福建山羊溶血性曼氏杆菌的分离鉴定及耐药性分析
|
摘要
【目的】对福清一山羊养殖场发生的山羊肺炎病原进行确诊,为福建山羊溶血性曼式杆菌的分离鉴定及检测提供研究基础。【方法】对剖检采取的组织进行细菌分离培养,随后对溶血性曼氏杆菌、羊口疮病毒、绵羊肺炎支原体和丝状支原体簇等羊常见呼吸道病原进行PCR检测,对分离菌进行16S rRNA基因克隆测序,并对分离株进行药物敏感性研究。【结果】细菌分离培养结果显示:从病死羊肺脏中分离到1株细菌;PCR检测结果显示,除溶血性曼氏杆菌有扩增条带外,未检测到其他病原;16S rRNA测序结果显示:分离株16S rRNA基因序列与GenBank上公布的其他溶血性曼氏杆菌的同源性为97.1%～100.0%,与溶血性曼氏杆菌美国D171参考株同源性高达100%;以上结果表明分离菌为溶血性曼氏杆菌,命名为FJ-FQ2018。药物敏感性试验表明分离株对氟苯尼考、恩诺沙星、头孢噻吩、头孢唑啉、环丙沙星等高敏,对氨苄西林中敏,对多粘菌素B、林可霉素低敏。【结论】福建某养殖场引进的山羊发生肺炎后经病原分离鉴定确诊为溶血性曼氏杆菌感染,分离菌株对8种抗生素的耐药性存在差异。
【Objective】To identify and study the pathogen that caused the goat pneumonia in a farm in Fuqing,Fujian,and provide reference for the isolation,identification and detection of goat Mannheimia. 【Method】Bacterial isolation and culture were conducted on the tissues taken by autopsy on the diseased goats. ORF virus,mycoplasma,ovipneumonia and mycoplasma cluster were performed by PCR. A microorganism was isolated and identified by 16 S rRNA gene cloning and sequencing combined with PCR. Subsequently,sensitivities of the isolated strain toward various drugs were tested. 【Result】The PCR detection showed an unique amplified band of the isolated strain that matched Mannheimia haemolytica. The 16 S rRNA sequencing of the strain shared 97.1%-100.0% similarities with those of other strains of M. haemolytica published in GenBank which included an 100% homology with the D171 strain. Positively confirmed to be of the M. haemolytica family,the isolate was code-named FJ-FQ2018. The drug sensitivity test showed the isolate to be highly sensitive to flufenicol,enoxacin,cephalothiophene,cefazoline,ciprofloxacin,et al,moderately sensitive to ampicillin,and slightly sensitive to polymyxin B and lincomycin. 【Conclusion】The pathogen of goat pneumonia isotaled from Fujian was a strain of M. haemolytica,and the drug sensitivities of the isolate was deemed varions toward 8 antibiotics.
【Objective】To identify and study the pathogen that caused the goat pneumonia in a farm in Fuqing,Fujian,and provide reference for the isolation,identification and detection of goat Mannheimia. 【Method】Bacterial isolation and culture were conducted on the tissues taken by autopsy on the diseased goats. ORF virus,mycoplasma,ovipneumonia and mycoplasma cluster were performed by PCR. A microorganism was isolated and identified by 16 S rRNA gene cloning and sequencing combined with PCR. Subsequently,sensitivities of the isolated strain toward various drugs were tested. 【Result】The PCR detection showed an unique amplified band of the isolated strain that matched Mannheimia haemolytica. The 16 S rRNA sequencing of the strain shared 97.1%-100.0% similarities with those of other strains of M. haemolytica published in GenBank which included an 100% homology with the D171 strain. Positively confirmed to be of the M. haemolytica family,the isolate was code-named FJ-FQ2018. The drug sensitivity test showed the isolate to be highly sensitive to flufenicol,enoxacin,cephalothiophene,cefazoline,ciprofloxacin,et al,moderately sensitive to ampicillin,and slightly sensitive to polymyxin B and lincomycin. 【Conclusion】The pathogen of goat pneumonia isotaled from Fujian was a strain of M. haemolytica,and the drug sensitivities of the isolate was deemed varions toward 8 antibiotics.
引文
[1]RICE J A,CARRASCO-MEDINA L C,HODGINS D C,et al.Mannheimia haemolytica and bovine respiratory disease [J].Animal Health Research Reviews,2008,8(2):117-128.
