山羊皮下脓肿的病原分离与鉴定
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摘要
为了解引起山羊皮下脓肿的主要病原,本研究从四川乐至县、金堂县和蓬溪县3个山羊养殖场无菌采集患皮下脓肿病羊脓液样本19份,通过细菌分离培养、生化试验及特异性PCR方法对其病原进行检测和鉴定,并通过药敏试验测定分离病原的药物敏感性。结果显示,PCR扩增法共检测出病原菌19株,其中伪结核棒状杆菌、金黄色葡萄球菌及化脓隐秘杆菌分离率分别为57.9%(11/19)、26.3%(5/19)及15.8%(3/19);采用细菌分离培养法分离到3种形态不同的病原菌共计17株,其中革兰氏阳性短杆菌经鉴定为伪结核棒状杆菌,分离率57.9%(11/19),革兰氏阳性球菌经鉴定为金黄色葡萄球菌,分离率为21.1%(4/19),革兰氏阳性杆菌经鉴定为化脓隐秘杆菌,分离率为10.5%(2/19),PCR鉴定较细菌分离培养法灵敏度更高。3种病原菌对丁胺卡那、氟苯尼考等多种药物敏感。结果表明,伪结核棒状杆菌是引起该地区山羊皮下脓肿的主要病原,金黄色葡萄球菌和化脓隐秘杆菌同样可引起山羊皮下脓肿,特异性PCR方法较细菌分离培养更为高效、准确。本研究结果可为后续进一步分析其生物学特性,以及四川地区山羊皮下脓肿的综合性防制提供依据。
To identify the main pathogens of goat subcutaneous abscess,a total of 19 abscess samples were collected from three goat farms in Lezhi county,Jintang county and Pengxi county in Sichuan province.The pathogens were detected and identified by bacterial isolation,biochemical test and specific PCR methods,and the drug sensitivity of the pathogens isolated was determined.The results showed that a total of 19 strains of 3 pathogens were isolated by PCR amplification method,and the isolation rate of Corynebacterium pseudotuberculos,Staphylococcus aureus and Arcanobacterium pyogenes was 57.9%(11/19),26.3%(5/19) and 15.8%(3/19),respectively.A total of 17 strains of 3 pathogens were isolated by isolation and identification method,in which 11 strains of Gram-positive Brevibacterium were identified as Corynebacterium pseudotuberculosis whose isolation rate was 57.9%(11/19),4 strains of Gram-positive cocci were identified as Staphylococcus aureus,and isolation rate was 21.1%(4/19),while 2 strains of Gram-positive bacilli were identified as Arcanobacterium pyogenes whose isolation rate was 10.5%(2/19).PCR was more sensitive than bacterial isolation.Three pathogens were sensitive to many drugs such as amikacin and florfenicol,etc.The results of this study provided a useful basis for further analysis of its biological characteristics and the development of comprehensive control measures for subcutaneous abscess in these areas.
To identify the main pathogens of goat subcutaneous abscess,a total of 19 abscess samples were collected from three goat farms in Lezhi county,Jintang county and Pengxi county in Sichuan province.The pathogens were detected and identified by bacterial isolation,biochemical test and specific PCR methods,and the drug sensitivity of the pathogens isolated was determined.The results showed that a total of 19 strains of 3 pathogens were isolated by PCR amplification method,and the isolation rate of Corynebacterium pseudotuberculos,Staphylococcus aureus and Arcanobacterium pyogenes was 57.9%(11/19),26.3%(5/19) and 15.8%(3/19),respectively.A total of 17 strains of 3 pathogens were isolated by isolation and identification method,in which 11 strains of Gram-positive Brevibacterium were identified as Corynebacterium pseudotuberculosis whose isolation rate was 57.9%(11/19),4 strains of Gram-positive cocci were identified as Staphylococcus aureus,and isolation rate was 21.1%(4/19),while 2 strains of Gram-positive bacilli were identified as Arcanobacterium pyogenes whose isolation rate was 10.5%(2/19).PCR was more sensitive than bacterial isolation.Three pathogens were sensitive to many drugs such as amikacin and florfenicol,etc.The results of this study provided a useful basis for further analysis of its biological characteristics and the development of comprehensive control measures for subcutaneous abscess in these areas.
