伪狂犬病毒流行毒株的抗原性和血清中和特性分析

2011年以来,我国多个省份伪狂犬病免疫猪群相继暴发伪狂犬病疫情。为了探明伪狂犬病毒(PRV)流行毒株是否存在抗原变异,本研究从3个规模猪场3种PRV商品疫苗免疫的健康猪群中采集30份血清,经检测PRV gB ELISA抗体均为阳性,gE ELISA抗体均为阴性。采用PRV新流行毒株ZJ-01株和传统的LA毒株分别进行血清中和抗体测定,结果显示,3个毒株活疫苗免疫血清与ZJ-01株中和抗体效价明显低于其与LA株中和抗体效价。同时,ZJ-01株抗血清与ZJ-01株和LA株的中和抗体效价相似,其变异系数R值为0.279⁰.413,证明PRV分离毒株ZJ-01抗原性发生明显变异。本研究为伪狂犬病的防控提供了重要依据。     

竞争性ELISA检测猪瘟病毒抗原

猪瘟（Classical swine fever，CSF）是由猪瘟病毒（Classical swine fever virus，CSFV）引起，主要侵袭家猪及野猪，引起高发病率、高死亡率的烈性传染病。由于其危害严重、流行分布广泛，成为国内外分子病毒学及兽医防疫研究的热点之一。CSFV属于黄病毒科（Flaviviridae）瘟病毒属（Pestvirus）成员，与同属的牛病毒性腹泻病毒（Bovineviral diarrhea virus，BVDV）、羊边界病病毒（Borderdiseasevirus，BDV）、长颈鹿瘟病毒（Giraffepestvirus），在病毒结构、抗原性和遗传特性等方面密切相关。     

猪乙脑乳胶凝集试验抗体检测试剂盒保存期试验

乙脑是经虫媒传播的人畜共患病毒病，该病引起种猪繁殖障碍，给畜牧业带来很大的经济损失，还严重危害人民身体健康，控制猪乙脑是防制乙脑、保障养猪业发展的重要措施，为此进行了猪乙脑乳胶凝集试验的研究，并在此基础上通过组装乙脑乳胶抗原、乙脑阳性血清、乙脑阴性血清和稀释液，研制出猪乙脑乳胶凝集试验抗体检测试剂盒，猪乙脑     

Immune response of sows and their offspring to pseudorabies virus: serum neutralization response to vaccination and field virus challenge.

One month prior to breeding, sows were vaccinated with an attenuated pseudorabies virus vaccine or challenged with a field strain of pseudorabies virus. A third group of sows were not vaccinated or challenged before breeding. Pigs from these sows were vaccinated at 3, 6, or 12 weeks of age and challenged with virulent virus three weeks later. One pig from each litter served as an unvaccinated, unchallenged control. Serum neutralization titers of these pigs were monitored from birth until 22 weeks of age. Titers of the sows were monitored through breeding, gestation and farrowing. The maximum prefarrowing anti-pseudorabies virus titer in the field virus challenged sows occurred four weeks following challenge. A significant decline in titers occurred at farrowing. Titers rose from one week postfarrowing and then declined. Titers in the field virus infected sows were consistently two to threefold greater than those of the vaccinated sows. The maximum prefarrowing anti-pseudorabies virus titer in the vaccinated sows occurred six weeks following vaccination. The geometric mean titer in these sow's then decreased and increased for two weeks after farrowing. The results in the pigs can be summarized as follows: Pigs from control sows had a greater serological response following field virus challenge than following vaccination with a modified live virus. Pigs from control sows responded serologically to vaccination at 3, 6 and 12 weeks of age. Pigs from control sows which were challenged at 6, 9 and 15 weeks of age had similar antibody responses. Pigs from vaccinated sows had no increase in titer following vaccination at three and six weeks of age. Titers increased when these pigs were vaccinated at 12 weeks of age. There was no significant increase in mean titers of pigs from challenged sows following vaccination at 3, 6 and 12 weeks of age. Vaccinated pigs from control and vaccinated sows had a secondary response following challenge three weeks after vaccination.(ABSTRACT TRUNCATED AT 250 WORDS)     

