Comparison of five diagnostic methods for detecting bovine viral diarrhea virus infection in calves.

Five diagnostic techniques performed on skin biopsies (shoulder region) and/or serum were compared for detection of bovine viral diarrhea virus infection in 224 calves 0-3 months of age, 23 calves older than 3 months but younger than 7 months, and 11 cattle older than 7 months. The diagnostic methods used were immunohistochemistry (IHC), 2 commercial antigen ELISAs, 1 commercial antibody ELISA, and real-time RT-PCR. Results of 249 out of 258 skin and serum samples were identical and correlated within the 3 antigen detection methods and the real-time RT-PCR used. Twenty-six of these 249 samples were BVDV-positive with all antigen detection methods and the real-time RT-PCR. Nine out of 258 samples yielding discordant results were additionally examined by RT-PCR, RT-PCR Reamplification (ReA), and antigen ELISA I on serum and by immunohistochemistry on formalin fixed and paraffin-embedded skin biopsies. Virus isolation and genotyping was performed as well on these discordant samples. In 3 cases, transiently infected animals were identified. Two samples positive by real-time RT-PCR were interpreted as false positive and were ascribed to cross-contamination. The antigen ELISA II failed to detect 2 BVDV-positive calves due to the presence of maternal antibodies; the cause of 2 false-positive cases in this ELISA remained undetermined. Only persistently infected animals were identified in skin samples by IHC or antigen ELISA I. The 3 antigen detection methods and the real-time RT-PCR used in parallel had a high correlation rate (96.5%) and similar sensitivity and specificity values.     

进口奶牛病毒性腹泻病毒快速检测鉴定

采用ELISA方法对3 073头奶牛全血进行牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)检测,结果发现编号a889的血样呈阳性反应,另外有3份血样(a126、a803、b1277)呈可疑反应;进一步的白细胞抽提物ELISA反应表明,a889血样呈阳性反应,其余呈阴性反应。对a889血样进行RT-PCR扩增,得到1条310 bp大小的特异性条带,与预期片段大小吻合;经序列同源性比较分析,确定a889血样中存在牛病毒性腹泻病毒。     

An immunodiffusion test for detection of bovine viral diarrhea virus antibodies in bovine serum.

An immunodiffusion test (IDT) was developed for detecting bovine viral diarrhea virus antibodies in bovine serum. The antigen utilized in the IDT was prepared from bovine viral diarrhea virus-infected monolayer cultures. Results of the IDT were obtained within 48 hours and correlated with the virus-neutralization test.     

Methods for detection and frequency of contamination of fetal calf serum with bovine viral diarrhea virus and antibodies against bovine viral diarrhea virus.

Methods used by the National Animal Disease Center to test fetal calf serum for contamination with bovine viral diarrhea virus (BVDV) and antibodies against BVDV are described. Using those methods, virus was isolated from 332 of 1,608 (20.6%) lots of raw fetal calf serum obtained specifically for the Center and 93 of 190 (49%) lots of commercially available fetal calf serum. Virus neutralization and immunoperoxidase staining tests were used to detect antibodies against BVDV in 224 of the 1,608 (13.9%) lots of raw fetal calf serum. Both BVDV and antibodies against BVDV were detected in 50 lots of raw serum. The molecular specificity of antibodies against BVDV was determined by radioimmunoprecipitation. Lots of fetal calf serum that contained BVDV-specific antibodies that did not neutralize virus were identified.     

Evolution of bovine viral diarrhea virus vaccines.

Control of bovine viral diarrhea virus (BVDV) infection is economically important to the cattle industry because the virus causes a variety of clinical diseases that adversely affect essentially all stages of the production cycle. Production losses primarily stem from reproductive failure and from immunosuppression during acute BVDV infection, which predisposes calves to respiratory or enteric diseases. Control is achieved by implementing herd health pro-grams focused on limiting exposure by avoiding persistently infected (PI) carrier cattle and by optimizing protective immunity through immunization. Vaccination cannot be relied upon solely to protect against fetal infection and losses due to BVD. This is because no single BVDV vaccine has been shown to give complete fetal protection. In addition to strategic use of vaccines, herd management practices should also be implemented to identify and eliminate PI carrier cattle and to avoid exposure to BVDV infection.     

Structural proteins of bovine viral diarrhea virus.

