奶牛群口蹄疫疫苗接种与抗体检测

为了解贵阳某公司A、B、C三个规模奶牛场牛群口蹄疫病毒隐性感染状况,制定科学有效的免疫计划,在2010年10月～2011年11月期间,对上述奶牛场约1 700头奶牛进行了3次口蹄疫O型、亚洲I型二价灭活疫苗的免疫接种试验,对其抗体水平、隐性感染及带毒状况进行了跟踪监测。结果表明,抗体有效持续时间为120～130d,即现行使用的口蹄疫灭活疫苗免疫保护期大约为4个月;C场奶牛血清中未检出口蹄疫病毒的隐性感染抗体,OP液中未检测出口蹄疫病毒核酸,属于口蹄疫比较洁净的奶牛场。A、B场奶牛不同程度地检测出口蹄疫病毒的隐性感染抗体,在部分隐性感染抗体阳性奶牛的OP液中检测出口蹄疫病毒核酸,属于受口蹄疫威胁的潜在污染场。上述数据为后续的口蹄疫防控计划奠定了基础。     

口蹄疫诊断技术研究进展

口蹄疫的有效控制关键在于早期检测 ,然而有很多疾病症状与口蹄疫相似 ,仅靠临床症状难以确诊 ,因此必须进行实验室诊断。实验室诊断包括病毒学诊断和血清学诊断。病毒学诊断方法有病毒分离、补体结合试验、酶联免疫吸附试验 ( EL ISA)、RT-PCR以及乳胶凝集试验 ( L AT)。 RT-PCR有待进一步完善 ,而用于野外检测的现场诊断方法已取得可喜进展。血清学诊断包括中和试验和 EL ISA,中和试验已经被 EL ISA方法取代 ,并且通过检测非结构蛋白的抗体可以区分感染动物和免疫动物。更加快速、敏感、可靠以及用于检测潜伏感染的诊断技术将是今后研究的热点。     

口蹄疫病毒3ABC抗体检测结果分析

口蹄疫(FMD)是一种高度接触性传染病,对国际贸易具有深远的影响,世界动物卫生组织(OIE)和联合国粮农组织(FAO)将口蹄疫列为A类烈性传染病之首.控制和消灭FMD策略包括疫苗免疫和屠宰可疑畜群.可疑畜群就是被FMDV感染动物和带毒动物.只有将感染动物和隐性带毒动物与疫苗免疫动物加以区分才能为FMD的控制和消灭提供科学的依据.3ABC-I-ELISA方法不但可以检测隐性感染动物而且可以区分感染动物和免疫动物.     

澳大利亚研制出新的低成本口蹄疫检测方法

英联邦科学和工业研究组织（CSIRO）澳大利亚动物卫生实验室的研制出一种新的低成本的口蹄疫检测方法，这种方法使用非传染性病毒材料，可以区分口蹄疫病毒的所有7的毒株和区分感染动物与接种动物。该法使用重组抗体区分感染动物与接种动物，重组抗体可以以低成本大量生产。     

口蹄疫3ABC非结构蛋白抗体检测试剂盒适用范围探讨

为了调查口蹄疫3ABC非机构蛋白抗体在临床诊断健康的动物体内是否存在,以及探索3ABC抗体检测试剂盒的适用范围,设计此试验。随机选取新乡市辖区范围内的猪场4个、牛场12个、羊场9个,采集动物全血,分离血清,利用间接ELISA方法(3ABC-I-ELISA)和荧光PCR方法分别检测免疫过口蹄疫疫苗的健康猪、牛、羊体内非结构蛋白3ABC抗体存在情况和血清中口蹄疫病毒存在情况。结果发现,临床健康动物也会产生口蹄疫3ABC非结构蛋白抗体,且动物日龄越大,产生抗体的比例越高。表明口蹄疫3ABC非结构蛋白抗体不仅在野毒感染动物(包括隐性感染动物)体内产生,而且在健康动物中由于免疫频率的增加和免疫剂量的加大也会产生。因此,口蹄疫3ABC非结构蛋白抗体检测试剂盒只适用于对口蹄疫疫情的初筛,且在日龄较小或免疫口蹄疫疫苗次数较少的动物更加适用。     