Radial immunodiffusion enzyme assay for detection of antibodies to pseudorabies virus in swine serum

A radial immunodiffusion enzyme assay for the detection and quantitation of antibodies to pseudorabies virus in swine sera was developed and the methods were standardized. The assay combined the principle of radial immunodiffusion with enzyme-linked immunosorbent assay. Quantitation of pseudorabies virus antibody titers was determined by measuring the diameter of a colored circular zone after overnight incubation of antibody with antigen. The specificity and sensitivity of the radial immunodiffusion enzyme assay were compared with that of the standard virus-neutralization test, and the results were determined to be correlated highly (r = 0.694, P less than 0.0001). The assay also appeared to be highly reproducible and simple to perform.     

Role of memory B-cell responses in serum and mucosal fluids of swine for protective immunity against pseudorabies virus.

We examined primary and memory isotype-specific antibody responses directed against pseudorabies virus in serum and mucosal fluids of pigs with and without passively acquired maternal antibody, and we studied the relationship between these responses and protection against virus challenge. Pigs were inoculated intranasally with the virulent NIA-3 strain or the avirulent Bartha strain, or they were inoculated IM with an inactivated vaccine containing the Phylaxia strain. Ten weeks later, all pigs were challenge-exposed intranasally with strain NIA-3. Only pigs that were without passively acquired antibody at the time they were inoculated with virulent virus appeared to have complete protective immunity against challenge exposure, as evidenced by lack of clinical signs of pseudorabies and lack of virus excretion. In contrast, pigs inoculated with strain Bartha or with the inactivated vaccine developed fever, had a period of growth arrest, and excreted virus after challenge exposure. In pigs without passively acquired antibody, intranasal inoculation with strains NIA-3 or Bartha was followed by primary IgM and IgA responses in serum and in oropharyngeal fluid as well as primary IgG1 and IgG2 responses in serum. Intramuscular inoculation with the inactivated vaccine induced primary serum IgM, IgG1, and IgG2 responses, but no mucosal responses. Challenge exposure of pigs that had been inoculated with the Bartha strain or the inactivated vaccine was followed by clear memory responses in serum and in oropharyngeal fluid. In contrast, challenge exposure of pigs that had been inoculated by the virulent NIA-3 strain was not followed by memory responses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Latent infection and subsequent reactivation of pseudorabies virus in swine exposed to pseudorabies virus while nursing immune dams

The ability of pseudorabies virus (PRV) to infect and establish latency in pigs with passively acquired (maternal) antibody for PRV was tested by exposing such pigs to the virus and subsequently attempting to reactivate latent virus by administering large doses of dexamethasone. Pigs of each of 4 litters that had nursed gilts with relatively high (512, gilts 1 and 2), moderate (32, gilt 3), and no (less than 2, gilt 4) serum titers of virus-neutralizing (VN) antibodies for PRV were allotted to 3 treatment groups (A, B, C) when they were 2 weeks old. Group-A pigs were separated from littermates and dam and thereafter kept in isolation; group-B pigs were experimentally exposed oronasally to PRV and 1 hour later returned to their dam; group-C pigs were kept with their dam and potentially exposed to PRV by contact with littermates of group B. Sera obtained from pigs at selected intervals until they were 17 weeks old were tested for VN activity and for precipitating activity for radiolabeled viral proteins. All group-A pigs remained clinically normal throughout the experiment. Depending on the initial amount of passively acquired antibody, little or no serum VN or precipitating activity remained by the time these pigs were 17 weeks old. Group-B and -C pigs, with relatively high amounts of passively acquired antibody when exposed to PRV, also remained clinically normal. However, most became latently infected as subsequently evidenced by either dexamethasone-induced or noninduced virus reactivation. Noninduced reactivation may have been initiated by weaning the pigs when they were about 8 weeks old.(ABSTRACT TRUNCATED AT 250 WORDS)     

