动物布鲁氏菌病实验室诊断与检测技术进展

实验室诊断与检测对防治动物布鲁氏菌病(brucello-sis)至关重要,其不仅可早期发现传染源和隐性感染动物,而且可检测动物通过疫苗免疫后产生的抗体水平,为合理制定免疫程序提供科学依据.实验室诊断与检测动物布鲁氏菌病常规方法包括:一般实验室检查、免疫血清学试验和病原学检查;高新生物技术包括:应用分子生物学技术检测及荧光偏振试验(FPA).目前,我国对该病诊断的国家标准主要是依据细菌学和血清学检测结果来确定.其中免疫血清学试验是我国最常用的诊断与检测方法,该方法不仅可用于动物布鲁氏菌病的诊断与检测,而且还用于人感染布鲁氏菌病的诊断与检测.但是,这种诊断与检测方法在我国已经使用半个多世纪,并且存在着许多弊端,诸如:非特异性反应、假阳性、前带现象或动物自身免疫抑制等,因此还远不能满足对动物布鲁氏菌病的防控需求.探索动物布鲁氏菌病的实验室诊断与检测新技术以及高新分子生物学技术的研究进展,对于早期开展动物布鲁氏菌病的分子流行病学调查、早期确诊和防控均具有重要意义.     

绵羊衣原体病抗体监测及两种衣原体病间接血凝试剂盒的比较

2004—2005年,笔者有机会为北京郊区的某些羊场做几种传染病的抗体监测,其中包括衣原体病的监测。世界动物卫生组织(O IE)《诊断试验和疫苗标准手册》(2000版)和《陆地动物病的诊断和疫苗手册》(2004版)介绍的羊衣原体病血清学诊断是CF(国际贸易替代试验)和EL ISA(处于研究阶段     

动物布鲁氏菌病实验室诊断与检测技术进展

实验室诊断与检测对防治动物布鲁氏菌病（brucellosis）至关重要,其不仅可早期发现传染源和隐性感染动物,而且可检测动物通过疫苗免疫后产生的抗体水平,为合理制定免疫程序提供科学依据。实验室诊断与检测动物布鲁氏菌病常规方法包括：一般实验室检查、免疫血清学试验和病原学检查;高新生物技术包括：应用分子生物学技术检测及荧光偏振试验（FPA）。目前,我国对该病诊断的国家标准主要是依据细菌学和血清学检测结果来确定。其中免疫血清学试验是我国最常用的诊断与检测方法,该方法不仅可用于动物布鲁氏菌病的诊断与检测,而且还用于人感染布鲁氏菌病的诊断与检测。但是,这种诊断与检测方法在我国已经使用半个多世纪,并且存在着许多弊端,诸如：非特异性反应、假阳性、前带现象或动物自身免疫抑制等,因此还远不能满足对动物布鲁氏菌病的防控需求。探索动物布鲁氏菌病的实验室诊断与检测新技术以及高新分子生物学技术的研究进展,对于早期开展动物布鲁氏菌病的分子流行病学调查、早期确诊和防控均具有重要意义。     

ELISA在猪伪狂犬病诊断中的应用

近年来 ,猪伪狂犬病不断传播蔓延 ,给世界养猪业带来了巨大危害 ,因此开发特异性强、敏感性高、简便、快速、能区别疫苗毒和野毒、能检出潜伏感染的诊断方法是防制该病的关键之一。EL ISA方法具有特异、敏感、快速、稳定等特点 ,为广大兽医工作者所关注。目前 ,国内外关于 EL ISA在猪伪狂犬病诊断中的应用方面的研究已有不少 ,已有试剂盒出售。笔者综述了应用于猪伪狂犬病诊断的多种 EL ISA方法 ,如直接 EL ISA、间接 EL ISA、竞争 EL ISA、斑点EL ISA、SPA-EL ISA、双抗体夹心 EL ISA、单抗夹心 L AB-EL ISA、血清学鉴别 EL ISA等。这些方法中 ,有测病毒的 ,有测抗体的 ;有单抗介导的 ,有多抗介导的 ;有的还引入了 SPA和 L AB;有的以分子生物学为基础并能够鉴别疫苗毒和野毒。此外 ,还总结了目前所用 EL ISA诊断方法的优点和缺点 ,并预测了其发展前景     