持续感染黄牛体内口蹄疫病毒抗原性和血清中和特性分析

以口蹄疫病毒O/Akesu/58毒株感染黄牛获得口蹄疫病毒持续带毒动物,定期分离黄牛食道/咽喉部黏性液体（O/P液）和血清,研究病毒分离毒株抗原基因变异及其血清中和特性,从基因水平和血清学方面研究口蹄疫病毒持续感染分离株的抗原变异性。用RT-PCR扩增分离株抗原基因VP1,分析VP1基因变异情况;用微量血清中和试验检测了口蹄疫病毒持续感染血清与对应动物分离毒株的中和特性,并用液相阻断ELISA方法检测了口蹄疫病毒持续感染血清的抗体水平变化,并对其相关性进行了分析。结果发现所有持续感染分离毒株的VP1基因核苷酸和氨基酸同源性都在98%以上,没有碱基缺失或插入现象;与O/Akesu/58的核苷酸同源性仅为85%左右,氨基酸同源性也仅为90%。持续感染分离株VP1基因有多处位点发生突变,其中有16个核苷酸位点发生一致突变,但只有2个位点造成氨基酸突变（I 56→T、A 210→E）;而持续感染分离毒株有4个核苷酸位点和3个氨基酸位点发生了颠换突变;同时证实不同时间分离株与对应血清都能相互中和,且中和作用能力随着时间延续呈下降趋势,最低为1∶80,具有较强的中和能力,这与LPB-ELISA检测结果基本一致。这说明口蹄疫病毒持续感染分离株抗原变异不显著,没有变异到动物自身血清不能识别的程度,而且分离毒株与自身动物血清具有较强的中和特性;在持续感染过程中,动物血清保持高水平抗体滴度,且持续感染毒株的分离与否与抗体水平没有显著相关性。     

A型口蹄疫病毒抗体化学发光免疫分析检测方法的建立

为建立动物血清中的A型口蹄疫病毒抗体化学发光免疫分析检测方法,通过获得的A型口蹄疫病毒VP1融合蛋白与相关单克隆抗体建立了A型口蹄疫病毒抗体竞争化学发光检测试剂盒。该方法能够特异性的定量检测猪、牛、羊血清中的A型口蹄疫病毒VP1蛋白抗体,敏感性高,特异性强,对其他相关的偶蹄类动物病原无交叉反应,批内变异系数小于10%,批间变异系数小于15%,具有良好的重复性。对该方法的特异性指标和敏感性指标进行了验证,同国产的A型口蹄疫抗体检测试剂盒进行了比较,阳性样品符合率为92.63%,阴性样品符合率为93.98%,总符合率为93.17%。建立的A型口蹄疫病毒抗体化学发光免疫分析检测方法,特异性高,敏感性强,操作简单、快速,具有较高的应用推广价值。     

孵蕴春荡——访北京市海江孵化设备制造有限公司董事长王复生

伪狂犬病(Pseudorabies PR)是由疱疹病毒科的伪狂犬病病毒引起的一种家畜及野生动物的急性传染病，在世界各地广泛流行。在我国，该病对猪的危害很严重，引起仔猪的高发病率，成年猪症状微轻，常呈现隐性带毒，成为新的传染源。用血清学方法检测猪血清中伪狂犬病病毒抗体，在猪伪狂犬病的诊断、流行病学调查及疫苗免疫效果的检测上有一定的意义。     

O型口蹄疫病毒抗体化学发光免疫分析检测方法的建立

为建立动物血清中O型口蹄疫病毒抗体化学发光免疫分析检测方法,通过获得的O型口蹄疫病毒VP1融合蛋白与相关单克隆抗体,制备了O型口蹄疫病毒抗体竞争化学发光检测试剂盒。结果表明:该方法能够定量检测猪、牛、羊血清中的O型口蹄疫病毒VP1蛋白抗体,与其他偶蹄类动物病原无交叉反应,具有良好的重复性;对该方法的特异性指标、敏感性指标进行了验证,同国产O型口蹄疫抗体检测试剂盒进行了比较,阳性样品符合率92.62%,阴性样品符合率92.14%,总符合率92.45%;重复性试验组内与组间变异系数分别低于10%和15%。综上,该研究建立的O型口蹄疫病毒抗体化学发光免疫分析检测方法特异性高、敏感性强,操作简单、快速,具有较高的应用推广价值。     

用天然口蹄疫病毒非结构蛋白2C鉴别感染与接种家畜

用天然口蹄疫病毒非结构蛋白２Ｃ鉴别感染与接种家畜李凯年摘译口蹄疫病毒（ＦＭＤＶ）感染康复的动物可以在感染后几个月甚至几年内带毒。在控制本病中，一个重要问题就是无法区分接种的和恢复期可能带毒的动物。尽管带毒动物可能间歇排毒并且可能把新的遗传变型引进易感...     