H9N2亚型猪流感病毒血凝素基因的表达及LAT诊断方法的研究

根据H9N2亚型猪流感病毒血凝素基因序列设计并合成引物,从本室分离保存的H9N2亚型猪流感病毒中扩增出血凝素基因,并将其克隆进pMD18-T载体,限制性酶切及序列测定后,进一步将其信号肽缺失并亚克隆到pGEX-KG中,使其在E.coli BL21（DE3）中与GST融合表达。SDS-PAGE显示表达的融合蛋白的相对分子质量约为90 000。Western印迹表明表达的蛋白质具有免疫学活性。表达产物位于包涵体中,包涵体经变性、复性处理,利用复性产物作为抗原,经碳二亚胺（EDAC）将表达产物和羧基化的乳胶共价偶联,建立了H9亚型猪流感病毒抗体的乳胶凝集试验的检测方法,结果表明,应用HA重组蛋白作为诊断抗原具有特异性强、敏感性高、重复性好的特点,可用于H9亚型猪流感抗体水平监测、流行病学调查和现地疫病普查。     

Development and evaluation of a microimmunodiffusion test for detection of antibodies to pseudorabies virus in swine serum.

A microimmunodiffusion test (MIDT) was developed for the detection of pseudorabies virus (PRV) antibodies in swine serum. The optimal medium for the MIDT was determined to contain 0.69% agarose in 0.05 M tris buffer (pH 7.2) with 0.025% sodium azide and no NaCl. The PRV antigen prepared by (NH4)2SO4 precipitation of viral fluids (42.5 g/100 ml), dialyzed against distilled water, and concentrated to approximately 100-fold of the original volume with polyethylene glycol (mol wt 20,000) provided a good reproducible antigen. The sensitivity of the MIDT was compared with the microtitration procedure of the virus-neutralization (VN) test by assaying 2,203 swine serums for PRV antibodies. An equal percentage of serums was positive in both tests; 419 had VN titers of greater than or equal to 4, and 421 were MIDT positive. Serums (314) that had VN titers of greater than or equal to 16 were all positive by the MIDT. Of serum samples with a VN titer of 8 (53), 50 were MIDT positive, a 94% correlation, and of 52 serums that had VN titers of 4, 36 were MIDT positive, a 69% correlation. In addition, 8 serums that had titer of less than 4 by VN test were positive by MIDT. Seventy-one serum samples were too cytotoxic, markedly hemolyzed, or contaminated to evaluate properly in the VN test; of these serums, 13 were MIDT positive. The MIDT is an accurate, rapid, economical, and sensitive diagnostic test for the detection of PRV antibodies in swine serums.     

Detection of pseudorabies virus antibody in swine serum and oral fluid specimens using a recombinant gE glycoprotein dual-matrix indirect ELISA

Pseudorabies (Aujeszky disease) virus (PRV) was eliminated from domestic swine in many countries using glycoprotein E (gE)-deleted vaccines and serum antibody gE ELISAs, but PRV continues to circulate in some regions and in most feral swine populations in the world. We created a dual-matrix (serum and oral fluid) indirect IgG gE ELISA (iELISA) and evaluated its performance using samples from 4 groups of 10 pigs each: negative control (NC), vaccination (MLV), PRV inoculation (PRV), and vaccination followed by challenge (MLV-PRV). All serum and oral fluid samples collected before PRV challenge and all NC samples throughout the study were negative for gE antibodies by commercial blocking ELISA (bELISA) and our iELISA. Nasal swab samples from 9 of 10 animals in the PRV group were gB quantitative PRC (qPCR) positive at 2 days post-inoculation (dpi). The oral fluid iELISA detected a significant S/P response in the PRV (p = 0.03) and MLV-PRV (p = 0.01) groups by 6 dpi. ROC analyses of serum bELISA (n = 428), serum iELISA (n = 426), and oral fluid iELISA (n = 247) showed no significant differences in performance (p > 0.05). Our data support the concept of PRV surveillance based on oral fluid samples tested by an indirect gE ELISA.     

