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Flow cytometric detection of bovine viral diarrhea virus in peripheral blood leukocytes of persistently infected cattle. 总被引：3，自引：0，他引：3 
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下载免费PDF全文 P Qvist B Aasted B Bloch A Meyling L Rnsholt H Houe 《Canadian journal of veterinary research》1990,54(4):469-472
Flow cytometry was investigated for detection of bovine viral diarrhea virus (BVDV) in peripheral blood mononuclear leukocytes of persistently infected cattle. The mononuclear leukocytes were purified by sedimentation in a gradient of Ficoll-Paque, fixed, permeabilized, and then labelled by indirect immunofluorescence using biotinylated immunoglobulins from a porcine antiserum to BVDV. Flow cytometric analysis of blood samples obtained from persistently infected cattle revealed virus in 3.0-21.0% (mean +/- SD, 11.2% +/- 6.4%) of the mononuclear leukocytes. Fluorescent cells were not observed in controls. Flow cytometric detection of BVDV in blood cells of persistently infected bovines is a rapid and objective technique which does not require cell culture facilities. 相似文献
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Transmission of Bovine viral diarrhea virus 1b to susceptible and vaccinated calves by exposure to persistently infected calves 下载免费PDF全文
下载免费PDF全文 Robert W. Fulton Robert E. Briggs Julia F. Ridpath Jeremiah T. Saliki Anthony W. Confer Mark E. Payton Glenn C. Duff D.L. Step D.A. Walker 《Canadian journal of veterinary research》2005,69(3):161-169
Bovine viral diarrhea virus (BVDV) persistently infected (PI) calves represent significant sources of infection to susceptible cattle. The objectives of this study were to determine if PI calves transmitted infection to vaccinated and unvaccinated calves, to determine if BVDV vaccine strains could be differentiated from the PI field strains by subtyping molecular techniques, and if there were different rates of recovery from peripheral blood leukocytes (PBL) versus serums for acutely infected calves. Calves PI with BVDV1b were placed in pens with nonvaccinated and vaccinated calves for 35 d. Peripheral blood leukocytes, serums, and nasal swabs were collected for viral isolation and serology. In addition, transmission of Bovine herpes virus 1 (BHV-1), Parainfluenza-3 virus (PI-3V), and Bovine respiratory syncytial virus (BRSV) was monitored during the 35 d observation period. Bovine viral diarrhea virus subtype 1b was transmitted to both vaccinated and nonvaccinated calves, including BVDV1b seronegative and seropositive calves, after exposure to PI calves. There was evidence of transmission by viral isolation from PBL, nasal swabs, or both, and seroconversions to BVDV1b. For the unvaccinated calves, 83.2% seroconverted to BVDV1b. The high level of transmission by PI calves is illustrated by seroconversion rates of nonvaccinated calves in individual pens: 70% to 100% seroconversion to the BVDV1b. Bovine viral diarrhea virus was isolated from 45 out of 202 calves in this study. These included BVDV1b in ranch and order buyer (OB) calves, plus BVDV strains identified as vaccinal strains that were in modified live virus (MLV) vaccines given to half the OB calves 3 d prior to the study. The BVDV1b isolates in exposed calves were detected between collection days 7 and 21 after exposure to PI calves. Bovine viral diarrhea virus was recovered more frequently from PBL than serum in acutely infected calves. Bovine viral diarrhea virus was also isolated from the lungs of 2 of 7 calves that were dying with pulmonary lesions. Two of the calves dying with pneumonic lesions in the study had been BVDV1b viremic prior to death. Bovine viral diarrhea virus 1b was isolated from both calves that received the killed or MLV vaccines. There were cytopathic (CP) strains isolated from MLV vaccinated calves during the same time frame as the BVDV1b isolations. These viruses were typed by polymerase chain reaction (PCR) and genetic sequencing, and most CP were confirmed as vaccinal origin. A BVDV2 NCP strain was found in only 1 OB calf, on multiple collections, and the calf seroconverted to BVDV2. This virus was not identical to the BVDV2 CP 296 vaccine strain. The use of subtyping is required to differentiate vaccinal strains from the field strains. This study detected 2 different vaccine strains, the BVDV1b in PI calves and infected contact calves, and a heterologous BVDV2 subtype brought in as an acutely infected calf. The MLV vaccination, with BVDV1a and BVDV2 components, administered 3 d prior to exposure to PI calves did not protect 100% against BVDV1b viremias or nasal shedding. There were other agents associated with the bovine respiratory disease signs and lesions in this study including Mannheimia haemolytica, Mycoplasma spp., PI-3V, BRSV, and BHV-1. 相似文献
