中欧预防控制口蹄疫2011年培训3.ppt
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1、Diagnostic Tests for FMD and New Developments in Diagnosis of FMD 口蹄疫诊断方法与最新进展Training to Improve Control and Prevention of Foot and Mouth Disease(FMD),旨在提高口蹄疫预防与控制行动的培训旨在提高口蹄疫预防与控制行动的培训Lanzhou,China,8-12 August 2011Chris MorrissyCSIRO Livestock Industries,Australian Animal Health Laboratory(AAHL),G
2、eelong,Australia.Outline(概要)Disease Diagnosis&Control(疫病诊断与控制)Detection of FMD Virus/Viral Antigen(病毒抗原检测)FMD Serology(口蹄疫血清学检测)Quality Control(质量控制)New Developments in Diagnosis(诊断技术的新进展)Summary(总结)Disease Diagnosis and Control疫病诊断与控制疫病诊断与控制Animal Health Network(Outbreak)动物健康工作网络(暴发疫情)动物健康工作网络(暴发疫情
3、)Field Veterinarians(田间兽医)(田间兽医)clinical diagnosis(临床诊断)(临床诊断)collect information/history of disease,vaccination&recent animal movement(收集信息收集信息/病史,免疫与最近病史,免疫与最近动物的移动情况)动物的移动情况)collect samples for laboratory(采集实验室诊断样品)采集实验室诊断样品)implement disease control(采取控制措施)(采取控制措施)quarantine&movement control(检疫与
4、移动控制)(检疫与移动控制)Disinfection(消毒)(消毒)Disease Diagnosis and ControlAnimal Health Network(Outbreak)Laboratory(实验室)confirm clinical diagnosis(临床诊断的确诊)Sero-typying ELISA(血清定型ELISA)PCR&sequencing(PCR与序列测定)virus isolation(病毒分离)molecular epidemiology&characterisation(分子流行病学与特征分析)vaccine matching 疫苗配比 send sam
5、ples to RRL&WRL (向RRL与WRL发送样本)share information 信息共享Diagnosis of FMD(诊断)(诊断)Clinical Diagnosis(临床诊断)(临床诊断)initial diagnosis based on clinical signs by a trained or experienced investigator 由专业人员根据临床症状作出的初步诊断)important to send history of disease outbreak and description of lesions(重点是对病史与病灶的描述)should
6、 also involve an epidemiological investigation to connect to other outbreaks 同时联系其他疫情开展流行病学调查 samples should be collected to confirm the serotype of FMD in the outbreak especially in new outbreak areas对新发地区尤其应当采集样品以确定FMD血清型Diagnosis of FMDLaboratory Diagnosis(实验室诊断)the role of the laboratory is gene
7、rally to confirm the clinical diagnosis 实验室诊断是为了进一步确认临床诊断实验室诊断是为了进一步确认临床诊断it may also confirm a suspicion of disease activity(PCR or serology)也可以用于对也可以用于对PCRPCR与血清学阳性疑似疫情进行确诊与血清学阳性疑似疫情进行确诊and to provide additional information about the agent involved(进一步了解病原因子的其他信息)进一步了解病原因子的其他信息)serotype of FMD(血清型
8、)(血清型)molecular epidemiology(分子流行病学)(分子流行病学)vaccine matching(疫苗配比)(疫苗配比)Send large tissue samples:1 2 grams送检送检1-21-2克的发病组织样品克的发病组织样品Disease Diagnosis and ControlAnimal Health Network(surveillance)Animal Health Network(surveillance)(疫情监测)(疫情监测)(疫情监测)(疫情监测)Field Veterinarians 田间兽医田间兽医 serosurveillanc