A procedure for the purification of radioactively labeled bovine viral diarrhea virus was critically evaluated. Purification of virus from artificial mixtures of unlabeled infected and labeled noninfected cells indicated that the extent of purification was approximately 100-fold with respect to host proteins. Residual host proteins were found to contaminate the viral preparation even after extensive purification by differential and isopycnic zonal centrifugation. Co-electrophoresis of 3H-labeled virus with 14C-labeled host cell material in neutral sodium dodecyl sulfate-7.5% polyacrylamide gels provided a means to distinguish viral specific proteins from host cell protein contaminants. Four major electrophoretic components were identified as being of viral origin; molecular weights of the components were estimated from their migration rates relative to protein markers of known molecular weight. Two viral components (VC), VC 1 and VC 3, migrated heterogeneously and had molecular weights of 93,000 to 110,000 and 50,000 to 59,000 daltons, respectively. Molecular weights of VC 2 and VC 4 were 70,000 and 25,000 daltons, respectively.     

Enzyme-linked immunosorbent assay for the detection of antibodies to bovine viral diarrhea virus in bovine sera

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was established for the detection of antibodies to bovine viral diarrhea virus (BVDV) in bovine sera. Polyethylene-glycol concentrated, equilibrium density gradient purified BVDV was used as test antigen at an optimal amount of 1 microgram/well, whereas the optimal concentration of conjugate was at 1/2000 dilution. The standardized test encountered no non-specific reaction with test sera at a starting dilution of 1/10. A total of 50 bovine serum samples was assayed for the presence of antibodies against BVDV by ELISA and serum neutralization test (SNT). A positive correlation between the 2 tests was found. However, ELISA could be as much as 500-fold more sensitive than SNT in detecting low levels of BVDV antibodies.     

牛病毒性腹泻病病毒荧光定量PCR检测体系的建立与评价

基于实时荧光定量PCR技术建立了一种有效地检测牛病毒性腹泻病病毒(Bovine viral diarrhea virus,BVDV)核酸的方法.对BVDV基因组进行同源比对,选取5'UTR区作为扩增目的区,经软件分析后设计特异扩增引物,扩增片段长度为203 bp.选用SYBR染料作为扩增时信号指示剂,经扩增曲线分析表明,建立的方法可有效地检测BVDV.检测体系可检测到10~2 copies/μL的样品拷贝数.故本研究建立的BVDV实时定量检测体系可用于易感动物,牛源血液生物制品及其他可能感染或污染BVDV样品的检测.     

基因2型牛病毒性腹泻病毒RT-PCR检测方法的建立和初步应用

根据已发表的牛病毒性腹泻病毒基因1型(BVDV-1)和2型(BVDV-2)5非编码区(5-UTR)的序列,分别设计了针对BVDV-1型、BVDV-2型以及针对BVDV-1和BVDV-2两型的特异性引物和通用引物,以标准参考株BVDV-1 NADL株和BVDV-2890 株为对照,建立了分别能检测BVDV-1、BVDV-2和BVDV-1/BVDV-2的RT-PCR 检测方法.在此基础上,进一步对疑似BVDV-2感染牛的临床病料组织(脾、淋巴结、心肌、血液等)检测结果表明,在所检测的18份样品中,BVDV-2阳性8份(44%).经RT-PCR鉴定为阳性的组织病料,进一步通过MDBK 细胞进行病毒分离,病毒分离率为100%;结合感染细胞病变观察,间接免疫荧光试验RT-PCR和序列测定鉴定,所分离的病毒均为BVDV-2.上述研究表明,该RT-PCR检测方法敏感、特异;并证实我国牛群已存在BVDV-2的污染或感染.     

Response of cattle persistently infected with noncytopathic bovine viral diarrhea virus to vaccination for bovine viral diarrhea and to subsequent challenge exposure with cytopathic bovine viral diarrhea virus

Nine steers persistently infected with noncytopathic bovine viral diarrhea (BVD) virus were allotted into 3 groups (3 cattle/group). Cattle in group A were vaccinated with a modified-live BVD virus vaccine of porcine cell origin, cattle in group B with a modified-live BVD virus vaccine of bovine cell origin, and cattle in group C with a killed BVD virus vaccine of bovine cell origin. Detrimental effects due to vaccination were not seen. Six weeks after vaccination, the steers were challenge exposed with a cytopathic BVD virus. All steers developed mucosal disease after challenge exposure, produced antibodies that neutralized various isolates of BVD virus, and remained persistently infected until death. Steers given killed virus vaccine had a minimal neutralizing-antibody response and developed mucosal disease as quickly as reported for challenge-exposed, nonvaccinated, persistently infected cattle. Steers given modified-live virus vaccines had higher neutralizing-antibody response and longer intervals from challenge exposure to development of mucosal disease. The specificity of the neutralizing-antibody response differed between groups of vaccinated cattle.     