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Julia F Ridpath Sharon K Hietala Steve Sorden John D Neill 《Journal of veterinary diagnostic investigation》2002,14(4):303-307
Bovine viral diarrhea viruses (BVDV) cause both acute and persistent infections. While diagnostic tests have been designed to detect animals persistently infected (PI) with BVDV, the reliability of these tests in detecting acute BVDV infections is not known. It is also possible that acute BVDV infections may be confused with persistent infections in surveys for PI animals. In this study, 2 tests presently in use in diagnostic laboratories to test for PI animals, polymerase chain reaction amplification followed by probe hybridization (RT-PCR/probe) of serum samples and immunohistochemical detection of viral antigen in skin biopsies (IHC), were evaluated for their ability to detect acute BVDV infections. Sixteen colostrum-deprived, BVDV-free, and BVDV-antibody-free calves were infected with 6 different BVDV strains. Clinical signs, seroconversion, and virus isolation indicated that inoculated animals did replicate virus. Virus could be detected in 19% (3/16) of acutely infected animals by the RT-PCR/probe technique. No acutely infected animals were positive by IHC. 相似文献
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Bovine viral diarrhea virus (BVDV) 1b: predominant BVDV subtype in calves with respiratory disease 总被引：1，自引：1，他引：0 下载免费PDF全文
下载免费PDF全文 Robert W. Fulton Julia F. Ridpath Jeremiah T. Saliki Robert E. Briggs Anthony W. Confer Lurinda J. Burge C. W. Purdy Raymond W. Loan Glenn C. Duff Mark E. Payton 《Canadian journal of veterinary research》2002,66(3):181-190
The prevalence of bovine viral diarrhea virus (BVDV) infections was determined in 2 groups of stocker calves with acute respiratory disease. Both studies used calves assembled after purchase from auction markets by an order buyer and transported to feedyards, where they were held for approximately 30 d. In 1 study, the calves were mixed with fresh ranch calves from a single ranch. During the studies, at day 0 and at weekly intervals, blood was collected for viral antibody testing and virus isolation from peripheral blood leukocytes (PBLs), and nasal swabs were taken for virus isolation. Samples from sick calves were also collected. Serum was tested for antibodies to bovine herpesvirus-1 (BHV-1), BVDV1a, 1b, and 2, parainfluenza 3 virus (PI3V), and bovine respiratory syncytial virus (BRSV). The lungs from the calves that died during the studies were examined histopathologically, and viral and bacterial isolation was performed on lung homogenates. BVDV was isolated from calves in both studies; the predominant biotype was noncytopathic (NCP). Differential polymerase chain reaction (PCR) and nucleic acid sequencing showed the predominant subtype to be BVDV1b in both studies. In 1999, NCP BVDV1b was detected in numerous samples over time from 1 persistently infected calf; the calf did not seroconvert to BVDV1a or BVDV2. In both studies, BVDV was isolated from the serum, PBLs, and nasal swabs of the calves, and in the 1999 study, it was isolated from lung tissue at necropsy. BVDV was demonstrated serologically and by virus isolation to be a contributing factor in respiratory disease. It was isolated more frequently from sick calves than healthy calves, by both pen and total number of calves. BVDV1a and BVDV2 seroconversions were related to sickness in selected pens and total number of calves. In the 1999 study, BVDV-infected calves were treated longer than noninfected calves (5.643 vs 4.639 d; P = 0.0902). There was a limited number of BVDV1a isolates and, with BVDV1b used in the virus neutralization test for antibodies in seroconverting calves' serum, BVDV1b titers were higher than BVDV1a titers. This study indicates that BVDV1 strains are involved in acute respiratory disease of calves with pneumonic Mannheimia haemolytica and Pasteurella multocida disease. The BVDV2 antibodies may be due to cross-reactions, as typing of the BVDV strains revealed BVDV1b or 1a but not BVDV2. The BVDV1b subtype has considerable implications, as, with 1 exception, all vaccines licensed in the United States contain BVDV1a, a strain with different antigenic properties. BVDV1b potentially could infect BVDV1a-vaccinated calves. 相似文献
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B J Hooft van Iddekinge J L van Wamel H G van Gennip R J Moormann 《Veterinary microbiology》1992,30(1):21-34