[2]陈艳红,颜忠,查振林,等.溶血性曼氏杆菌致病机制的研究进展[J].中国畜牧兽医,2010,37(12):153-155.CHEN Y H,YAN Z,CHA Z L,et al.Progress on the study of Mannheimia haemolytica [J].China Animal Husbandry & Veterinary Medicine,2010,37(12):153-155.(in Chinese)
[3]李基棕,李文良,毛立,等.1株绵羊溶血性曼氏杆菌的分离鉴定及致病性研究[J].江苏农业科学,2017,45(7):156-158.LI J Z,LI W L,M L,et al.Isolation,identification and pathogenicity of a strain of Mannheimia haemolytica [J].Jiangsu Agriculture Science,2017,45(7):156-158.(in Chinese)
[4]ALEXANDER T W,COOK S R,YANKE L J,et al.A multiplex polymerase chain reaction assay for the identification of Mannheimia haemolytica,Mannheimia glucosidal and Mannheimia ruminalis [J].Veterinary Microbiology,2008,130(1/2):165-175.
[5]韩猛立,康立超,钟发刚,等.细菌性牛呼吸道疾病的研究进展[J].中国畜牧兽医,2013,40(5):165-172.HAN M L,KANG L C,ZHONG F G,et al.Research progress on bacterial pathogens of bovine respiratory disease [J].China Animal Husbandry & Veterinary Medicine,2013,40(5):165-172.(in Chinese)
[6]李娟,刘阳,彭欠欠,等.羊溶血性曼氏杆菌的分离与鉴定[J].黑龙江畜牧兽医,2013(20):96-97.LI J,LIU Y,PENG Q Q,et al.Isolation and identification of Mannheimia haemolytica [J].Heilongjiang Animal Science and Veterinary Medicine,2013(20):96-97.(in Chinese)
[7]徐慧,贺云霞,龚玉梅,等.溶血性曼氏杆菌的分离鉴定[J].黑龙江畜牧兽医,2009(12):97-98.XU H,HE Y X,GONG Y M,et al.Isolation and identification of Mannheimia haemolytica [J].Heilongjiang Animal Science and Veterinary Medicine,2009(12):97-98.(in Chinese)
[8]AYALEW S,CONFER A,HARTSON S,et al.Immunoproteomic analyses of outer membrane proteins of Mannheimia haemolytica and identification of potential vaccine candidates [J].Proteomics,2010,10(11):2151-2164.
[9]李雪霞,李富祥,赵文华,等.云南羊溶血性曼氏杆菌的分离鉴定及药敏试验[J].上海畜牧兽医通讯,2014(1):36-38.LI X X,LI F X,ZHAO W H,et al.Isolation,Identification and Antibiotics Resistance Analysis of Mannheimia Haemolytica from Yunnan [J].Shanghai Journal of Animal Husbandry and Veterinary Medicine,2014(1):36-38.(in Chinese)
[10]AYALEW S,SHRESTHA B,MONTELONGO M,et al.Immunogenicity of Mannheimia haemolytica recombinant outer membrane proteins serotype 1-specific antigen,OmpA,OmpP2,and OmpD15 [J].Clinical Vaccine Immunology,2011,18:2067-2074.
[11]WEISBURG W G,BARNS S M,PELLETIER D A,et al.16S ribosomal DNA amplification for phylogenetic study [J].Journal of Bacteriology,1991,173(2):697-703.
[12]林裕胜,江锦秀,江斌,等.羊口疮病毒和丝状支原体簇双重PCR检测方法的建立及应用[J].中国预防兽医学报,2017,39(4):292-295.LIN Y S,JIANG J X,JIANG B,et al.Establishment and application of a duplex PCR assay for detection of Orf virus and Mycoplasma mycoides cluster [J].Chinese Journal of Preventive Veterinary Medicine,2017,39(4):292-295.(in Chinese)
[13]林裕胜,江锦秀,江斌,等.绵羊肺炎支原体、丝状支原体山羊亚种和山羊支原体山羊肺炎亚种多重PCR检测方法的建立 [J].中国预防兽医学报,2018,40(9):812-817.LIN Y S,JIANG J X,JIANG B,et al.Development of a triplex PCR assay for detection of Mycoplasma ovipneumoniae,M.mycoides subsp.Capri and M.capricolum subsp.capripneumoniae [J].Chinese Journal of Preventive Veterinary Medicine,2018,40(9):812-817.(in Chinese)
[14]DASSANAYAKE R P,SHANTHALINGAM S,HERNDON C N,et al.Mycoplasma ovipneumoniae can predispose bighorn sheep to fatal Mannheimia haemolytica pneumonia [J].Veterinary Microbiology,2010,145(3-4):354-359.
