从进口种用奶牛中分离传染性牛鼻气管炎病毒

在对2865头进口种用奶牛隔离检疫期间,从1头IBR中和抗体阳性奶牛中分离1株病毒。该分离株表现类似于IBR病毒特征的细胞病变(CPE),细胞圆缩,聚集成葡萄串样群落,在单层细胞上形成空洞,有时会发现有几个细胞核的巨大细胞。用特异性抗IBRV阳性血清与之进行中和试验,发现IBRV标准阳性血清对分离株的中和抗体滴度为26.5,与对IBR标准株的中和抗体滴度相差不到一个滴度。调取IBRVLA株gB基因序列,设计出1对IBR特异性的引物进行PCR,能扩增出与目的基因片段大小一致的特异性条带。结果表明:分离到的病毒为IBRV。     

牛传染性鼻气管炎病毒的分离鉴定与灭活疫苗免疫效果研究

由2例疑似牛传染性鼻气管炎（IBR）病例的荷斯坦奶牛分离到一株病毒,命名为IBRV—C1株。该病毒可被IBR标准阳性血清完全中和;接种MDBK细胞可出现IBR病毒典型细胞病变效应;选取IBR病毒gB蛋白基因序列设计引物进行PCR检测和基因测序,结果可扩增出特异性目的片段;动物回归试验显示,3头牛均可见体温升高、鼻流粘液、呼吸困难等典型的IBR临床症状。在此基础上制备了三批牛传染性鼻气管炎灭活疫苗,并进行了疫苗安全性和效力试验,结果表明三批疫苗对靶动物安全,免疫效果较好,免疫牛中和抗体效价几何平均值可达1：41以上,攻毒保护率达5／5。     

牛传染性鼻气管炎病毒单克隆抗体的制备及阻断ELISA方法的建立

为建立检测牛传染性鼻气管炎病毒(IBRV)血清抗体的阻断ELISA方法,本研究以经蔗糖密度梯度离心法纯化的IBRV作为免疫原制备1株单克隆抗体(MAb),命名为cp-1-1。经间接ELISA、IFA和western blot鉴定,该MAb与IBRV呈阳性反应,与牛病毒性腹泻病毒(BVDV)及牛副流感病毒3型(BPIV3)呈阴性反应,具有较强的特异性。质谱分析结果显示MAb cp-1-1识别的表位位于IBRV VP8蛋白。以纯化的IBRV作为包被抗原、MAb cp-1-1作为检测抗体,建立检测IBRV血清抗体的阻断ELISA方法。该检测方法的抗原包被量为0.89μg/孔,样品稀释度为12,检测抗体MAb量为1.3μg/孔,二抗稀释度为15000。利用50份IBRV抗体呈弱阳性的牛血清(中和抗体效价为14~116)作为标准参考血清,确定该检测方法的阻断率Cut Off值为52.06%,即阻断率高于52.06%时判为阳性,低于52.06%时判为阴性。阻断ELISA方法特异性试验显示仅IBRV阳性血清检测为阳性,而BVDV、BPIV3、牛腺病毒3型(BADV-3)和O型口蹄疫病毒(O-FMDV)阳性牛血清均检测为阴性,表明该方法具有较强的特异性;该方法可检测的最低中和抗体效价为14,与病毒中和试验的敏感性一致,表明该方法具有较高的敏感性;重复性试验显示该方法批内、批间变异系数均小于10%,显示较好的重复性。对130份现地牛血清检测结果显示,该方法与病毒中和试验的符合率为98.46%。用该方法对某牛场接种IBRV灭活疫苗的牛血清进行检测,抗体阳性率为99.51%(205/206)。另外,采用该方法对我国8个省(市、自治区)的801份牛血清进行检测,IBRV的抗体阳性率为41.6%(333/801)。本研究建立的阻断ELISA方法可以用于IBRV疫苗免疫监测和血清流行病学调查,为我国IBR的防控提供技术支持。     

河北省奶牛传染性鼻气管炎病毒的分离鉴定与系统进化分析

为了分离与鉴定河北省牛传染性鼻气管炎病毒(Infectious bovine rhinotracheitis virus, IBRV),试验对河北省某规模化奶牛场IBRV初检阳性病料进行处理,采用病毒分离培养、分离毒毒价测定、中和试验、PCR扩增、目的基因片段测序、同源性分析及系统进化树构建的方法对病毒进行鉴定和分析。结果表明:筛选出一株病变毒株,其TCID_(50)为1×10~(-4.7)/0.1 mL,分离株抗原能够被IBRV阳性血清中和,PCR扩增出大小为314 bp的特异性片段,扩增的gB基因片段共编码316个核苷酸,与GenBank中部分IBRV毒株的同源性为99.3%～100%,该毒株与BH81株(DQ006854)亲源关系较近,将其命名为HBXT-IBRV。     