We used the polymerase chain reaction (PCR) technique to detect bovine viral diarrhea virus (BVDV) infections in cattle. Of 120 cattle screened in this study, 29 were scored positive for BVDV with both PCR and conventional virus isolation. Ninety cattle were negative in both assays. One cow was scored positive for BVDV with the PCR but was negative with virus isolation. In dilution experiments PCR analysis was at least 10 times more sensitive than BVDV isolation. 相似文献
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K L Roberts J K Collins J Carman C D Blair 《Journal of veterinary diagnostic investigation》1991,3(1):10-15
A ribonucleic acid (RNA) hybridization assay to identify cattle infected by bovine viral diarrhea virus (BVDV) is described. The RNA probe was derived from the coding region at the 3' end of the genome of the NADL strain of BVDV. Total RNA from infected cell cultures or peripheral blood leukocytes from suspect animals was extracted and applied to nylon membranes with a slot blot apparatus. Peripheral blood leukocytes were tested concurrently for BVDV by virus isolation. The results of hybridization and virus isolation were in agreement for 92% of the cases. When compared with virus isolation, hybridization had a sensitivity of detection of 59.5% and a specificity of 95%. Cross-reactivity to RNA extracts of border disease virus-infected cells was noted. No cross-reactivity was detected to other common bovine viruses (bovine herpesvirus-1, bovine respiratory syncytial virus, parainfluenza-3 virus, and bluetongue virus), to viruses classified in related families (equine arteritis virus and Venezuelan equine encephalitis virus), or to viruses having similar genomic organization (dengue virus type 2 and Japanese encephalitis virus). 相似文献
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牛病毒性腹泻病病毒荧光定量PCR检测体系的建立与评价 总被引：2，自引：0，他引：2
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. 相似文献
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L L Bissey A K Williams S Bolin E W Collisson 《Journal of veterinary diagnostic investigation》1991,3(1):16-21
The molecular technique of RNA fingerprinting was used to characterize the genomes of 5 isolates of bovine viral diarrhea virus (BVDV): 2 viral pairs from the same animal, BVD-ILN/BVD-ILC and BVD-TGAN/BVD-TGAC, and the cytopathic viral prototype, BVD-NADL. Oligonucleotide patterns from the viruses were compared, and unique and overlapping oligonucleotides were identified. A comparison of the fingerprints indicated that the genome of each virus was distinguishable by the T1 RNase oligonucleotide fingerprinting technique. The greatest similarity observed was between oligonucleotides from BVD-ILC and BVD-ILN. Eighteen large oligonucleotides were conserved in all 5 BVDV isolates studied. We found that within a pair of BVDV, the cytopathic fingerprint was different from the noncytopathic fingerprint, indicating that cytopathic and noncytopathic BVDV may be distinct viruses. 相似文献
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Stokstad M Niskanen R Lindberg A Thorén P Belák S Alenius S Løken T 《Journal of veterinary medicine. B, Infectious diseases and veterinary public health》2003,50(9):424-429
Nineteen pregnant cows were experimentally infected with bovine viral diarrhoea virus (BVDV) between day 74 and 81 of pregnancy. All cows became infected and developed serum antibodies. Sixteen of the cows delivered persistently infected (PI) offspring, whereas the remaining three gave birth to calves with detectable serum antibodies and free from BVDV. The 16 cows with PI foetuses developed higher levels of antibodies in serum during pregnancy than did their three peers carrying non-PI calves. Multivariate analysis showed that the antibody levels in these two groups of cows were significantly different from day 135 of pregnancy. Foetal fluid was successfully collected from 18 of the 19 infected cows and from five uninfected control cows between 10 and 24 days before delivery by use of a percutaneous, blind puncture technique. No negative effects were observed in the cows or their offspring. BVDV was isolated and detected with an immunoperoxidase test in foetal fluid from 13 of the 16 cows carrying PI foetuses, and from 15 of the cows when a quantitative fluorescent polymerase chain reaction (PCR) technique was used. The negative sample in the PCR assay was positive for BVDV antibodies. The number of viral copies per microlitre in foetal fluids varied between 103 and 1080 in the positive samples. All samples taken from the cows carrying non-PI foetuses were negative for BVDV in both assays. In this experiment, examination of either serum or foetal fluids could identify the cows carrying a PI foetus. Examination of serum for BVDV antibodies was a reliable indicator of a PI foetus if the serum was collected during the last 2 months of pregnancy. For examination of foetal fluids, both viral and serological analyses should be performed. For viral analysis, PCR should be the test of choice. High levels of BVDV antibodies in conjunction with a negative result in the PCR may be indicative of a false-negative virus result. Further experience with the method of collection of foetal fluids is necessary for evaluation of its safety. Investigation of pregnant cows in order to discover a PI offspring before it is born could be a useful tool in control and eradication of BVDV. 相似文献
