J,Zhong Z J,Peng G N,et al.ERIC-PCR fingerprinting analysis of intestinal flora in different feeding animals[J].Chinese Journal of Veterinary Science,2014,34(11):1764-1769.
[8] Shannon C E,Weaver W.The mathematical theory of communication[M].Urbana:University of Illinois Press,1998.
[9] Hunter P R,Gaston M A.Numerical index of the discriminatory ability of typing systems:an application of Simpson’s index of diversity[J].Journal of Clinical Microbiology,1988,26(11):2465-2466.
[10]陈祥,高崧,王雷,等.华东地区致初生仔猪腹泻大肠杆菌的O血清型和毒力因子[J].微生物学报,2004,44(1):96-100.Chen X,Gao S,Wang L,et al.Serogroups and virulence factors of Escherichia coli associated with diarrhea in piglets in eastern China[J].Acta Microbiologica Sinica,2004,44(1):96-100.
[11]倪莉,徐丽敏,马宁,等.山羊奇异变形杆菌毒力因子zapA基因的克隆及蛋白结构预测[J].西南师范大学学报(自然科学版),2012,37(6):46-50.Ni L,Xu L M,Ma N,et al.Cloning and prediction of protein structure of virulence factors zapAgene of Proteus mirabilis from goat[J].Journal of Southwest China Normal University(Natural Science Editon),2012,37(6):46-50.
[12]李云飞,秦翠丽,王忠泽,等.兔粪中微生物区系ERIC-PCR DNA指纹图谱的建立及分析[J].畜牧兽医学报,2016,47(4):716-722.Li Y F,Qin C L,Wang Z Z,et al.Establishment and analysis of ERIC-PCR DNA fingerprinting of microbial flora in rabbit feces[J].Acta Veterinaria et Zootechnica Sinica,2016,47(4):716-722.
[13]潘玉善.禽源大肠杆菌、奇异变形杆菌多重耐药的分子特征及CTX-M基因传播扩散机制[D].郑州:河南农业大学,2012.Pan Y S.Molecular characterization and mechanism of dissemination of CTX-M-type extended-spectrum-β-lactamase genes in multidrug-resistant isolates of Escherichia coli and Proteus mirabilis from poultry[D].Zhengzhou:Henan Agricultural University,2012.
[14]田存锋,吴同山,王敬军,等.仔猪腹泻的病因分析及防控措施探索[J].广东畜牧兽医科技,2015(3):48-50.Tian C F,Wu T S,Wang J J,et al.Pathogeny analysis and prevention and control measures of piglet diarrhea[J].Guangdong Journal of Animal and Veterinary Science,2015(3):48-50.
[15] Ruiz V L A,Bersano J G,Carvalho A F,et al.Case-control study of pathogens involved in piglet diarrhea[J].BMC Research Notes,2016,9(1):22.
[16] Khosravi A D,Hoveizavi H,Mohammadian A,et al.Genotyping of multidrug-resistant strains of Pseudomonas aeruginosa isolated from burn and wound infections by ERIC-PCR[J].Acta Cirurgica Brasileira,2016,31(3):206-211.
[17] Madhusudana Rao B,Surendran P K.Genetic heterogeneity of non-O1and non-O139 Vibrio cholerae isolates from shrimp aquaculture system:a comparison of RS-,REP-and ERICPCR fingerprinting approaches[J].Letters in Applied Microbiology,2010,51(1):65-74.
[18]陈玲,张菊梅,杨小鹃,等.南方食品中沙门氏菌污染调查及分型[J].微生物学报,2013,53(12):1326-1333.Chen L,Zhang J M,Yang X J,et al.Investigation and typing of Salmonellacontamination in food in South China[J].Acta Microbiologica Sinica,2013,53(12):1326-1333.
[19] Chen L,Li Y,Yu Y.Soil bacterial and fungal community successions under the stress of chlorpyrifos application and molecular characterization of chlorpyrifos-degrading isolates using ERIC-PCR[J].Journal of Zhejiang University(Science B),2014,15(4):322.
[20] Tungao W,Nimnoi P,Lumyong S,et al.Determination of bacterial diversity in bulk soil and rhizospheres using molecular fingerprintings and phenotypic features[J].World Applied Sciences Journal,2012,19(11):1613-1620.
[21] Pieper R,Janczyk P,Urubschurov V,et al.Effect of a single oral administration of Lactobacillus plantarum DSMZ 8862/8866before and at the time point of weaning on intestinal microbial communities in piglets[J].International Journal of Food Microbiology,2009,130(3):227-232.
[22] Bhandari S K,Xu B,Nyachoti C M,et al.Evaluation of alternatives to antibiotics using an Escherichia coli K88model of piglet diarrhea:effects on gut microbial ecology[J].Journal of Animal Science,2008,86(4):836-847.
[23] Deng H,Zhuang Y,Cai Y,et al.Isolation and molecular biological analysis of pig Proteus mirabilis[J].Chinese Journal of Veterinary Medicine,2015,3:6.
[24] Chen X,Wang Y W,Wang Y,et al.Isolation,identification and phylogenetic analysis of Proteus mirabilis from goats with diarrhea[J].Chin Anim Husb Vet Med,2014,41(8):215-218.
[25]张博.城市污水中肠杆菌的抗生素耐药性和多种耐药基因的检测[D].哈尔滨:东北农业大学,2013.Zhang B.Detection of antibiotic resistance and multidrug resistance genes of Enterobacteriaceae isolated from municipal wastewater[D].Harbin:Northeast Agricultural University,2013.