Laboratory diagnosis of bovine viral diarrhea virus infections.

The past 20 years have witnessed dramatic improvements in laboratory methods for diagnosing bovine viral diarrhea virus(BVDV) infections. However, improvements in diagnostic technology have not necessarily led to improved diagnosis of BVDV at the individual animal or herd level. This article reviews BVDV laboratory diagnostic methods in the context of their rational application for improved detection of BVDV in the field.     

The many faces of bovine viral diarrhea virus.

The complex and unique nature of bovine viral diarrhea virus(BVDV) continues to present challenges to infectious disease re-searchers, veterinarians, and the cattle industry. In addition, the BVDV pathogen will undoubtedly continue to change and present itself in many different configurations.     

Effect of bovine viral diarrhea virus in the feedlot.

It could be argued that bovine viral diarrhea virus (BVDV) is one of the most economically significant infectious pathogens of feedlot cattle. Although the direct economic losses caused by this virus have not been well quantified, the role it plays as an immunosuppressive agent and as a potentiator for other diseases, most notably bovine respiratory disease, have been well documented. It is also a difficult disease for the feedlot veterinarian to control effectively.Individual cattle persistently infected with BVDV often serve as the source of infectious virus within a group of feedlot cattle, and the ultimate responsibility for preventing persistent infections in cattle rests with the cow-calf producer and not with the feedlot owner. The enormous impact of the virus on the livestock industry has led the Academy of Veterinary Consultants to draft a position statement that resolves that the beef and dairy industries adopt measures to control and target eventual eradication of BVDV from North America.     

Biosecurity and biocontainment of bovine viral diarrhea virus.

Infection of cattle with BVDV results in a variety of clinical illnesses costly to the cattle industry worldwide. The reservoir and primary source of transmission is cattle born PI with BVDV after transplacental infection in early gestation. It is a challenge to determine with certainty whether or not BVDV is circulating among a herd of cattle. If the virus is present in a herd,then biocontainment strategies are used to eliminate the virus by testing to removing PI cattle, preventing exposure of pregnant cattle to the virus, and increasing resistance to infection using vaccination. If it is clear that the virus is not present in a herd then, biosecurity actions must be taken to prevent introducing the virus into the herd.     

Monoclonal antibody-based immunohistochemical detection of bovine viral diarrhea virus in formalin-fixed, paraffin-embedded tissues.

Thirty-two monoclonal antibodies directed against epitopes on bovine viral diarrhea virus proteins and glycoproteins were tested for immunohistochemical reactivity with bovine viral diarrhea virus in formalin-fixed and paraffin-embedded tissues from 45 cases of bovine viral diarrhea virus-associated mucosal disease. Only one antibody, designated 15C5, which reacts with the 48-kD glycoprotein of bovine viral diarrhea virus, detected an epitope preserved in these specimens. Monoclonal antibody 15C5 and a polyclonal antibody to bovine viral diarrhea virus successfully detected bovine viral diarrhea viral antigens in 44/45 cases of mucosal disease and did not react with formalin-fixed tissues from 30 uninfected cattle. Monoclonal antibody-based immunohistochemical detection of bovine viral diarrhea virus is routinely fixed tissue specimens has advantages over other currently available techniques in terms of the convenience of specimen submission, the relative ease of method standardization, and the rapidity of the test, and by enabling identification of the virus in association with specific tissues, cell types, and histologic lesions.     

Anti-idiotypic antibodies mimic bovine viral diarrhea virus antigen.

Polyclonal rabbit anti-idiotypic antibodies (anti-ids) against two neutralizing murine monoclonal antibodies (mAbs) specific to a bovine viral diarrhea virus (BVDV) glycoprotein, 53 kDa, were produced, purified, and characterized. Each anti-id inhibited the binding of its respective mAb to BVDV antigen in a competitive ELISA and blocked the immunoprecipitation of the 53 kDa protein by the mAb. The anti-ids also inhibited the virus-neutralizing activity of their homologous mAbs. These results suggest that the anti-ids bear an internal image of a BVDV antigen and mimic neutralizing epitopes on the 53 kDa protein. Treatment of MDBK cells with the anti-ids inhibited BVDV infection, indicating that they block a cellular component, such as a virus receptor, required for virus adsorption or entry. Inhibition of the homologous mAb and lack of inhibition of the heterologous mAb indicate that the anti-ids are specific for the unique antigen-binding sites on the mAbs.