用酶联免疫吸附试验检测牛传染性鼻气管炎病毒抗体的敏感性和特异性

应用酶联免疫吸附试验(ELISA)检测牛血清中牛传染性鼻气管炎病毒(IBRV)抗体的技术已经得到了发展。我们用ELISA方法检测从无IBR病毒牛群中采集的304份血清,其特异性为100%;检测经鼻内疫苗接种免疫后采集的62份牛血清,其诊断敏感性在免疫后第一个月为27.4%,第六个月为100%,检测标准血清中和抗体滴度≥1:2的303份牛血清,其敏感性为100%;463份随意诊断样品经比较试验证明ELISA检出血清阳性牛(61.6%)比标准血清中和试验(49.9%)要高。因此,我们认为ELISA具有技术上的优越性,可以作为一种常规诊断试验来检测牛血清中IBR病毒抗体。     

IBRV重组gD蛋白的真核表达与间接ELISA检测方法的建立

通过优化牛传染性鼻气管炎病毒（IBRV）gD基因序列为Sf9细胞偏好密码子,转座形成穿梭载体,将质粒瞬时转染Sf9昆虫细胞,利用昆虫杆状病毒表达系统表达纯化重组IBRV gD蛋白;以制备的重组IBRV gD蛋白为包被抗原,建立检测IBRV血清中和抗体的间接ELISA方法,并通过比对检测已知血清中和抗体效价的牛血清,验证所建立的间接ELISA方法的敏感性、特异性与重复性等指标。结果显示：本研究建立的间接ELISA方法对相关的牛病毒血清抗体无交叉反应,敏感性可达1:512,组内和组间变异系数均低于10%;使用建立的间接ELISA检测方法结合血清中和试验,对480份临床血清样品进行检测,发现两者敏感性符合率为98.18%,特异性符合率为93.33%,总符合率为96.67%。结果表明,本研究建立的检测IBRV血清中和抗体的间接ELISA方法具有良好的特异性、敏感性和重复性,可开发为临床适应的检测试剂盒。     

江汉平原—规模化猪场暴发伪狂犬病的调查研究(Ⅲ)——猪伪狂犬病毒HS-9304株的克隆纯化及检定

应用细胞培养病毒蚀斑技术,将分离的伪狂犬病毒(PrV)HS-9304株在鸡胚成纤维细胞(CEF)单层培养上覆以营养琼脂培养基,连续挑斑纯化3次,成功地获得了纯化病毒克隆株.对病毒克隆株在传代细胞上复壮增殖后进行毒价测定.接种试验动物人工造病并进行血清中和试验以及外源性污染的检定.该病毒克隆株对BHK-21细胞感染的滴度为10~(8.5)TClD_50,能使小鼠和家兔出现特异性的症状和死亡,病毒对PrV标准阳性血清的中和效价达到1:195.病毒纯净无外源性污染.     

牛传染性鼻气管炎间接ELISA诊断方法的建立

以牛肾细胞系（MDBK）培养牛传染性鼻气管炎病毒（IBRV）Bartha Nu/67株,经超速离心纯化病毒,再经超声破碎处理后作为诊断抗原,建立了检测牛血清IBRV抗体的间接酶联免疫吸附试验.该ELISA的判定标准为：血清D490 nm值大于0.369的判为阳性,小于0.295的判为阴性,在0.295与0.369之间的为可疑.特异性和重复性试验结果表明,该方法特异性高、重复性好.与法国进口ELISA抗体诊断试剂盒比较,其符合率为96.3%;与中和试验比较,符合率为95.8%,且敏感性更高.应用该诊断方法调查了我国部分地区IBRV的感染情况,结果显示,这些地区的IBRV感染率为67.1%.     

猪繁殖与呼吸综合征病毒地方株的分离与鉴定

从河南省某猪场发病仔猪体内分离到1株病毒,该病毒能在 Marc-145细胞上增殖并产生特征性的细胞病变 (CPE),在 Vero、PK-15细胞上不出现CPE.该病毒能被猪繁殖与呼吸综合征病毒阳性血清特异性地中和,用 PCR反应能扩增出 720 bp的特异性片段.分离病毒回归 30日龄仔猪可出现高热和呼吸道症状.初步鉴定该分离株为猪繁殖与呼吸综合征病毒.     

