屠宰生猪副猪嗜血杆菌的分离鉴定及药物敏感性分析
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摘要
为调查屠宰生猪副猪嗜血杆菌(Hps)的隐性感染情况,本研究从陕西省某规模化生猪屠宰场采集80份病变肺脏,应用PCR方法进行Hps核酸检测,无菌取阳性肺脏,通过平板划线分离获得疑似Hps,进一步对分离菌进行形态观察、培养特性分析、卫星现象观察、生化试验和分离菌核酸片段序列分析,纯化获得Hps分离菌;用药敏纸片法分析Hps分离菌株对常用药物的敏感性。结果显示,从80份病变肺脏中检出39份Hps阳性,阳性率48.75%(39/80);从39份Hps阳性肺脏中分离获得3株疑似Hps,分离菌在显微镜下呈革兰氏阴性、短杆状;在添加有胎牛血清和NAD的TSA平板上生长良好;在金黄色葡萄球菌周围呈现出典型的"卫星现象"生长;能发酵葡萄糖、蔗糖、果糖、半乳糖、麦芽糖和阿拉伯糖,不发酵甘露醇;PCR扩增序列与Hps同源性在98%以上。药物敏感性试验结果显示,3株分离菌对环丙沙星、恩诺沙星和氟苯尼考等抗菌药物高度敏感,对青霉素、林可霉素和甲氧苄啶等抗菌药物耐药。本研究获得的Hps分离株为疫苗研制提供了基础材料,为生猪养殖场选择敏感药物防控Hps感染提供了参考依据。
In order to investigate the hidden infection of Haemophilus parasuis(Hps) in slaughtered pigs,this study collected 80 diseased lungs from a large-scale pig slaughterhouse in Shaanxi province.The positive lungs were aseptically sampled,and the suspected Hps was obtained by streaking through the plate.The morphology of the isolates,analysis of the culture characteristics,observation of the satellite phenomenon,biochemical test and sequence analysis of the isolates nucleic acid fragments were carried out,and the Hps isolates were purified.The sensitivity of Hps isolates to commonly used drugs was analyzed by drug sensitive paper method.The results showed that 39 Hps positive samples were detected from 80 lungs,and the positive rate was 48.75%(39/80).Three suspected Hps were isolated from 39 Hps-positive lungs.The isolate was Gram-negative,short rod under the microscope,grew well on TSA plates supplemented with fetal bovine serum and NAD,exhibited a typical "satellite phenomenon" growth around S.aureus,could ferment glucose,sucrose,fructose,galactose,maltose and arabinose without fermenting mannitol.The PCR amplification sequence was more than 98% homologous to Hps.The results of drug susceptibility test showed that the three isolates were highly sensitive to antibacterial drugs such as ciprofloxacin,enrofloxacin and florfenicol,and were not sensitive to antibacterial drugs such as penicillin,lincomycin and trimethoprim.The Hps isolates obtained in this study provided the basic materials for vaccine development,and provided a reference for the selection of sensitive drugs to prevent and control Hps infection in pig farms.
In order to investigate the hidden infection of Haemophilus parasuis(Hps) in slaughtered pigs,this study collected 80 diseased lungs from a large-scale pig slaughterhouse in Shaanxi province.The positive lungs were aseptically sampled,and the suspected Hps was obtained by streaking through the plate.The morphology of the isolates,analysis of the culture characteristics,observation of the satellite phenomenon,biochemical test and sequence analysis of the isolates nucleic acid fragments were carried out,and the Hps isolates were purified.The sensitivity of Hps isolates to commonly used drugs was analyzed by drug sensitive paper method.The results showed that 39 Hps positive samples were detected from 80 lungs,and the positive rate was 48.75%(39/80).Three suspected Hps were isolated from 39 Hps-positive lungs.The isolate was Gram-negative,short rod under the microscope,grew well on TSA plates supplemented with fetal bovine serum and NAD,exhibited a typical "satellite phenomenon" growth around S.aureus,could ferment glucose,sucrose,fructose,galactose,maltose and arabinose without fermenting mannitol.The PCR amplification sequence was more than 98% homologous to Hps.The results of drug susceptibility test showed that the three isolates were highly sensitive to antibacterial drugs such as ciprofloxacin,enrofloxacin and florfenicol,and were not sensitive to antibacterial drugs such as penicillin,lincomycin and trimethoprim.The Hps isolates obtained in this study provided the basic materials for vaccine development,and provided a reference for the selection of sensitive drugs to prevent and control Hps infection in pig farms.