9、e for disease prevalence 疫病发生状况的血清学监测疫病发生状况的血清学监测 serosurveillance post vaccination免疫后的血清学监测免疫后的血清学监测 collect information/history of disease&vaccination&vaccine used 收集信息、病史与免疫与疫苗使用情况收集信息、病史与免疫与疫苗使用情况 investigate possible vaccine failure 调查疫苗免疫失败的可能性调查疫苗免疫失败的可能性 collect samples for laboratory 采集样本用于
10、实验室诊断采集样本用于实验室诊断Disease Diagnosis and ControlAnimal Health Network(surveillance)动物健康工作网络(监测)动物健康工作网络(监测)Laboratory(实验室)(实验室)confirm disease prevalence 确认疫病流行状况确认疫病流行状况 confirm post vaccination antibody 确认免疫后抗体产生情况确认免疫后抗体产生情况 virus characterisation(carrier animals)病毒特征分析(带毒动物)病毒特征分析(带毒动物)Disease Diag
11、nosis(诊断)(诊断)Panel of Tests(检测方法)(检测方法)Detection of Virus/Viral Antigen 检测病毒抗原检测病毒抗原 Detection of Antibody 检测抗体检测抗体 Production of reagents 试剂生产试剂生产 Rapid,Sensitive and Specific 快速,敏感和特快速,敏感和特异异 Choose Test fit for purpose 根据目的选择方法根据目的选择方法Diagnostics for Detection of Virus/Viral Antigen(病原(病原检测方法)方法)
12、Virus Isolation 病毒分离 Molecular Techniques 分子检测技术 Electron Microscopy 电子显微镜 Antigen Capture ELISA 抗原捕获ELISA Quality Assurance 质量保证Virus Isolation 病毒分离病毒分离 Virus isolation requires specialized facilities for cell culture&handling of virus 要求具有专门用于操作病毒与细胞培养的设施要求具有专门用于操作病毒与细胞培养的设施 Cell culture is expens
13、ive to maintain 细胞培养是昂贵的细胞培养是昂贵的 Reference laboratories need to grow virus to allow further characterization 参考实验室需要培养病毒用于进一步研究参考实验室需要培养病毒用于进一步研究 FMD virus isolates important in comparing current field isolates against vaccines being used.(R-Value/vaccine matching)病毒分离是进行流行毒与疫苗毒抗原比较的基础病毒分离是进行流行毒与疫苗毒
14、抗原比较的基础(R R值值/疫苗配比)疫苗配比)FMD virus isolates used for molecular epidemiology 用于分子流行病学研究用于分子流行病学研究Molecular Technologies 分子分子检测技技术 PCRPCR Conventional PCR/RT-PCR 传统PCR group&specific primers(FMD serotyping)群与型特异性引物群与型特异性引物 (鉴定血清型)(鉴定血清型)Real-time PCR 实时荧光实时荧光PCRPCR group primers&probe:FMD(群引物与探针)(群引物与探
15、针)Pirbright&Plum Island test specific primers&probe for sero-typing are needed 需要血清型特异性的引物与探针需要血清型特异性的引物与探针 Sequencing PCR productSequencing PCR product (PCRPCRPCRPCR产物测序)产物测序)产物测序)产物测序)confirm PCR product is from FMDV 为为FMDVFMDV病毒基因病毒基因 sero-typing FMDV 血清型鉴定血清型鉴定 molecular epidemiology 分子流行病学分析分子流
16、行病学分析Molecular Technologies 分子分子检测技技术Technology is used in reference laboratories to Technology is used in reference laboratories to undertake genetic studies to link viruses into undertake genetic studies to link viruses into familiesfamilies(参考实验室利用该方法主要是进行遗传关系研(参考实验室利用该方法主要是进行遗传关系研(参考实验室利用该方法主要是进
17、行遗传关系研(参考实验室利用该方法主要是进行遗传关系研究,以确定病毒类型)究,以确定病毒类型)究,以确定病毒类型)究,以确定病毒类型)sequencing&sequence analysis is best carried out at a Reference laboratory(测序与序列分析最好是在(测序与序列分析最好是在参考实验室进行)参考实验室进行)Epidemiological studies-molecular epidemiologyEpidemiological studies-molecular epidemiology 流行病学研究流行病学研究分子流行病学分子流行病学co
