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。     

Evaluation of the rabbit as a laboratory model for infectious bovine rhinotracheitis virus infection

Experimental infection of rabbits with infectious bovine rhinotracheitis virus (IBRV) produced diverse manifestations of disease which included abortion, conjunctivitis, dermatitis, vulvovaginitis, systemic infection, neonatal death and respiratory tract infection. Each disease syndrome was studied using virus isolation, fluorescent antibody examination and histologic examination. Conjunctivitis, dermatitis and vulvovaginitis lesions were characterized by edema, infiltration of mucosa and submucosa with inflammatory cells and ulceration of epithelium. Systemic infection resulted in severe necrosis of liver and adrenal glands with large numbers of cells containing intranuclear inculsions. Pregnant rabbits aborted within 48 hours following inoculation of IBRV. Virus infection and viral lesions were not demonstrated in aborted fetuses.     

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%.     

Enhancement of infectious bovine keratoconjunctivitis by modified-live infectious bovine rhinotracheitis virus vaccine

The effects of a modified-live infectious bovine rhinotracheitis virus vaccine (administered ocularly or intranasally) on experimentally induced infectious bovine keratoconjunctivitis were evaluated. The modified-live infectious bovine rhinotracheitis virus vaccine was administered to 13 male Holstein calves (intranasally in 4 and ocularly in 9; day 0). Five calves were not vaccinated and served as controls. Calves were examined daily and, starting on day 4, Moraxella bovis was administered ocularly to all 18 calves once daily for 4 days. The eyes of all calves were assigned a clinical score, and the ocular secretions were evaluated for presence of infectious bovine rhinotracheitis virus and M bovis daily until day 19. The severity of the ocular lesions was estimated by scoring the lesions clinically and by determining the protein concentration, myeloperoxidase activity, and WBC count in the tears. By day 5, conjunctivitis, chemosis, and epiphora were observed in all of the calves vaccinated ocularly. The calves vaccinated intranasally developed conjunctival plaques, but did not develop chemosis or photophobia. All of the calves developed keratitis after inoculation with M bovis. The median lesion scores were greater in both groups of vaccinated calves than in the controls. Corneal perforations developed exclusively in the vaccinated calves. The frequency of M bovis isolation from ocular secretions was significantly (P less than 0.05) greater in the vaccinated calves than in the controls. The tears from the intranasally vaccinated calves contained the highest myeloperoxidase activity and WBC count. The mean protein concentration in the tears of vaccinated calves was not significantly different from that in tears of controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reactivation of infectious bovine rhinotracheitis virus by transport

Transport was studied as a cause of reactivation of infectious bovine rhinotracheitis virus (Bovine herpesvirus-1; BHV-1) in heifers vaccinated 2-6 months before transport, using a double dose of the thermosensitive (ts) vaccine strain (Tracherine). Eight out of 19 animals showed ts strain re-excretion over a period of 1-3 days, beginning, in 5 out of the 8 heifers, the day after transport. In 14 other heifers, only sera were examined by sero-neutralisation: only 1 out of these 14 animals showed a rise in BHV-1 neutralising antibodies. Transport can therefore be considered as a stimulus of BHV-1 reactivation.     

Isolation of infectious bovine rhinotracheitis virus in Tanzania

Summary Infectious bovine rhinotracheitis (IBR) virus was identified for the first time in Tanzania. The virus isolations were made from cattle affected with respiratory diseases. Concurrent infection with foot-and-mouth disease was observed and enhanced the severity of the illness.

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Sumario El virus de la rinotraqueitis infecciosa de los bovinos (I.B.R.) ha sido identificado por primera vez en Tanzania. Los aislamientos de virus fueron hechos de bovinos afectados con enfermedades respiratorias. Se observó infección concurrente con fiebre aftosa y este hecho exacerbaba la severidad de la enfermedad.

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Résumé Ce virus a été isolé pour la première fois en Tanzanie, à partir de bétail atteint d'affections respiratoires. Cette maladie a sévi concurremment avec la fièvre aphteuse, ce qui explique sa sévérité.

     

Microimmunodiffusion test for detection of antibodies to infectious bovine rhinotracheitis virus in bovine serum.

A microimmunodiffusion test (MIDT) specific for detection of antibodies to infectious bovine rhinotracheitis (IBR) in bovine serum has been developed. The antigen used in the MIDT was prepared from IBR virus-infected Madin-Darby bovine kidney cells grown in tissue culture. The antigen was stable, and relatively high yields were obtained readily. Results of the MIDT were obtained within 48 hours and agreed with those of the serum-neutralization test.     

Survival of infectious bovine rhinotracheitis virus in stored bovine semen

No loss in the titre of infectious bovine rhinotracheitis virus was found during storage in semen at –196°C for 1 year.     

Evaluation of inactivated infectious bovine rhinotracheitis vaccines.

Sixty-five calves of approximately three months of age and of mixed sex were vaccinated twice at four week intervals with either attenuated or inactivated infectious bovine rhinotracheitis vaccines. Following initial vaccination there was no demonstrable serum infectious bovine rhinotracheitis titer in any of the calves receiving the inactivated vaccine with 20.7% of the calves receiving the attenuated vaccines having demonstrable titers. Following a second administration of vaccine at eight weeks post-initial vaccination 63.9% of the calves receiving the inactivated vaccine had no demonstrable titer with 72.4% of the calves receiving the attenuated vaccine exhibiting a blood titer of four or greater.     

Enhancement of infectious bovine rhinotracheitis virus replication with corticosterone

Infectious bovine rhinotrachetis virus (IBRV) progeny was increased ten to 12 fold in bovine turbinate (BT) cells treated with 10^?3 M corticosterone acetate (CCA) as demonstrated by plaque assay. Autoradiographic studies demonstrated an increased binding of ³H-corticosterone (³H-CS) in IBRV infected cells and the fractionation of labelled cells revealed 78–80% of the total hormone associated with the cytoplasmic components. Incorporation of ³H-uridine and ³H-valine precursors into cells treated with the hormone demonstrated up to 16-fold increase in RNA and protein synthesis which was inhibited by the addition of actinomycin D. The data suggest that increased rate of macromolecular synthesis in IBRV infected cells treated with the corticosteroid may result in the enhancement of virus production.