引文
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[2] 李伯森.副猪嗜血杆菌病的病理学诊断及防制[J].中国畜牧兽医,2010,37(7):192-194.LI B S.Pathological diagnosis and prevention of Haemophilus parasuis[J].China Animal Husbandtry & Veterinary Medicine,2010,37(7):192-194.(in Chinese)
[3] 徐惠娟,薛云,赵战勤,等.副猪嗜血杆菌国内流行血清型菌株的生物学特性比较研究[J].中国兽医学报,2014,34(5):729-735.XU H J,XUE Y,ZHAO Z Q,et al.Comparative study on biological characterization of the epidemic serotypes Haemophilus suis in China[J].Chinese Journal of Veterinary Science,2014,34(5):729-735.(in Chinese)
[4] 苗立中,李书光,胡琳琳,等.副猪嗜血杆菌病发病概况及诊断方法[J].动物医学进展,2014,35(6):140-144.MIAO L Z,LI S G,HU L L,et al.Introduction of haemophilosis and diagnostic methods[J].Progress in Veterinary Medicine,2014,35(6):140-144.(in Chinese)
[5] 王建,邵卫星,吕占军,等.2012年我国部分省市规模化猪场副猪嗜血杆菌分离鉴定及菌株血清分型[J].动物医学进展,2014,35(3):48-52.WANG J,SHAO W X,LYU Z J,et al.Isolation,identification and serotyping of Haemophilus parasuis in parts of large scale pig farms in China 2012[J].Progress in Veterinary Medicine,2014,35(3):48-52.(in Chinese)
[6] 李明波,库旭钢,赵海忠,等.猪繁殖与呼吸综合征病毒(PRRSV)协同感染细菌性疾病的分析研究[J].中国畜牧兽医,2018,45(3):781-791.LI M B,KU X G,ZHAO H Z,et al.Study on co-infection analysis of porcine reproductive and respiratory syndrome virus (PRRSV) and bacterial [J].China Animal Husbandtry & Veterinary Medicine,2018,45(3):781-791.(in Chinese)
[7] MACEDO N,ROVIRA A,TORREMORELL M.Haemophilus parasuis:Infection,immunity and enrofloxacin[J].Veterinary Research,2015,46(1):128.
[8] 华升,梅书棋,曾德芳,等.副猪嗜血杆菌病研究进展[J].动物医学进展,2006,27(11):7-10.HUA S,MEI S Q,ZENG D F,et al.Advance in Haemophilus parasuis[J].Progress in Veterinary Medicine,2006,27(11):7-10.(in Chinese)
[9] 米丁丹,张艳禾,张万江,等.副猪嗜血杆菌临床分离株耐药性与耐药机制的研究[J].中国预防兽医学报,2015,37(7):524-527.MI D D,ZHANG Y H,ZHANG W J,et al.Investigation of drug resistance and resistance mechanism ofHaemophilus parasuis[J].Chinese Journal of Preventive Veterinary Medicine,2015,37(7):524-527.(in Chinese)
[10] 代军,刘君,黄倩,等.副猪嗜血杆菌分型血清制备及其四川地区流行病学[J].中国兽医学报,2016,36(1):24-29.DAI J,LIU J,HUANG Q,et al.Preparation of antisera to different serotype of Haemophilus parasuis and application in epidemiology study of Sichuan province[J].Chinese Journal of Veterinary Science,2016,36(1):24-29.(in Chinese)
[11] 李曦婷,董俏,李化生,等.副猪嗜血杆菌检测技术及耐药性研究进展[J].动物医学进展,2016,37(10):85-89.LI X T,DONG Q,LI H S,et al.Advances in detection technology and drug resistance of Haemophilus parasuis[J].Progress in Veterinary Medicine,2016,37(10):85-89.(in Chinese)
[12] HOWELL K J,PETERS S E,WANG J,et al.Development of a multiplex PCR assay for rapid molecular serotyping of Haemophilus parasuis[J].Journal of Clinical Microbiology,2015,53(12):3812-3821.