引文
[1]李乡城.四川部分地区山羊皮下脓肿的细菌学分析[D].成都:四川农业大学,2016.LI X C.Goat’s bacteriological investigation of subcutaneous abscess in Sichuan area[D].Chengdu:Sichuan Agricultural University,2016.(in Chinese)
[2]赵宏坤,范伟兴,胡敬东,等.羊伪结核病研究进展[J].中国预防兽医学报,2000,22(3):77-78.ZHAO H K,FAN W X,HU J D,et al.Research progress for Corynebacterium pseudotuberculosis disease in goats[J].Chinese Journal of Preventive Veterinary Medicine,2000,22(3):77-78.(in Chinese)
[3]COLOMCADENA A,VELARDE R,SALINAS J,et al.Management of a caseous lymphadenitis outbreak in a new Iberian ibex(Capra pyrenaica)stock reservoir[J].Acta Veterinaria Scandinavica,2014,56(1):83-93.
[4]GEZON H M,BITHER H D,HANSON L A,et al.Epizootic of external and internal abscesses in a large goat herd over a 16-year period[J].Journal of The American Veterinary Medical Association,1991,98(2):257-263.
[5]霍宁宁.奶山羊干酪性淋巴结炎抗体ELISA检测方法研究及血清流行病学初步调查[D].杨凌:西北农林科技大学,2015.HUO N N.Study on the diagnosis methods of ELISAfor antibody against Corynebacterium pseudotuberculosis and serological epidemiology of caseous lymphadenitis[D].Yangling:Northwest A&F University,2015.(in Chinese)
[6]廖飞,杨先富,张冠华,等.一起山羊伪结核感染的诊断及防治[J].黑龙江畜牧兽医,2018,10:145-146.LIAO F,YANG X F,ZHANG G H,et al.Diagnosis and treatment of Corynebacterium pseudotuberculosis infection in goats[J].Heilongjiang Animal Science and Veterinary Medicine,2018,10:145-146.(in Chinese)
[7]郑国英,马玉馨,李太仙,等.昆明市西山区山羊伪结核棒状杆菌的分离鉴定[J].动物医学进展,2017,38(9):32-37.ZHENG G Y,MA Y X,LI T X,et al.Isolation and identification of goat Corynebacterium pseudotuberculosis in Xishan Distract Kunming[J].Progress in Veterinary Medicine,2017,38(9):32-37.(in Chinese)
[8]徐志豪,黄诗琴,李乡城,等.四川省山羊皮下脓肿的流行病学调查及病原体分析[J].中国兽医科学,2016,46(8):991-996.XU Z H,HUANG S Q,LI X C,et al.Epidemiological survey and etiological analysis in goat with subcutaneous abscess in Sichuan province[J].Chinese Veterinary Science,2016,46(8):991-996.(in Chinese)
[9]曾泽,韦雷飞,黄秀君,等.一起山羊伪结核棒状杆菌感染的诊断[J].动物医学进展,2014,35(3):131-133.ZENG Z,WEI L F,HUANG X J,et al.Diagnosis of Corynebacterium pseudotuberculosis infection in goats[J].Progress in Veterinary Medicine,2014,35(3):131-133.(in Chinese)
[10]DE L F R,BALLESTEROS C,BAUTISTA V,et al.Staphylococcus aureus subsp.anaerobius isolates from different countries are clonal in nature.[J].Veterinary Microbiology,2011,150(1):198-202.
[11]RIBEIRO M G,RISSETI R M,BOLAOS C A D,et al.Trueperella pyogenes multispecies infections in domestic animals:A retrospective study of 144cases(2002 to 2012)[J].Veterinary Quarterly,2015,35(2):82-87.
[12]周作勇,李和贤,杨浩钺,等.伪结核棒状杆菌毒力因子的研究进展[J].中国人兽共患病学报,2017,33(12):1115-1119.ZHOU Z Y,LI H X,YANG H Y,et al.Research advances on the virulence factors of Corynebacterium pseudotuberculosis[J].Chinese Journal of Zoonoses,2017,33(12):1115-1119.(in Chinese)
[13]SZALUSJORDANOW O,KANBAR T,SOEDAR-MANTO I,et al.Phenotypic and genotypic properties of Staphylococcus aureus subsp.anaerobius isolated from lymph node abscesses of goats[J].Berliner Und Munchener Tierarztliche Wochenschrift,2011,124(3-4):123-127.