Developments in diagnostic techniques for differentiating infection from vaccination in foot-and-mouth disease

Foot-and-mouth disease (FMD) is a highly contagious and economically significant disease of cattle, pigs, sheep, goats and wild ruminant species. The FMD virus genome encodes a unique polyprotein from which the different viral polypeptides are cleaved by viral proteases, including eight different non-structural proteins (NSPs). Both structural and non-structural antigens induce the production of antibodies in infected animals. In contrast, vaccinated animals which have not been exposed to replicating virus will develop antibodies only to the viral antigens in the inactivated material. Vaccination against FMD is a key element in the control of the disease in addition to slaughter and movement restrictions. However, countries that vaccinate in the event of an outbreak will have to re-establish their FMD free status to the satisfaction of their trading partners.Because currently available vaccines stimulate the production of antibodies indistinguishable from those produced by infected animals in response to live virus and because vaccinated animals can be infected and become carriers of FMD virus, efforts have been made to develop diagnostic test that can differentiate vaccinated animals from those that are convalescent and from those that have been vaccinated and become carriers following subsequent contact with live virus. Currently the detection of antibodies to non-structural protein's (NSPs) is the preferred diagnostic method to distinguish virus infected, carrier, animals from vaccinated animals. However this is currently only possible at the herd level because of the great variability in the initiation, specificity and duration of the immune response in individual animals to the NSPs shown in many studies. Considerable effort and attention is now being directed toward the development of new methods and techniques for the rapid and accurate detection of anti-NSP antibodies, harmonization and standardization of current diagnostic techniques, as well as the production of defined reagents.     

Evaluation of a novel proximity ligation assay for the sensitive and rapid detection of foot-and-mouth disease virus

A novel proximity ligation assay (PLA) using a pan-serotype reactive monoclonal antibody was developed and evaluated for the detection of foot-and-mouth disease virus (FMDV) in clinical samples collected from field cases of disease. The FMDV-specific PLA was found to be 100 times more sensitive for virus detection than the commonly used antigen capture-ELISA (AgELISA). As few as five TCID50 were detected in individual assays, which was comparable with the analytical sensitivity of real-time RT-PCR. Although this assay was capable of detecting diverse isolates from all seven FMDV serotypes, the diagnostic sensitivity of the PLA assay was lower than real-time RT-PCR mainly due to a failure to detect some SAT 1, SAT 2 and SAT 3 FMDV strains. In conclusion, this new PLA format has high analytical sensitivity for the detection of FMDV in clinical samples and may prove valuable as a rapid and simple tool for use in FMD diagnosis.     

Microarray-based detection and typing of foot-and-mouth disease virus

Foot-and-mouth disease virus (FMDV) is the most economically important veterinary pathogen because of its highly infectious nature and the devastating effects the virus has on the livestock industry. Rapid diagnostic methods are needed for detection and typing of FMDV serotypes and differentiation from other viruses causing vesicular diseases. We developed a microarray-based test that uses a FMD DNA chip containing 155 oligonucleotide probes, 35-45 base pair (bp) long, virus-common and serotype-specific, designed from the VP3-VP1-2A region of the genome. A set of two forward primers and one reverse primer were also designed to allow amplification of approximately 1100 bp of target sequences from this region. The amplified target was labelled with Alexa-Fluor 546 dye and applied to the FMD DNA chip. A total of 23 different FMDV strains representing all seven serotypes were detected and typed by the FMD DNA chip. Microarray technology offers a unique capability to identify multiple pathogens in a single chip.     