[13] 王超,陈欣,张梦南,等.副猪嗜血杆菌的分离鉴定及其OmpP5基因序列分析[J].动物医学进展,2018,39(1):16-21.WANG C,CHEN X,ZHANG M N,et al.Isolation and identification of Haemophilus parasuis and sequence analysis of it’s OmpP5 gene[J].Progress in Veterinary Medicine,2018,39(1):16-21.(in Chinese)
[14] KAVANOVá L.Immune response of porcine alveolar macrophages to a concurrent infection with porcine reproductive and respiratory syndrome virus and Haemophilus parasuis in vitro[J].Veterinary Microbiology,2015,180(1-2):28-35.
[15] 林初文,苗立中.副猪嗜血杆菌病疫苗研究进展[J].中国畜牧兽医,2013,40(5):203-206.LIN C W,MIAO L Z.Progress onvaccine of Haemophilus parasuis[J].China Animal Husbandtry & Veterinary Medicine,2013,40(5):203-206.(in Chinese)
[16] 陈同煜.泰安及周边地区屠宰生猪肺脏健康状况评估及相关病原感染调查分析[D].泰安:山东农业大学,2017.CHEN T Y.Taian and surrounding areas slaughter pigs lung health assessment and related pathogenic infection investigation and analysisea virus[D].Tai’an:Shandong Agricultural University,2017.(in Chinese)
[17] 张悦,李蓓蓓,魏建超,等.上海副猪嗜血杆菌分离鉴定及其耐药性分析[J].中国动物传染病学报,2015,23(4):25-30.ZHANG Y,LI B B,WEI J C,et al.Isolation and antibiotic resistance of Haemophilus parasuis in Shanghai[J].Chinese Journal of Animal Infectious Diseases,2015,23(4):25-30.(in Chinese)
[18] 陆秀玉.丹东地区屠宰生猪肺脏细菌的分离鉴定[J].中国兽医杂志,2016,52(8):97-99.CHEN X Y.Isolation and identification of lung bacteria from slaughter pigs in Dandong area[J].Chinese Journal of Veterinary Medicine,2016,52(8):97-99.(in Chinese)
[19] 蔡旭旺.副猪嗜血杆菌的分离鉴定及诊断方法与灭活疫苗的研究[D].武汉:华中农业大学,2006.CAI X W.Isolation and characterization of Haemophilus parasuis and development of its diagnostic method and inactivated bacterin[D].Wuhan:Huazhong Agricultural University,2006.(in Chinese)
[20] CERDà-CUéLLAR M,NARANJO J F,VERGE A,et al.Sow vaccination modulates the colonization of piglets by Haemophilus parasuis[J].Veterinary Microbiology,2010,145(3-4):315-320.