[14]冉多良,王爱冬,戴志刚,等.野生岩羊金黄色葡萄球菌感染的诊断[J].动物医学进展,2007,28(12):114-115.RAN D L,WANG A D,DAI Z G,et al.Diagnosis of Staphylococcus aureus infection in Pseudois nayaur[J].Progress in Veterinary Medicine,2007,28(12):114-115.(in Chinese)
[15]马利青,王戈平.羊葡萄球菌病的诊断[J].中国兽医杂志,2003,39(3):54-55.MA L Q,WANG G P.Diagnosis of staphylocosis in lamb[J].Chinese Journal of Veterinary Medicine,2003,39(3):54-55.(in Chinese)
[16]JOST B H,BILLINGTON S J.Arcanobacterium pyogenes:Molecular pathogenesis of an animal opportunist[J].Antonie Van Leeuwenhoek,2005,88(2):87-102.
[17]张素辉,付利芝,黄勇富,等.山羊化脓隐秘杆菌的分离鉴定与分析[J].中国兽医杂志,2015,51(6):37-39.ZHANG S H,FU L Z,HUANG Y F,et al.Separation identification and analysis of Trueperella(Arcanobacterium)pyogenes in goats[J].Chinese Journal of Veterinary Medicine,2015,51(6):37-39.(in Chinese)
[18]张明富.牛源化脓隐秘杆菌的分离鉴定及其PCR检测方法的建立[D].哈尔滨:东北农业大学,2012.ZHANG M F.Isolation and identification of Arcanobacterium pyogenes and establishment of PCR detection method[D].Harbin:Northeast Agricultural University,2012.(in Chinese)
[19]许国洋,付利芝,杨金龙,等.山羊淋巴结炎病原茵的分离鉴定及多重PCR检测方法的建立[J].畜牧兽医学报,2017,48(2):324-330.XU G Y,FU L Z,YANG J L,et al.Isolation and identification of pathogenic bacteria of lymphadenitisin goat and establishment of multiplex PCR detection method[J].Acta Veterinaria et Zootechnica Sinica,2017,48(2):324-330.(in Chinese)
[20]BRAKSTAD O G,AASBAKK K,MAELAND J A.Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene[J].Journal of Clinical Microbiology,1992,30(7):1654-1660.
[21]DORELLA F A,PACHECO L G,OLIVEIRA S C,et al.Corynebacterium pseudotuberculosis:Microbiology,biochemical properties,pathogenesis and molecular studies of virulence[J].Veterinary Research,2006,37(2):201-218.
[22]ELLIS T M,SUTHERLAND S S,WILKINSON F C,et al.The role of Corynebacterium pseudotuberculosis lung lesions in the transmission of this bacterium to other sheep[J].Australian Veterinary Journal,1987,64(9):261-263.
[23]SZALUS'JORDANOW O,KABA J,CZOPOWICZ M,et al.Epidemiological features of Morel’s disease in goats[J].Polish Journal of Veterinary Sciences,2010,13(3):437-445.
[24]GARINO J F,MATOS R A T,MIRANDA NETO E G,et al.Clinical mastitis in goat due to Arcanobacterium pyogenes[J].Arquivo Brasileiro De Medicina Veterinária E Zootecnia,2012,64(4):1070-1073.