检测非洲猪瘟病毒的新方法

正非洲猪瘟(ASF)是一种高死亡率的病毒性传染病,各品种及年龄阶段的猪群都具易感性。对此迄今还没有有效的治疗方法。非洲猪瘟的持续蔓延已对全球养猪业构成严重威胁,故在当前无有效疫苗的条件下,对被感染动物的早期检测对于控制该病的暴发及蔓延显得极其重要。目前实验室诊断主要是根据病毒学方法(抗原和基因检测)和血清学诊断。在目前的工作中,针对非洲猪瘟病毒的侧流试验(LFA)     

口蹄疫检测技术的研究进展与应用

从生物学试验、血清学诊断技术和分子生物学检测技术等方面概述了目前口蹄疫检测技术的研究进展及其应用。其中生物学试验包括动物试验和细胞培养;血清学诊断技术包括补体结合试验、中和试验、凝集试验、酶联免疫吸附试验和免疫胶体金快速检测技术等;分子生物学检测技术包括反转录聚合酶链式反应、核酸探针、核酸序列分析和等电聚焦等。     

间接ELISA检测禽流感抗体方法的建立

应用醛化的鸡红细胞经吸附释放方法获得纯化的禽流感病毒 (AIV) ,经 triton X-1 0 0处理并反复冻融制备禽流感 EL ISA抗原。应用抗鸡 Ig轻链单抗 1 B7辣根过氧化物酶结合物建立了定量检测禽流感抗体水平的 EL ISA方法。确立了将鸡血清 1 0 0倍稀释监测 EL ISA效价(ET)的回归方程 y=0 .77452 x 0 .8793 5(r=0 .9466) ,可用于定量测定。血凝抑制 (HI)抗体与 EL ISA方法的比较试验表明 ,EL ISA法比血凝抑制试验敏感 1 .5倍以上     

快速诊断血清和牛奶中的牛白血病病毒

最近,我们建立了一种快速、简便的血清学诊断牛白血病的酶联免疫方法。在牛奶中对白血病的控制是非常重要的,由于出口受到限制和屠宰时胴体的污染(最后形成淋巴组织瘤),从而造成经济上的损失。牛白血病(BLV)的诊断试验,包括各种酶联免疫吸附试验(EL ISA),放射     

反转录环介导等温扩增技术快速检测猪水疱病毒方法的建立

为了建立一种针对猪水泡病毒(SVDV)的反转录环介导等温扩增技术(RT-LAMP),用于减少猪水泡病与口蹄疫因临床症状相似而造成的误诊,试验针对SVDV VP1基因序列设计4个LAMP引物,其中外侧引物用于SVDV PCR检测,并利用RT-LAMP和RT-PCR方法进行敏感性试验、特异性试验以及临床样品检测。结果表明:针对SVDV的RT-LAMP检测方法的敏感性是RT-PCR的10倍; RT-LAMP方法的特异性良好,与口蹄疫病毒无交叉反应;两种方法对临床样品检测的符合率为100%。说明试验建立的RT-LAMP方法具有敏感性高、特异性强、快速等特点,适合实验室和临床对SVDV的快速诊断。     

检测用抗体标准样品的研究现状和展望

正血清学检测方法是利用抗原抗体在体外的免疫反应,实现疫病感染检测和免疫效果评估,已在人类和动物疫病的监测和诊断中得到了普遍使用。常规的血清学方法主要包括有琼脂免疫扩散试验、血凝抑制试验、补体结合试验、病毒中和试验和酶联免疫吸附试验等。抗体标准样品是用于血清学检测方法质量控制和评价的重要材料,对于保证抗体检测结果的准确性、一致性和可比性,实现检测     

Laboratory decision-making during the classical swine fever epidemic of 1997-1998 in The Netherlands

The National Reference Laboratory for classical swine fever (CSF) virus in the Netherlands examined more than two million samples for CSF virus or serum antibody during the CSF epizootic of 1997–1998. The immense amount of samples and the prevalence of border disease (BD) virus and bovine viral diarrhoea (BVD) virus infections in Dutch pig herds necessitated the diagnostic efforts of the laboratory to be focused on generating CSF specific test results throughout the eradication campaign.