[21] 池晶晶,杨春蕾,路超,等.天津地区副猪嗜血杆菌的分离鉴定及敏感药物筛选[J].中国畜牧兽医,2015,42(5):1306-1310.CHI J J,YANG C L,LU C,et al.Isolation,identification and sensitive drug screening of Haemophilus parasuis in Tianjin[J].China Animal Husbandry & Veterinary Medicine,2015,42(5):1306-1310.(in Chinese)
[22] 靳国旺,蔡敏.副猪嗜血杆菌的分离鉴定及药敏试验[J].黑龙江畜牧兽医,2016,5:131-132.JIN G W,CAI M.Isolation,identification and drug sensitivity test of Haemophilus parasuis[J].Heilongjiang Animal Science and Veterinary Medicine,2016,5:131-132.(in Chinese)
[23] 陈鹏强,章秋月,马华魁,等.闽北地区副猪嗜血杆菌的分离鉴定及药敏试验[J].畜牧与兽医,2016,48(4):116-118.CHEN P Q,ZHANG Q Y,MA H K,et al.Isolation,identification and drug sensitivity of Haemophilus parasuis in the area of North Fujian[J].Animal Husbandry & Veterinary Medicine,2016,48(4):116-118.(in Chinese)
[2] 李伯森.副猪嗜血杆菌病的病理学诊断及防制[J].中国畜牧兽医,2010,37(7):192-194.LI B S.Pathological diagnosis and prevention of Haemophilus parasuis[J].China Animal Husbandtry & Veterinary Medicine,2010,37(7):192-194.(in Chinese)
[3] 徐惠娟,薛云,赵战勤,等.副猪嗜血杆菌国内流行血清型菌株的生物学特性比较研究[J].中国兽医学报,2014,34(5):729-735.XU H J,XUE Y,ZHAO Z Q,et al.Comparative study on biological characterization of the epidemic serotypes Haemophilus suis in China[J].Chinese Journal of Veterinary Science,2014,34(5):729-735.(in Chinese)
[4] 苗立中,李书光,胡琳琳,等.副猪嗜血杆菌病发病概况及诊断方法[J].动物医学进展,2014,35(6):140-144.MIAO L Z,LI S G,HU L L,et al.Introduction of haemophilosis and diagnostic methods[J].Progress in Veterinary Medicine,2014,35(6):140-144.(in Chinese)
[5] 王建,邵卫星,吕占军,等.2012年我国部分省市规模化猪场副猪嗜血杆菌分离鉴定及菌株血清分型[J].动物医学进展,2014,35(3):48-52.WANG J,SHAO W X,LYU Z J,et al.Isolation,identification and serotyping of Haemophilus parasuis in parts of large scale pig farms in China 2012[J].Progress in Veterinary Medicine,2014,35(3):48-52.(in Chinese)
[6] 李明波,库旭钢,赵海忠,等.猪繁殖与呼吸综合征病毒(PRRSV)协同感染细菌性疾病的分析研究[J].中国畜牧兽医,2018,45(3):781-791.LI M B,KU X G,ZHAO H Z,et al.Study on co-infection analysis of porcine reproductive and respiratory syndrome virus (PRRSV) and bacterial [J].China Animal Husbandtry & Veterinary Medicine,2018,45(3):781-791.(in Chinese)
[7] MACEDO N,ROVIRA A,TORREMORELL M.Haemophilus parasuis:Infection,immunity and enrofloxacin[J].Veterinary Research,2015,46(1):128.
[8] 华升,梅书棋,曾德芳,等.副猪嗜血杆菌病研究进展[J].动物医学进展,2006,27(11):7-10.HUA S,MEI S Q,ZENG D F,et al.Advance in Haemophilus parasuis[J].Progress in Veterinary Medicine,2006,27(11):7-10.(in Chinese)
[9] 米丁丹,张艳禾,张万江,等.副猪嗜血杆菌临床分离株耐药性与耐药机制的研究[J].中国预防兽医学报,2015,37(7):524-527.MI D D,ZHANG Y H,ZHANG W J,et al.Investigation of drug resistance and resistance mechanism ofHaemophilus parasuis[J].Chinese Journal of Preventive Veterinary Medicine,2015,37(7):524-527.(in Chinese)
[10] 代军,刘君,黄倩,等.副猪嗜血杆菌分型血清制备及其四川地区流行病学[J].中国兽医学报,2016,36(1):24-29.DAI J,LIU J,HUANG Q,et al.Preparation of antisera to different serotype of Haemophilus parasuis and application in epidemiology study of Sichuan province[J].Chinese Journal of Veterinary Science,2016,36(1):24-29.(in Chinese)
[11] 李曦婷,董俏,李化生,等.副猪嗜血杆菌检测技术及耐药性研究进展[J].动物医学进展,2016,37(10):85-89.LI X T,DONG Q,LI H S,et al.Advances in detection technology and drug resistance of Haemophilus parasuis[J].Progress in Veterinary Medicine,2016,37(10):85-89.(in Chinese)
[12] HOWELL K J,PETERS S E,WANG J,et al.Development of a multiplex PCR assay for rapid molecular serotyping of Haemophilus parasuis[J].Journal of Clinical Microbiology,2015,53(12):3812-3821.