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AIM: To assess the ability of two commercial bovine viral diarrhoea (BVD) virus (BVDV) antigen-capture enzyme-linked immunosorbent assays (ELISAs) to detect virus in serum and skin biopsies. METHODS: Thirty cattle persistently infected (PI) with BVDV were identified using routine diagnostic laboratory testing. Additional ear-notch skin biopsies and blood samples were collected from these animals to confirm the diagnosis, and from 246 cohorts, to determine their BVDV status. Skin biopsies were soaked overnight in buffer and the eluate collected. All sera and eluate were tested using two commercially available ELISAs for detecting BVDV antigen, and a subsample of positive and negative sera was tested using a polymerase chain reaction (PCR) test. A study was also performed to ascertain the risk of cross contamination occurring during the collection and processing of skin biopsies. RESULTS: Both serum and skin samples tested using either ELISA resulted in the detection of all cattle identified as PI and no non-infected cattle were incorrectly classified as infected using either method. Agreement between all assays (ELISAs, whether performed on serum or skin, and PCR) was 100%. No cross-contamination of skin samples between animals was evident using routine biopsy methods. CONCLUSIONS: Viraemic cattle infected with BVDV were accurately identified using either of the two commercial ELISAs evaluated on either serum or skin samples. CLINICAL RELEVANCE: Either skin biopsies or serum samples can be collected from cattle to determine their BVDV status. This should overcome problems in accurately identifying the infection status of young calves in which colostral antibodies might interfere with the antigen-capture ELISA. 相似文献
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M. Stokstad R. Niskanen A. Lindberg P. Thorn S. Belk S. Alenius T. Lken 《Zoonoses and public health》2003,50(9):424-429
Nineteen pregnant cows were experimentally infected with bovine viral diarrhoea virus (BVDV) between day 74 and 81 of pregnancy. All cows became infected and developed serum antibodies. Sixteen of the cows delivered persistently infected (PI) offspring, whereas the remaining three gave birth to calves with detectable serum antibodies and free from BVDV. The 16 cows with PI foetuses developed higher levels of antibodies in serum during pregnancy than did their three peers carrying non‐PI calves. Multivariate analysis showed that the antibody levels in these two groups of cows were significantly different from day 135 of pregnancy. Foetal fluid was successfully collected from 18 of the 19 infected cows and from five uninfected control cows between 10 and 24 days before delivery by use of a percutaneous, blind puncture technique. No negative effects were observed in the cows or their offspring. BVDV was isolated and detected with an immunoperoxidase test in foetal fluid from 13 of the 16 cows carrying PI foetuses, and from 15 of the cows when a quantitative fluorescent polymerase chain reaction (PCR) technique was used. The negative sample in the PCR assay was positive for BVDV antibodies. The number of viral copies per microlitre in foetal fluids varied between 103 and 1080 in the positive samples. All samples taken from the cows carrying non‐PI foetuses were negative for BVDV in both assays. In this experiment, examination of either serum or foetal fluids could identify the cows carrying a PI foetus. Examination of serum for BVDV antibodies was a reliable indicator of a PI foetus if the serum was collected during the last 2 months of pregnancy. For examination of foetal fluids, both viral and serological analyses should be performed. For viral analysis, PCR should be the test of choice. High levels of BVDV antibodies in conjunction with a negative result in the PCR may be indicative of a false‐negative virus result. Further experience with the method of collection of foetal fluids is necessary for evaluation of its safety. Investigation of pregnant cows in order to discover a PI offspring before it is born could be a useful tool in control and eradication of BVDV. 相似文献