[25]蔡俊波,王爱玲,李海敏,等.陕南白山羊伪结核棒状杆菌分离鉴定及灭活铝胶疫苗研制[J].动物医学进展,2016,37(7):49-52.CAI J B,WANG A L,LI H M,et al.Isolation and identification of Corynebacterium pseudotuberculosis from white goat in South Shaanxi,and development of an inactivated vaccine with aluminum hydroxide adjuvant[J].Progress in Veterinary Medicine,2016,37(7):49-52.(in Chinese)
[26]冯洁,谢建云,胡建华,等.3种条件性致病菌三重PCR检测方法的建立及初步应用[J].中国畜牧兽医,2015,42(6):1389-1395.FENG J,XIE J Y,HU J H,et al.Establishment and preliminary application of triple PCR to identify three kinds of conditional pathogenic bacteria[J].China Animal Husbandry&Veterinary Medicine,2015,42(6):1389-1395.(in Chinese)
[2]赵宏坤,范伟兴,胡敬东,等.羊伪结核病研究进展[J].中国预防兽医学报,2000,22(3):77-78.ZHAO H K,FAN W X,HU J D,et al.Research progress for Corynebacterium pseudotuberculosis disease in goats[J].Chinese Journal of Preventive Veterinary Medicine,2000,22(3):77-78.(in Chinese)
[3]COLOMCADENA A,VELARDE R,SALINAS J,et al.Management of a caseous lymphadenitis outbreak in a new Iberian ibex(Capra pyrenaica)stock reservoir[J].Acta Veterinaria Scandinavica,2014,56(1):83-93.
[4]GEZON H M,BITHER H D,HANSON L A,et al.Epizootic of external and internal abscesses in a large goat herd over a 16-year period[J].Journal of The American Veterinary Medical Association,1991,98(2):257-263.
[5]霍宁宁.奶山羊干酪性淋巴结炎抗体ELISA检测方法研究及血清流行病学初步调查[D].杨凌:西北农林科技大学,2015.HUO N N.Study on the diagnosis methods of ELISAfor antibody against Corynebacterium pseudotuberculosis and serological epidemiology of caseous lymphadenitis[D].Yangling:Northwest A&F University,2015.(in Chinese)
[6]廖飞,杨先富,张冠华,等.一起山羊伪结核感染的诊断及防治[J].黑龙江畜牧兽医,2018,10:145-146.LIAO F,YANG X F,ZHANG G H,et al.Diagnosis and treatment of Corynebacterium pseudotuberculosis infection in goats[J].Heilongjiang Animal Science and Veterinary Medicine,2018,10:145-146.(in Chinese)
[7]郑国英,马玉馨,李太仙,等.昆明市西山区山羊伪结核棒状杆菌的分离鉴定[J].动物医学进展,2017,38(9):32-37.ZHENG G Y,MA Y X,LI T X,et al.Isolation and identification of goat Corynebacterium pseudotuberculosis in Xishan Distract Kunming[J].Progress in Veterinary Medicine,2017,38(9):32-37.(in Chinese)
[8]徐志豪,黄诗琴,李乡城,等.四川省山羊皮下脓肿的流行病学调查及病原体分析[J].中国兽医科学,2016,46(8):991-996.XU Z H,HUANG S Q,LI X C,et al.Epidemiological survey and etiological analysis in goat with subcutaneous abscess in Sichuan province[J].Chinese Veterinary Science,2016,46(8):991-996.(in Chinese)
[9]曾泽,韦雷飞,黄秀君,等.一起山羊伪结核棒状杆菌感染的诊断[J].动物医学进展,2014,35(3):131-133.ZENG Z,WEI L F,HUANG X J,et al.Diagnosis of Corynebacterium pseudotuberculosis infection in goats[J].Progress in Veterinary Medicine,2014,35(3):131-133.(in Chinese)
[10]DE L F R,BALLESTEROS C,BAUTISTA V,et al.Staphylococcus aureus subsp.anaerobius isolates from different countries are clonal in nature.[J].Veterinary Microbiology,2011,150(1):198-202.
[11]RIBEIRO M G,RISSETI R M,BOLAOS C A D,et al.Trueperella pyogenes multispecies infections in domestic animals:A retrospective study of 144cases(2002 to 2012)[J].Veterinary Quarterly,2015,35(2):82-87.
[12]周作勇,李和贤,杨浩钺,等.伪结核棒状杆菌毒力因子的研究进展[J].中国人兽共患病学报,2017,33(12):1115-1119.ZHOU Z Y,LI H X,YANG H Y,et al.Research advances on the virulence factors of Corynebacterium pseudotuberculosis[J].Chinese Journal of Zoonoses,2017,33(12):1115-1119.(in Chinese)
[13]SZALUSJORDANOW O,KANBAR T,SOEDAR-MANTO I,et al.Phenotypic and genotypic properties of Staphylococcus aureus subsp.anaerobius isolated from lymph node abscesses of goats[J].Berliner Und Munchener Tierarztliche Wochenschrift,2011,124(3-4):123-127.