[13] 王超,陈欣,张梦南,等.副猪嗜血杆菌的分离鉴定及其OmpP5基因序列分析[J].动物医学进展,2018,39(1):16-21.WANG C,CHEN X,ZHANG M N,et al.Isolation and identification of Haemophilus parasuis and sequence analysis of it’s OmpP5 gene[J].Progress in Veterinary Medicine,2018,39(1):16-21.(in Chinese)
[14] KAVANOVá L.Immune response of porcine alveolar macrophages to a concurrent infection with porcine reproductive and respiratory syndrome virus and Haemophilus parasuis in vitro[J].Veterinary Microbiology,2015,180(1-2):28-35.
[15] 林初文,苗立中.副猪嗜血杆菌病疫苗研究进展[J].中国畜牧兽医,2013,40(5):203-206.LIN C W,MIAO L Z.Progress onvaccine of Haemophilus parasuis[J].China Animal Husbandtry & Veterinary Medicine,2013,40(5):203-206.(in Chinese)
[16] 陈同煜.泰安及周边地区屠宰生猪肺脏健康状况评估及相关病原感染调查分析[D].泰安:山东农业大学,2017.CHEN T Y.Taian and surrounding areas slaughter pigs lung health assessment and related pathogenic infection investigation and analysisea virus[D].Tai’an:Shandong Agricultural University,2017.(in Chinese)
[17] 张悦,李蓓蓓,魏建超,等.上海副猪嗜血杆菌分离鉴定及其耐药性分析[J].中国动物传染病学报,2015,23(4):25-30.ZHANG Y,LI B B,WEI J C,et al.Isolation and antibiotic resistance of Haemophilus parasuis in Shanghai[J].Chinese Journal of Animal Infectious Diseases,2015,23(4):25-30.(in Chinese)
[18] 陆秀玉.丹东地区屠宰生猪肺脏细菌的分离鉴定[J].中国兽医杂志,2016,52(8):97-99.CHEN X Y.Isolation and identification of lung bacteria from slaughter pigs in Dandong area[J].Chinese Journal of Veterinary Medicine,2016,52(8):97-99.(in Chinese)
[19] 蔡旭旺.副猪嗜血杆菌的分离鉴定及诊断方法与灭活疫苗的研究[D].武汉:华中农业大学,2006.CAI X W.Isolation and characterization of Haemophilus parasuis and development of its diagnostic method and inactivated bacterin[D].Wuhan:Huazhong Agricultural University,2006.(in Chinese)
[20] CERDà-CUéLLAR M,NARANJO J F,VERGE A,et al.Sow vaccination modulates the colonization of piglets by Haemophilus parasuis[J].Veterinary Microbiology,2010,145(3-4):315-320.
[21] 池晶晶,杨春蕾,路超,等.天津地区副猪嗜血杆菌的分离鉴定及敏感药物筛选[J].中国畜牧兽医,2015,42(5):1306-1310.CHI J J,YANG C L,LU C,et al.Isolation,identification and sensitive drug screening of Haemophilus parasuis in Tianjin[J].China Animal Husbandry & Veterinary Medicine,2015,42(5):1306-1310.(in Chinese)
[22] 靳国旺,蔡敏.副猪嗜血杆菌的分离鉴定及药敏试验[J].黑龙江畜牧兽医,2016,5:131-132.JIN G W,CAI M.Isolation,identification and drug sensitivity test of Haemophilus parasuis[J].Heilongjiang Animal Science and Veterinary Medicine,2016,5:131-132.(in Chinese)
[23] 陈鹏强,章秋月,马华魁,等.闽北地区副猪嗜血杆菌的分离鉴定及药敏试验[J].畜牧与兽医,2016,48(4):116-118.CHEN P Q,ZHANG Q Y,MA H K,et al.Isolation,identification and drug sensitivity of Haemophilus parasuis in the area of North Fujian[J].Animal Husbandry & Veterinary Medicine,2016,48(4):116-118.(in Chinese)