Detection of 82% of the 429 outbreaks was achieved through the combined use of a direct immunofluorescence and peroxidase assay (FAT/IPA) with samples (tonsils) collected from clinically-suspected pigs. This suggests that in the majority of the outbreaks, the pigs had clinical signs that were recognised by the farmer and/or veterinarians, indicating the presence of CSF virus in a pig herd. A positive diagnosis of 74% of all the tissue samples (tonsils) collected at infected pig holdings was established by FAT. More than 140,000 heparinised blood samples were examined by virus isolation, resulting in the detection of 4.5% of the infected herds. CSF virus was isolated in approximately 29% of all the blood samples collected from pigs at infected or suspected farms.

Several serological surveys — each done within a different framework — led to the detection of 13.5% of the total number of outbreaks. The detection of CSF virus antibody in serum was carried out by semi-automated blocking ELISA. Approximately 28.5% of the sera which reacted in the ELISA were classified as CSF virus-neutralising antibody positive and 26.5% as positive for other pestiviruses following the virus neutralisation test (VNT).

We concluded that two of the CSF laboratory diagnostic methods described were determinative in the eradication campaign: first, the FAT for the screening of diseased pigs; and second, the ELISA and VNT when millions of predominantly healthy pigs needed to be screened for the presence of CSF serum antibody. Decision-making on the basis of results generated by either method can, however, be seriously hindered when samples are examined from pig herds with a high prevalence of non-CSF pestiviruses.     

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.     

Detection of foot-and-mouth disease antigen in bovine epithelial samples: comparison of sites of sample collection by an enzyme linked immunosorbent assay (ELISA) and complement fixation test

Serially collected epithelial samples from lesions in the mouth and on the feet of calves experimentally infected with foot-and-mouth disease (FMD) type O1 BFS 1860 were assayed for the presence of FMD viral antigen using a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) and a complement fixation (CF) test. The amount of infectious virus in each sample was also determined. FMD viral antigen was detected by ELISA in 70 per cent of the mouth samples and 92 per cent of samples from the feet. The CF test was less sensitive; it detected antigen in 44 per cent of mouth and 85 per cent of foot samples. In mouth samples the amount of antigen decreased rapidly becoming undetectable by the fourth day of sampling whereas in foot samples the quantity of antigen declined more slowly, and could be detected until the seventh day of sampling. Therefore it was concluded that the age of lesion and the site from which epithelial samples are collected are both important determinants in the laboratory diagnosis of FMD. In cattle, foot lesions are more likely than mouth lesions to yield antigen and to remain positive for a longer period.     

Describing the within laboratory and between laboratory agreement of a serum ELISA in a national laboratory network

Results from laboratory assays for detection of animal disease are often assessed for repeatability (agreement within laboratory) and reproducibility (agreement between laboratories). This work aimed to understand the strengths and limitations of available methods for describing these quantities. Five major veterinary laboratories in Australia volunteered to participate in a designed evaluation based on repeat testing of twenty bovine sera. Sampling was stratified so that ten of the sera were negative to the virus neutralisation test (VNT) for antibody to bovine herpes virus 1 (BHV-1) and the remaining ten sera were VNT positive. Each serum was divided into 50 replicates and each laboratory assayed one replicate of each serum on a weekly basis using a commercial ELISA for BHV-1. Laboratories were blinded to the identity of sera. The data on sample to positive control ratio (S/P) for these 1000 individual assays were collated, sources of variance analysed using a random effects model, and reliability coefficients (ρ) obtained from the variance estimates as quantitative measures of within and between laboratory agreement. Coefficient of variation (CV) was calculated for combinations of sera and laboratory. CV was found to be higher for sera with the lowest mean S/P values (VNT -ve sera). For VNT -ve sera, agreement of S/P within laboratory was low to moderate (ρ: 0.01-0.27) and the agreement between all labs was low (ρ=0.02). Reliability coefficients for VNT +ve sera were very high for agreement within laboratories (ρ: 0.63-0.92) and moderate for agreement between laboratories (ρ=0.52). As well, simulation demonstrated that sero-prevalence has a dramatic affect on the reliability coefficient if sampling were to be irrespective of VNT status. We conclude that there are some limitations with the available approaches for assessing agreement within and between laboratories. Although reliability coefficients have some drawbacks they are an attractive way of reducing reliance on subjective assessment of agreement.     