[15]NIU Y D,COOK S R,WANG J,et al.Comparative analysis of multiple inducible phages from Mannheimia haemolytica [J].BMC Microbiology,2015,15:175.
[16]SINGH K,RITCHEY J W,CONFER A W.Mannheimia haemolytica:bacterial-host interactions in bovine pneumonia [J].Veterinary Pathology,2011,48(2):338-348.
[17]SHANTHALINGAM S,GOLDY A,BAVANANTHASIVAM J,et al.PCR assay detects Mannheimia haemolytica in culture negative pneumonic lung tissues of bighorn sheep (Ovis canadensis) from outbreaks in the western USA,2009 -2010 [J].Journal of Wild Disease,2014,50(1):1-10.
[18]冯旭飞,刀筱芳,王志敏,等.绵羊肺脏中溶血性曼氏杆菌的分离鉴定及其药物敏感性分析[J].中国畜牧兽医,2014,41(8):224-228.FENG X F,DAO X F,WANG Z M,et al.Isolation,identification and antibiotics susceptibility of Mannheimia haemolytica from sheep lungs [J].China Animal Husbandry & Veterinary Medicine,2014,41(8):224-228.(in Chinese)
[19]KATSUDA K,KOHMOTO M,MIKAMI O.Relationship between serotype and the antimicrobial susceptibility of Mannheimia haemolytica isolates collected between 1991 and 2010 [J].Research Veterinary Science,2013,94(2):205-208.
[20]LUBBERS B,HANZLICEK G.Antimicrobial multidrug resistance and coresistance patterns of Mannheimia haemolytica isolated from bovine respiratory disease cases-a three-year (2009-2011) retrospective analysis [J].Journal of Veterianry Diagnostic Investigation,2013,25(3):413-417.
[21]AYALEW S,CONFER A W,HARTSON S D,et al.Immunoproteomic analyses of outer membrane proteins of Mannheimia haemolytica and identification of potential vaccine candidates [J].Proteomics,2010(10):2151-2164.
[2]陈艳红,颜忠,查振林,等.溶血性曼氏杆菌致病机制的研究进展[J].中国畜牧兽医,2010,37(12):153-155.CHEN Y H,YAN Z,CHA Z L,et al.Progress on the study of Mannheimia haemolytica [J].China Animal Husbandry & Veterinary Medicine,2010,37(12):153-155.(in Chinese)
[3]李基棕,李文良,毛立,等.1株绵羊溶血性曼氏杆菌的分离鉴定及致病性研究[J].江苏农业科学,2017,45(7):156-158.LI J Z,LI W L,M L,et al.Isolation,identification and pathogenicity of a strain of Mannheimia haemolytica [J].Jiangsu Agriculture Science,2017,45(7):156-158.(in Chinese)
[4]ALEXANDER T W,COOK S R,YANKE L J,et al.A multiplex polymerase chain reaction assay for the identification of Mannheimia haemolytica,Mannheimia glucosidal and Mannheimia ruminalis [J].Veterinary Microbiology,2008,130(1/2):165-175.
[5]韩猛立,康立超,钟发刚,等.细菌性牛呼吸道疾病的研究进展[J].中国畜牧兽医,2013,40(5):165-172.HAN M L,KANG L C,ZHONG F G,et al.Research progress on bacterial pathogens of bovine respiratory disease [J].China Animal Husbandry & Veterinary Medicine,2013,40(5):165-172.(in Chinese)
[6]李娟,刘阳,彭欠欠,等.羊溶血性曼氏杆菌的分离与鉴定[J].黑龙江畜牧兽医,2013(20):96-97.LI J,LIU Y,PENG Q Q,et al.Isolation and identification of Mannheimia haemolytica [J].Heilongjiang Animal Science and Veterinary Medicine,2013(20):96-97.(in Chinese)
[7]徐慧,贺云霞,龚玉梅,等.溶血性曼氏杆菌的分离鉴定[J].黑龙江畜牧兽医,2009(12):97-98.XU H,HE Y X,GONG Y M,et al.Isolation and identification of Mannheimia haemolytica [J].Heilongjiang Animal Science and Veterinary Medicine,2009(12):97-98.(in Chinese)
[8]AYALEW S,CONFER A,HARTSON S,et al.Immunoproteomic analyses of outer membrane proteins of Mannheimia haemolytica and identification of potential vaccine candidates [J].Proteomics,2010,10(11):2151-2164.