牛传染性鼻气管炎病毒gB ELISA抗体与中和抗体相关性分析

牛传染性鼻气管炎病毒（IBRV）是牛传染性鼻气管炎（IBR）的病原体,常引起流产和呼吸道感染等综合征,给养牛业造成了巨大经济损失。gB作为IBRV中最保守的抗原蛋白,是IBRV诊断的重要靶蛋白。为探究牛gB–ELISA抗体水平与VNT效价的相关性,本研究采用阻断ELISA和VNT两种不同的方法对某一牛场50份血清进行IBRV gB ELISA抗体检测和中和抗体测定。gB ELISA结果显示,阳性血清19份,阴性血清31份;中和抗体结果阳性17份,阴性33份。通过统计学相关系数分析,gB抗体阻断率和中和抗体效价值的相关系数为1.0,从而说明IBRV gB ELISA抗体和中和抗体呈较强的正相关。本研究为使用IBRV gB ELISA抗体水平评估牛群中IBRV抗体保护效果提供理论依据。     

传染性牛鼻气管炎病毒分离鉴定及特性分析

传染性牛鼻气管炎病毒(infectious bovine rhinotracheitis virus,IBRV)是牛的重要传染病病原,除能引起呼吸道疾病外,还可引起结膜炎、流产、脑炎和全身性感染。本试验从广东某牛场疑似IBRV感染牛中采取鼻拭子样品,用PCR和荧光PCR方法,初步诊断病牛感染IBRV。用MDBK细胞对初诊阳性病料进行病毒分离和鉴定,分离出1株传染性牛鼻气管炎病毒,命名为GD0109。细胞感染试验表明,该株病毒可以使MDBK细胞产生典型的圆缩、呈葡萄样聚集以及空洞等细胞病变。将GD0109与强毒的ATCCVR-2112TM株感染细胞裂解液分别接种MDBK细胞并盲传5代,对5代、10代、15代的病毒分别进行TCID50测定及中和试验,结果表明分离株和参考株VR-2112TM相似,提示该分离株为强毒株,并且具有良好的传代稳定性,为进一步疫苗生产打下良好的基础。     

The influence of complement on the neutralization of infectious bovine rhinotracheitis virus by globulins derived from early and late bovine antisera.

Calves were inoculated at four week intervals with infectious bovine rhinotracheitis virus (IBRV). Sera were obtained at eight days (early sample) and 21 days (late sample) after each inoculation. Kinetic neutralization was carried out with 7S and 19S globulins derived from these sera, both in the presence and in the absence of guinea pig complement (C). In all instances the 19S neutralizing antibody (AB) was dependent on C for neutralization of IBVR. However, C dependency was not observed with any of the 7S preparations. Neutralizing activity was readily detected in 19S globulins from the early sera after primary and subsequent inoculations but not in any of the late sera. Early sera collected after primary inoculation did not contain any 7S neutralizing AB but it was present in all the other sera tested. The 7S AB when present was always at a considerably higher concentration than 19S AB. Thus it may be possible to determine whether cattle have recently been exposed to IBRV when paired serum samples are not available by determining the presence of C-dependent 19S globulins. In addition, by comparing 19S and 7S levels, a distinction may be made between primary and secondary responses to IBRV.     

新疆某规模化奶牛场牛传染性鼻气管炎病毒抗体水平检测

为了调查新疆地区某规模化奶牛场牛传染性鼻气管炎（IBR）发病情况,通过采集不同生长阶段牛群血清共计362 份,使用牛传染性鼻气管炎病毒gB（IBR-gB）抗体检测试剂盒检测牛传染性鼻气管炎病毒（IBRV）抗体效价,评估该奶牛场IBRV疫苗免疫效果。结果显示,后备牛中犊牛和青年牛IBRV抗体阳性率分别为88.57%（31/35）、75.00%（21/28）;成年母牛中泌乳期母牛、干奶期母牛IBRV抗体阳性率分别为81.46%（145/178）、95.04%（115/121）。阴性数共44 份,可疑数8 份,IBRV抗体平均阳性率为88.38%;结果表明,疫苗接种后,不同生产阶段牛群均可产生不错的抗体保护效果,为奶牛场防控IBR提供依据。     

Effect of infectious bovine rhinotracheitis virus immunization on viral shedding in challenge-exposed calves treated with dexamethasone