Evaluation of serological tests for H5N1 avian influenza on field samples from domestic poultry populations in Vietnam: consequences for surveillance

In Vietnam, serological post H5N1 vaccination surveillance using the HI test is applied to assess the efficiency of the vaccination in addition to virological monitoring. In this paper we report on the evaluations of the performances of the haemagglutination inhibition (HI) test and of a H5-ELISA, using chicken and duck field samples. The evaluations were conducted by comparison with a pseudotyped-based virus neutralization test (H5pp VNT) performed in a reference laboratory and considered as a "gold standard" and also by using methods developed for imperfect reference test. Their global accuracy and best cut-offs were also estimated. Results from the HI test for several haemagglutinin subtypes and from a commercial type A influenza competition ELISA were also compared. The results showed that performance of the HI test was very good in comparison with the H5pp VNT. Data also clearly supported the cut-off of ≥ 4 log(2) used for the HI test for chickens but, a 3 log(2) positivity cut-off would be more appropriate for ducks. When compared with the VNT, the H5-ELISA showed poor specificity when using the positivity cut-off specified by the manufacturer but could be used as a screening test if confirmed by the HI test or the H5ppVNT which presents some interests for large scale testing (no need for biosafety level 3 conditions and high performance). A general and highly sensitive pre-screening can also be achieved using the detection of NP-specific antibodies with a competition ELISA. This appears of little interest in a context of high subtypes diversity where only a subtype is targeted for surveillance and control.     

Antibodies to foot-and-mouth disease virus infection associated (VIA) antigen: use of a bioengineered VIA protein as antigen in an ELISA

An enzyme-linked immunosorbent assay (ELISA) to detect antibodies to foot-and-mouth disease (FMD) virus infection associated (VIA) antigen (viral RNA polymerase) in cattle sera, was developed using a bioengineered VIA (BioVIA) protein antigen. Compared with the classical immunodiffusion test, with viral RNA polymerase purified from infected cell cultures as antigen, this ELISA was more sensitive. However, depending on the cattle population examined, sera with antibodies to viral RNA polymerase, probably due to infection with other picornaviruses, were detected. Despite these observations, the ELISA using BioVIA provided a rapid answer as to whether or not FMD virus circulated in a given herd of cattle. The main advantage of this ELISA is its absolute safety, since in no step of the antigen production was infectious or uninfectious FMD virus involved. The test can therefore be performed under normal laboratory conditions and no isolation units are needed as they are for the immunodiffusion test.     

A simulation model of intraherd transmission of foot and mouth disease with reference to disease spread before and after clinical diagnosis.