[9]李雪霞,李富祥,赵文华,等.云南羊溶血性曼氏杆菌的分离鉴定及药敏试验[J].上海畜牧兽医通讯,2014(1):36-38.LI X X,LI F X,ZHAO W H,et al.Isolation,Identification and Antibiotics Resistance Analysis of Mannheimia Haemolytica from Yunnan [J].Shanghai Journal of Animal Husbandry and Veterinary Medicine,2014(1):36-38.(in Chinese)
[10]AYALEW S,SHRESTHA B,MONTELONGO M,et al.Immunogenicity of Mannheimia haemolytica recombinant outer membrane proteins serotype 1-specific antigen,OmpA,OmpP2,and OmpD15 [J].Clinical Vaccine Immunology,2011,18:2067-2074.
[11]WEISBURG W G,BARNS S M,PELLETIER D A,et al.16S ribosomal DNA amplification for phylogenetic study [J].Journal of Bacteriology,1991,173(2):697-703.
[12]林裕胜,江锦秀,江斌,等.羊口疮病毒和丝状支原体簇双重PCR检测方法的建立及应用[J].中国预防兽医学报,2017,39(4):292-295.LIN Y S,JIANG J X,JIANG B,et al.Establishment and application of a duplex PCR assay for detection of Orf virus and Mycoplasma mycoides cluster [J].Chinese Journal of Preventive Veterinary Medicine,2017,39(4):292-295.(in Chinese)
[13]林裕胜,江锦秀,江斌,等.绵羊肺炎支原体、丝状支原体山羊亚种和山羊支原体山羊肺炎亚种多重PCR检测方法的建立 [J].中国预防兽医学报,2018,40(9):812-817.LIN Y S,JIANG J X,JIANG B,et al.Development of a triplex PCR assay for detection of Mycoplasma ovipneumoniae,M.mycoides subsp.Capri and M.capricolum subsp.capripneumoniae [J].Chinese Journal of Preventive Veterinary Medicine,2018,40(9):812-817.(in Chinese)
[14]DASSANAYAKE R P,SHANTHALINGAM S,HERNDON C N,et al.Mycoplasma ovipneumoniae can predispose bighorn sheep to fatal Mannheimia haemolytica pneumonia [J].Veterinary Microbiology,2010,145(3-4):354-359.
[15]NIU Y D,COOK S R,WANG J,et al.Comparative analysis of multiple inducible phages from Mannheimia haemolytica [J].BMC Microbiology,2015,15:175.
[16]SINGH K,RITCHEY J W,CONFER A W.Mannheimia haemolytica:bacterial-host interactions in bovine pneumonia [J].Veterinary Pathology,2011,48(2):338-348.
[17]SHANTHALINGAM S,GOLDY A,BAVANANTHASIVAM J,et al.PCR assay detects Mannheimia haemolytica in culture negative pneumonic lung tissues of bighorn sheep (Ovis canadensis) from outbreaks in the western USA,2009 -2010 [J].Journal of Wild Disease,2014,50(1):1-10.
[18]冯旭飞,刀筱芳,王志敏,等.绵羊肺脏中溶血性曼氏杆菌的分离鉴定及其药物敏感性分析[J].中国畜牧兽医,2014,41(8):224-228.FENG X F,DAO X F,WANG Z M,et al.Isolation,identification and antibiotics susceptibility of Mannheimia haemolytica from sheep lungs [J].China Animal Husbandry & Veterinary Medicine,2014,41(8):224-228.(in Chinese)
[19]KATSUDA K,KOHMOTO M,MIKAMI O.Relationship between serotype and the antimicrobial susceptibility of Mannheimia haemolytica isolates collected between 1991 and 2010 [J].Research Veterinary Science,2013,94(2):205-208.
[20]LUBBERS B,HANZLICEK G.Antimicrobial multidrug resistance and coresistance patterns of Mannheimia haemolytica isolated from bovine respiratory disease cases-a three-year (2009-2011) retrospective analysis [J].Journal of Veterianry Diagnostic Investigation,2013,25(3):413-417.
[21]AYALEW S,CONFER A W,HARTSON S D,et al.Immunoproteomic analyses of outer membrane proteins of Mannheimia haemolytica and identification of potential vaccine candidates [J].Proteomics,2010(10):2151-2164.