18、mpare field strain to vaccine strain(比较田间毒株(比较田间毒株与疫苗毒株)与疫苗毒株)virus source and evolution(病毒来源与进化)(病毒来源与进化)FMD Antigen Capture ELISA(抗原捕获(抗原捕获ELISAELISA)Test of choose for countries endemic with FMD 该方法用于该方法用于FMDFMD流行国家流行国家 Pirbright reagents generally used(expensive)一般用英国参考实验室的试剂(较昂贵)一般用英国参考实验室的试剂(较
19、昂贵)Allow sero-typing of FMDV(need for PCR)(鉴定血清型)(鉴定血清型)Routinely test for serotypes O,A&Asia 1(通常测定(通常测定O O、A A、Asia1Asia1)Result in 3-4 hours(3 34 4小时出结果)小时出结果)Sero-typing important for control by vaccination 鉴定血清型是疫苗免疫的重要依据鉴定血清型是疫苗免疫的重要依据 Lesions older than 4 to 5 days reduced antigen 发病发病4 45 5天
20、的陈旧病料含毒量低天的陈旧病料含毒量低 Samples unsuitable for virus isolation(VI)&PCR can be used in the ELISA(不适于进行病毒分离和不适于进行病毒分离和PCRPCR的样品可以用于的样品可以用于ELISAELISA)FMD Antigen Capture ELISA(抗原捕抗原捕获ELISA)Sero-typing ELISA(定型ELISA)Serotype specific so 7 separate tests required on suspect sample(also VSV x 2&SVD)(每个可疑样本要进行
21、(每个可疑样本要进行7个个测试以确定血清型(也要测试测试以确定血清型(也要测试VSV与与SVD)Most countries test for circulating sero-types (O,A,Asia1)(Sat1,2,3 also in Africa)多数国家只测定流行的血清型(O,A,Asia1)(在非洲还要测南非在非洲还要测南非3个型)个型)Positive is regarded as diagnostic confirmation(有阳性结果(有阳性结果就可以作出确诊)就可以作出确诊)Electron Microscope&PCR used as well if availa
22、ble(电镜与PCR也可以)Confirm CPE result in cell culture with antigen detection ELISA(细胞培养出现CPE后要用抗原捕获ELISA进行确认)FMD Antigen Capture ELISA(抗原捕抗原捕获ELISA)Does not require infectious virus,but does require a good specimen from a fresh lesion(要求要求样品采自新鲜的病灶样品采自新鲜的病灶)Antigen can be destroyed by excessive heat or p
23、resence of disinfectant(高热或消毒剂都会破坏抗原)高热或消毒剂都会破坏抗原)PCR&VI combined with ELISA increases chance of a result PCR/PCR/病毒分离结合病毒分离结合ELISAELISA定型以提高确诊的机会定型以提高确诊的机会FMD Virus Isolation(病毒分离)(病毒分离)Cell Culture(细胞培养)(细胞培养)Primary bovine(calf)thyroid cell cultures considered most sensitive 小牛甲状腺细胞是分离病毒最敏感的小牛甲状腺
24、细胞是分离病毒最敏感的 Primary pig,calf or lamb kidney cell cultures 原代肾细胞原代肾细胞 Cell lines BHK(baby hamster kidney),IBRS2(swine kidney cell line)sometimes less sensitive for field strains and PK-15(BHK,IBRS2(BHK,IBRS2与与PK-15PK-15细胞对田间毒株略为不敏感细胞对田间毒株略为不敏感)FMD virus grows quickly and cytopathic effect can usually
25、 be seen at first or second pass in primary cells;can take longer in cell lines(FMDVFMDV在原代细胞生长迅速,通常在原代细胞生长迅速,通常1 12 2代就能出现明显的代就能出现明显的CPECPE,在传代细胞系所需时间会长一些),在传代细胞系所需时间会长一些)FMD Virus Isolation(病毒分离)(病毒分离)virus isolation requires live virus but can detect smaller amounts(病毒分离需要的活病毒的量较小)(病毒分离需要的活病毒的量较小
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