[14]冉多良,王爱冬,戴志刚,等.野生岩羊金黄色葡萄球菌感染的诊断[J].动物医学进展,2007,28(12):114-115.RAN D L,WANG A D,DAI Z G,et al.Diagnosis of Staphylococcus aureus infection in Pseudois nayaur[J].Progress in Veterinary Medicine,2007,28(12):114-115.(in Chinese)
[15]马利青,王戈平.羊葡萄球菌病的诊断[J].中国兽医杂志,2003,39(3):54-55.MA L Q,WANG G P.Diagnosis of staphylocosis in lamb[J].Chinese Journal of Veterinary Medicine,2003,39(3):54-55.(in Chinese)
[16]JOST B H,BILLINGTON S J.Arcanobacterium pyogenes:Molecular pathogenesis of an animal opportunist[J].Antonie Van Leeuwenhoek,2005,88(2):87-102.
[17]张素辉,付利芝,黄勇富,等.山羊化脓隐秘杆菌的分离鉴定与分析[J].中国兽医杂志,2015,51(6):37-39.ZHANG S H,FU L Z,HUANG Y F,et al.Separation identification and analysis of Trueperella(Arcanobacterium)pyogenes in goats[J].Chinese Journal of Veterinary Medicine,2015,51(6):37-39.(in Chinese)
[18]张明富.牛源化脓隐秘杆菌的分离鉴定及其PCR检测方法的建立[D].哈尔滨:东北农业大学,2012.ZHANG M F.Isolation and identification of Arcanobacterium pyogenes and establishment of PCR detection method[D].Harbin:Northeast Agricultural University,2012.(in Chinese)
[19]许国洋,付利芝,杨金龙,等.山羊淋巴结炎病原茵的分离鉴定及多重PCR检测方法的建立[J].畜牧兽医学报,2017,48(2):324-330.XU G Y,FU L Z,YANG J L,et al.Isolation and identification of pathogenic bacteria of lymphadenitisin goat and establishment of multiplex PCR detection method[J].Acta Veterinaria et Zootechnica Sinica,2017,48(2):324-330.(in Chinese)
[20]BRAKSTAD O G,AASBAKK K,MAELAND J A.Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene[J].Journal of Clinical Microbiology,1992,30(7):1654-1660.
[21]DORELLA F A,PACHECO L G,OLIVEIRA S C,et al.Corynebacterium pseudotuberculosis:Microbiology,biochemical properties,pathogenesis and molecular studies of virulence[J].Veterinary Research,2006,37(2):201-218.
[22]ELLIS T M,SUTHERLAND S S,WILKINSON F C,et al.The role of Corynebacterium pseudotuberculosis lung lesions in the transmission of this bacterium to other sheep[J].Australian Veterinary Journal,1987,64(9):261-263.
[23]SZALUS'JORDANOW O,KABA J,CZOPOWICZ M,et al.Epidemiological features of Morel’s disease in goats[J].Polish Journal of Veterinary Sciences,2010,13(3):437-445.
[24]GARINO J F,MATOS R A T,MIRANDA NETO E G,et al.Clinical mastitis in goat due to Arcanobacterium pyogenes[J].Arquivo Brasileiro De Medicina Veterinária E Zootecnia,2012,64(4):1070-1073.
[25]蔡俊波,王爱玲,李海敏,等.陕南白山羊伪结核棒状杆菌分离鉴定及灭活铝胶疫苗研制[J].动物医学进展,2016,37(7):49-52.CAI J B,WANG A L,LI H M,et al.Isolation and identification of Corynebacterium pseudotuberculosis from white goat in South Shaanxi,and development of an inactivated vaccine with aluminum hydroxide adjuvant[J].Progress in Veterinary Medicine,2016,37(7):49-52.(in Chinese)
[26]冯洁,谢建云,胡建华,等.3种条件性致病菌三重PCR检测方法的建立及初步应用[J].中国畜牧兽医,2015,42(6):1389-1395.FENG J,XIE J Y,HU J H,et al.Establishment and preliminary application of triple PCR to identify three kinds of conditional pathogenic bacteria[J].China Animal Husbandry&Veterinary Medicine,2015,42(6):1389-1395.(in Chinese)