副猪嗜血杆菌的分离鉴定和防控措施(恢复)(恢复)

I分类号：学校代码：UDC：研究生学号：密级：硕士专业学位论文副猪嗜血杆菌病分离鉴定和防治措施学位申请人：柏建东指导教师：副教授合作教师：研究员专业学位类别：兽医硕士专业学位领域：2015年4月2论文题目：副猪嗜血杆菌病分离鉴定及防治措施专业：兽医硕士研究生：柏建东指导教师：副教授3摘要副猪嗜血杆菌病,其英文名字是（Haemophilusparasuis，HPs），由副猪嗜血杆菌引起的重要的细菌性传染病，导致保育仔猪产生多发性浆膜炎与关节炎，使养殖业蒙受巨大的财产损失。为探索出其有效的防治措施，本研究利用临床诊断、病理解剖以及实验室诊断等方法，从几例临床症状、病例剖检疑似为副猪嗜血杆菌病猪体内分离到革兰氏阴性可疑菌，然后进行镜检、生化试验、基因测序等手段确定其为副猪嗜血杆菌。但由于副猪嗜血杆菌体外培养条件严格，生长繁殖受到抑制，严重影响了疫苗的大量生产。为了探索副猪嗜血杆菌培养的液体条件，本试验通过副猪嗜血杆菌的优化培养，确定该菌的最适培养条件为胰蛋白胨15.0g,大豆蛋白胨5.0g、氯化钠5.0g、葡萄糖2.5g、钙离子0.04-0.06%、磷离子0.02-0.03%、灭菌蒸馏水1000mL,pH7.2-7.4时在5%的CO2厌氧培养箱中培养16h。通过药敏试验，筛选出对该猪场的患病仔猪具有针对性的敏感药物，并通过对该菌的生物学特性、毒力、遗传特征、免疫原性进行检测，发现该菌株符合作为灭活疫苗的特征。并通过AlCl3作为佐剂来制备副猪嗜血杆菌自家灭活苗，最终结果显示物理性状符合AlCl3疫苗的特征，安全性好，免疫原性强，对断奶之后至保育阶段的仔猪有良好的保护性。关键词：副猪嗜血杆菌，分离鉴定药敏试验疫苗论文类型：其他4AbstractHaemophilusparasuisdisease,whosenameisHPs,causedbytheH.parasuisimportantbacterialdiseases,resultinginnurserypigsproducemultipleserositisandarthritisthatsufferedhugeaquaculturedamagetoproperty.Inordertoexploreitseffectivecontrolmeasures,thisstudy,clinicaldiagnosis,andanatomicpathologylaboratorydiagnosticmethods,clinicalsymptomsfromafewcases,suspectedcasesofnecropsyHaemophilusparasuisisolatedGram-negativepigsinvivotosuspiciousbacteriaandmicroscopicexamination,biochemicaltests,genesequencingandothermeansidentifiedasHaemophilusparasuis.However,duetoH.parasuisstrictconditionsinvitro,inhibitedgrowthandreproduction,seriouslyaffectingthemassproductionofthevaccine.Inordertoexploretheconditionsparasuisliquidculture,thetestbyoptimizingthecultureofH.parasuis,determinetheoptimalcultureconditionsforthebacteriatryptone15.0g,soypeptone5.0g,sodiumchloride,5.0g,glucose2.5g,0.04-0.06%calcium,0.02-0.03%phosphorusions,steriledistilledwater1000mL,pH7.2-7.416hincubationina5%CO2incubatoranaerobic.Bysusceptibilitytesting,screeningoutthesickpigfarmstargetedsensitivedrugs,andbythebiologicalcharacteristicsofthebacteria,virulence,geneticcharacteristics,immunogenicitytesting,thisstrainwasfoundtoqualifyasofffeatureslivevaccines.AndAlCl3asanadjuvantpreparedbyHaemophilusparasuisownvaccine,thefinalresultsshowedcharacteristicsconsistentwiththephysicalpropertiesofAlCl3vaccinesafety,immunogenicity,afterweaningofpigletstothenurserystagewithgoodprotectionsex.Keywords:Haemophilusparasuisisolationandidentificationdrugallergytestingvaccine5目录第1章文献综述.............................................................................................................................71.1病原学................................................................................................................................71.2流行病学............................................................................................................................71.3分离鉴定............................................................................................................................81.4防治措施............................................................................................................................91.5防治目的与意义..............................................................................................................11第二章病原菌的分离与鉴定.......................................................................................................122.1试验材料..........................................................................................................................122.1.1试验仪器.............................................................................................122.1.2试验试剂.............................................................................................132.1.3试验病料.............................................................................................132.2试验方法..........................................................................................................................132.2.1病原菌的初筛.....................................................................................132.2.2镜检.....................................................................................................142.2.3纯培养.................................................................................................142.2.4生化试验.............................................................................................152.2.5分子生物学鉴定.................................................................................162.3实验结果与分析..............................................................................................................162.3.1病理解剖.............................................................................................162.3.2镜检结果.............................................................................................172.3.3生化试验结果.....................................................................................172.3.4PCR鉴定结果.....................................................................................202.4讨论..................................................................................................................................212.4.1病料来源的选择.............................................................................