Intraherd transmission of foot and mouth disease virus (FMDV) was examined using a simulation model for a hypothetical 1,000-cow dairy, assuming clinical diagnosis was made when at least 1% (10 cows) or 5% (50 cows) had clinical signs of FMD, I index case cow, and transition state distributions for the latent, subclinically infectious, and clinically infectious periods of FMD calculated from published data. Estimates assumed for the number of animal-to-animal contacts (k) adequate for transmission ranged from 0.6 to 9.0 per hour (13.7-216.0 per day). A total of 40,000 iterations (5,000 for each scenario, assessing 4 adequate contact rates and 2 detection criteria) were run. The model predicted that FMD would not be diagnosed in the herd until 10.0-13.5 days after the index case cow had become infected, at which time between 65% and 97% of the cows (646-967 cows) to nearly 100% (978-996 cows) would already have become infected with the virus, if the number of cows showing clinical signs of FMD at the time of diagnosis were 10 or 50, respectively. At the time of diagnosis, the simulated number of infectious cattle varied substantially from 82-472 to 476-537 cows, depending on adequate contact rate and whether the diagnosis was made when 10 or 50 animals were showing clinical signs, respectively. The simulated number of infectious cows increased rapidly during the first few days after diagnosis. In the scenario where at least 10 cows showing clinical signs was necessary before a clinical diagnosis was made, each day after diagnosis, the number of infectious animals increased by nearly 100 to more than 200 cases per day up to day 5, assuming 0.57-9.0 animal-to-animal contacts per hour, respectively. Results obtained when it was assumed that at least 50 clinical cases were present at the time of diagnosis showed smaller relative increases because nearly one-half of the herd was projected to be infected at the time of diagnosis. From these results, it is clear that once an individual in a herd becomes infected with FMDV, herd infectivity is not static, rather it accelerates as would be expected as long as there are sufficient susceptible animals to sustain the increasing transmission rate, after which time the rate at which new infections occurs will diminish. Results indicate that biosecurity strategies aimed at minimizing both intraherd and interherd contact will be critical in minimizing the spread of FMD before the initial diagnosis is made. In addition, simulations suggest that very early clinical diagnosis of FMD and effective isolation or depopulation and disposal will be critical in limiting the number of infectious animals capable of transmitting the virus to other herds and thus in timely control of an epidemic. Early diagnosis will rely on early virus detection from animals in the preclinical phase of infection, rather than waiting for clinical signs to manifest in sufficient numbers to be noticed and to warrant investigation.     

牛传染性鼻气管炎诊断方法研究进展

进行牛传染性鼻气管炎分子流行病学调查时,首先通过临床症状观察进行初诊,然后再进行实验室确诊。目前实验室诊断主要包括病原学诊断和血清学诊断。病原学诊断方法包括包涵体检查、病毒分离和病毒核酸检测;血清学诊断方法包括中和试验、琼脂扩散试验、间接血凝试验、酶联免疫吸附试验和变态反应。有时还要进行鉴别诊断,鉴别诊断主要有单克隆抗体法、鉴别PCR等。牛传染性鼻气管炎在世界范围内流行,给全球的养牛业造成了很大的影响。论文综述了牛传染性鼻气管炎诊断方法研究进展,为预防和消除该病提供参考。     

应用固相竞争ELISA、液相阻断ELISA和中和试验检测口蹄疫抗体的比较研究

为了评价建立的口蹄疫固相竞争酶联免疫吸附试验(solid phase competition ELISA,SPC-ELISA)方法与传统的中和试验(VNT)和液相阻断酶联免疫吸附试验(liquid phase block ELISA,LPB-ELISA)检测方法之间的关系,本研究对野外采集的不同种类的猪、牛和牦牛共94份血清样品分别进行O型口蹄疫病毒SPC-ELISA、LPB-ELISA和VNT检测,并分别比较3种方法的符合率和阳性检出率。结果表明,建立的O型口蹄疫病毒SPC-ELISA方法与VNT 具有很好的符合率,达88.3%,其中,猪和牦牛血清符合率高达90%和89.5%,阳性检出率为91.2%比LPB-ELISA更符合VNT,因此建立的SPC-ELISA方法比LPB-ELISA方法更符合VNT。     

A Bayesian evaluation of four immunological assays for the diagnosis of clinical cryptosporidiosis in calves

A Bayesian approach was used to evaluate four immunological assays for the clinical diagnosis of cryptosporidiosis in calves: an immunofluorescence assay (IFA), two ELISA tests and an immunochromatographic (dipstick) assay. Faecal samples from 287 calves aged less than 6 weeks with clinical signs of gastrointestinal disease were examined for the presence of Cryptosporidium spp. The high prevalence (63%) of Cryptosporidium spp. indicated the relevance of this agent in the aetiology of diarrhoea in calves. All diagnostic assays were found to be relatively specific (IFA: 94.8%; Tetra ELISA: 95.9%; Techlab ELISA: 92.7%; dipstick assay: 91.5%) and sensitive (IFA: 97.4%; Tetra ELISA: 93.6%; Techlab ELISA: 95.4%; dipstick: 87.8%). Despite a lower sensitivity, the dipstick assay provided a practical alternative to laboratory diagnosis of clinical cryptosporidiosis in calves.