利用重组抗原建立间接酶联免疫吸附试验检测猪传染性胃肠炎血清抗体

随着分子生物学的发展 ,重组抗原用于疾病的血清学诊断的报道越来越多 [3 ] 。周仲芳等报道了采用杆状病毒表达系统生产的TGE病毒纤突蛋白 ( S)建立了一种竞争ELISA检测猪 TGE血清抗体 ,获得了较为理想的特异性和准确性。本文报道了采用同样的重组抗原建立了一种间接 ELISA用于TGE血清抗体的检测 ,同时由于在结果判定中采用终点滴度技术 ( End- titer) ,使得检测结果的判定更直观和迅速。1　材料与方法1.1　 EL ISA操作方法　本研究中的抗原制备和包被方法见周仲芳等 ( 1997)的报道。ELISA板经抗原包被后可立即使用 ,也可保存…     

Comparison of 16 chelonid herpesviruses by virus neutralization tests and restriction endonuclease digestion of viral DNA

A total of 16 chelonid herpesviruses that were isolated between 1992 and 1998 were compared with one another on the basis of serology and restriction enzyme digestion patterns of viral DNA. The viruses stem from tortoises of three different species in four different European countries and the United States of America. The majority of the isolates were similar to one another. One isolate, however, differed strongly from all others both serologically and in the restriction cleavage pattern of its DNA, showing that there are at least two different sero- and genotypes of herpesviruscs that infect tortoises.     

猪圆环病毒2型感染对伪狂犬疫苗免疫应答的影响

为明确PCV2感染对伪狂犬(PR)疫苗免疫应答的影响,本研究采用阻断ELISA方法对单独接种猪PR疫苗组(A组)及PCV2人工感染3周后接种猪PR疫苗组(PA组)不同时相血清中的猪PR病毒gB抗体进行检测;同时对不同时相前腔静脉血进行CD4+/CD8+流式细胞术及血常规分析。结果表明,在PCV2感染后2周至5周间,A组白细胞含量均高于PA组,随后PA组白细胞恢复至与A组略高的正常水平;在整个实验中,除接种猪PR疫苗后1周(WPI)和9周(WPI)外的所有时相PA组的淋巴细胞含量均略高于A组;PCV2感染后可使记忆/激活Th细胞数量略有升高,幼稚型Th细胞含量的下降;PCV2感染后2周～7周PA组Tc细胞均高于A组,在9WPIPA组Tc细胞数量显著下降(p0.05);除9WPI外,A组的S/N值均低于PA组。结果表明,PCV2感染看降低机体产生针对PRVgB特异性抗体水平,而且在一定程度上降低了幼稚型Th细胞及Tc细胞含量。     

猪传染性胃肠炎病毒核衣壳蛋白在ELISA中的应用

猪传染性胃肠炎(TGE)是引起猪腹泻的病毒性传染病,感染猪的主要临床症状是腹泻、呕吐和脱水,不同年龄的猪均易感,尤其两周龄以内的猪死亡率可达100%,给养猪业造成了巨大的经济损失.因此开展猪传染性胃肠炎诊断方法的研究对该病的防制具有重要意义.     

新城疫抗原凝集价影响因素的探讨

红细胞凝集试验 (HA)和红细胞凝集抑制试验(HI)是国际兽疫局 (OIE)指定或推荐用于多种畜禽传染病 ,如鸡新城疫、禽流感、鸡支原体感染、传染性支气管炎等的诊断和检测方法。由于效价的高低直接反映病毒抗原的浓度或动物体的免疫状态 ,加上检测所用的仪器简单、操作方便、试剂容易获得等因素 ,在基层单位或其它兽医检测机构的应用非常普遍。但是在检测过程中应明确影响试验结果的因素 [2 ,3,6] ,使该方法逐渐过度为标准化的程序 ,以便于获得的结果在不同的条件下具有可比性和重复性。本试验拟就影响检测结果的几个因素作一探讨。1　材料与…