Calves not vaccinated with infectious bovine rhinotracheitis virus (IBRV) became latently infected when challenge exposed and treated with dexamethasone (DM). Calves that shed IBRV after DM treatment were considered to be latently infected. Vaccination with a temperature-sensitive intranasal vaccine or with formalinized IBRV in Freund's complete adjuvant (IBRV-FCA) protected some, but not all, calves against latent infection--indicating a role for the immune response in preventing latent infection. That all latently infected calves were not detected after DM treatment was indicated by the fact that after a 2nd DM treatment of 3 calves treated 6 months previously and not found to shed virus, 1 of the calves was latently infected. Latently infected calves were inoculated with successive doses of IBRV-FCA and treated with DM. Nonvaccinated calves shed virus, whereas vaccinated calves similarly treated did not shed virus. Because both groups had a comparable cell-mediated immune response, as determined by blastogenic response to IBRV, but the vaccinated group had significantly higher virus-neutralizing antibody titers, a role for humoral antibody in preventing viral shedding was indicated.     

The effect of repeated treatment with dexamethasone on the re-excretion pattern of infectious bovine rhinotracheitis virus and humoral immune response

Two cattle, free of antibody to infectious bovine rhinotracheitis virus (IBRV) were infected intranasally with IBRV, and developed specific antibody to the virus. Ten weeks later, both animals were given an intravenous course of dexamethasone (DM). Nasal excretion of physical particles of virus, as judged by electron microscopy, occurred in both animals, as early as 24 h after the first DM injection, and high levels of infectious particles appeared several days later. Neutralizing antibody titre to IBRV increased following excretion of virus. Further courses of DM given at 20 and 32 weeks following initial infection were not associated with excretion of physical, ‘non-infectious’ particles or significant changes in specific antibody titre, although on each occasion one of the two animals excreted low levels of infectious particles.     

The incidence of antibody to infectious bovine rhinotracheitis virus in Kenyan cattle.

A total of 3204 cattle sera, collected between 1966 and 1974, from all seven provinces and 34 of the 42 districts of Kenya were screened in a neutralisation test for antibody to infectious bovine rhinotracheitis virus (IBRV). Antibody to IBRV was found in some sera from all the districts, with cattle over two years old showing the highest incidence. A small number of goat sera also showed some antibody. From the results obtained it is concluded that IBRV infection is widespread in Kenyan cattle.     

Comparative serological response in calves to eight commercial vaccines against infectious bovine rhinotracheitis, parainfluenza-3, bovine respiratory syncytial, and bovine viral diarrhea viruses

A field trial was conducted to compare the serological responses in calves to eight commercial vaccines against infectious bovine rhinotracheitis virus (IBRV), parainfluenza-3 virus (PI3V), bovine respiratory syncytial virus (BRSV), and/or bovine viral diarrhea virus (BVDV). Calves given IBRV, P13V, BRSV, and BVDV vaccines had significantly higher antibodies to these viruses than unvaccinated controls; however, serological responses to killed BVDV vaccines were low. Calves with preexisting antibodies to IBRV, PI3V, BRSV, and the Singer strain of BVDV had lower seroconversion rates following vaccination than calves that were seronegative initially.

Serological responses in calves to IBRV, PI3V, BRSV, and BVDV differed among various commercial vaccines. Antibody titers to IBRV were higher in calves vaccinated with modified-live IBRV vaccines than in those vaccinated with killed IBRV vaccines. Following double vaccination with modified-live IBRV and PI3V vaccines, seroconversion rates and antibody titers to IBRV and PI3V were higher in calves vaccinated intramuscularly than in those vaccinated intranasally. Calves given Cattlemaster 4 had significantly higher titers to BRSV and PI3V, and lower titers to BVDV, than calves given Cattlemaster 3, suggesting that the addition of BRSV to Cattlemaster 4 caused some interaction among antigens.

     

牛传染性鼻气管炎病毒gD基因的克隆与表达

构建pET32a重组表达载体,转化到BL21(DE3),诱导表达牛传染性鼻气管炎病毒重组gD蛋白。用已发表的IBRV的gD基因序列设计一对特异性引物,通过PCR方法扩增一条766bp的目的基因gD,将gD基因克隆到原核表达载体pet32a,得到重组表达载体pet32-gD,转化到BL21(DE)中,通过IPTG诱导获得融合蛋白。重组表达蛋白经纯化后,免疫印迹分析。结论证明表达的重组蛋白具有良好的免疫原性。