口蹄疫诊断技术的研究进展

综述了口蹄疫的诊断方法,包括临床诊断、病毒分离、血清学、分子生物学及一些新型的诊断技术,尤其对ELISA、PCR和基因芯片等快速、灵敏的诊断技术做以阐述,对其优缺点进行比较。     

Diagnosis and screening of foot-and-mouth disease

Foot-and-mouth disease (FMD) diagnostic methods are reviewed. As the presence of clinical signs alone is inconclusive, laboratory diagnosis should always be carried out. The presence of FMD virus can be demonstrated by cell culture isolation, complement fixation test, ELISA or the more recent polymerase chain reaction (PCR) method. Serological diagnosis is also a valuable tool. The virus neutralization test has been replaced by ELISA and the antibody response to some viral non-structural proteins allows to discriminate between vaccinated and infected animals on a herd basis. More rapid and accurate tests as well as an earlier detection system in preclinical state are still needed.

Résumé

Les différentes méthodes de diagnostic de la FA (fièvre aphteuse) sont exposées. Le recours au laboratoire est indispensable; la seule présence de signes cliniques ne permettant pas d'établir le diagnostic. Le virus peut être mis en évidence par isolement en culture cellulaire, par le test de fixation du complément, l'ELISA ou la technique récente de polymérisation en chaine (PCR). La recherche des anticorps est aussi reconnue comme outil diagnostic. Le test de séroneutralisation a été supplanté par l'ELISA et la réponse anticorps contre certaines des protéines virales non structurales permet la différenciation entre troupeaux vaccinés et troupeaux infectés. Des tests plus rapides et précis, la possibilité de détecter le virus lors de la phase préclinique seraient d'utilité certaine.     

Implementation in Australia of molecular diagnostic techniques for the rapid detection of foot and mouth disease virus

OBJECTIVE: To evaluate and implement rapid molecular diagnostic techniques for the detection of foot and mouth disease virus (FMDV) suitable for use in Australia. DESIGN: Two PCR TaqMan assays targeted to the FMDV internal ribosome entry site or the 3D polymerase coding region for the rapid detection of FMDV were evaluated using non-infectious materials to determine the test most appropriate for implementation as part of Australia's national preparedness for the rapid detection and diagnosis of FMD outbreaks. RESULTS: Two published tests (PCR TaqMan assays targeted to the FMDV IRES region or the FMDV 3D polymerase coding region) were evaluated for their ability to detect FMDV genetic material in non-infectious FMDV ELISA antigen stocks held at Australian Animal Health Laboratory. Both tests were able to detect FMDV genetic material from strains O1 Manisa, O-3039, A22, A24, A Malaysia, C, Asia 1 and SAT 1, 2 and 3. With the exception of Asia 1, the TaqMan assay targeted to the FMD 3D polymerase coding region had Ct values equal to or lower than for the TaqMan assay targeted to the IRES region suggesting that this test may provide broader serotype detection and sensitivity. However, the TaqMan assay directed to the FMDV IRES is the only one to date to have undergone substantial evaluation using clinical samples collected during an outbreak. The greatest differences observed were for O-3039, SAT 1, and 3. CONCLUSION: Given the ease of setting up both tests, AAHL currently runs both tests on highly suspect FMD investigations to provide independent confirmation of the absence of FMDV because the tests are focused on two independent regions of the FMDV genome. These tests add substantially to Australia's preparedness for FMD diagnosis complementing the already well-established virus isolation and antigen capture ELISA tests for index case diagnosis of FMD in Australia.     

Detection of foot-and-mouth disease virus by nucleic acid sequence-based amplification (NASBA)

A study was conducted to evaluate the performance of a nucleic acid sequence-based amplification (NASBA) assay for the detection of foot-and-mouth disease virus (FMDV). Two detection methods: NASBA-electrochemiluminescence (NASBA-ECL) and a newly developed NASBA-enzyme-linked oligonucleotide capture (NASBA-EOC) were evaluated. The diagnostic sensitivity of these assays was compared with other laboratory-based methods using 200 clinical samples collected from different regions of the world. Assay specificity was also assessed using samples (n=43) of other viruses that cause vesicular disease in livestock and genetic relatives of FMDV. Concordant results were generated for 174/200 (87.0%) of suspect FMD samples between NASBA-ECL and real-time RT-PCR. In comparison with the virus isolation (VI) data, the sensitivity of the NASBA-ECL assay was 92.9%, which was almost identical to that of the real-time RT-PCR (92.4%) for the same set of samples. There was broad agreement between the results of the NASBA-ECL and the simpler NASBA-EOC detection method for 97.1% of samples. In conclusion, this study provides further data to support the use of NASBA as a rapid and sensitive diagnostic method for the detection and surveillance of FMDV.