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.     

The prevalence of bovine viral diarrhea virus infection in a population of feedlot calves in western Canada.

The prevalence of bovine viral diarrhea virus (BVDV) infection was examined in a population of 5129 recently weaned steer calves entering a large feedlot in central Saskatchewan from September to December 1991. Serum samples were collected within 24 h of arrival at the feedlot from every fifth calf processed and again 96 d postarrival. A microtiter virus isolation test was used to determine the prevalence of calves viremic with BVDV on entry to the feedlot. An enzyme-linked immunosorbent assay (ELISA) which detects antibody against glycoprotein 53 of the BVDV was used on paired sera to determine the seroconversion risk during the first 96 d in the feedlot. A virus neutralization (VN) test for BVDV was conducted on a sub-sample of paired sera to measure agreement in determination of seroconversion risk with the ELISA. A polymerase chain reaction (PCR) test which detects BVDV was used to determine if cattle were acutely viremic when treated for disease. The estimated prevalence of persistently infected calves in this population was < 0.1%. The seroconversion risk for BVDV was 27% (236/864) according to the ELISA and it varied from 0 to 63% among the 20 pens sampled. According to the VN test, the seroconversion risk for BVDV was 40% (132/327) and it varied from 0 to 100% among the 11 pens tested. The agreement between the ELISA and VN tests in seroconversion risk to BVDV was very poor (kappa = 0.15 +/- 0.039 SE). The prevalence of acute viremia in calves treated at the feedlot hospital was low at 4% (6/149).(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional analysis of antibody responses of feedlot cattle to bovine respiratory syncytial virus following vaccination with mixed vaccines.

The antibody response of cattle to bovine respiratory syncytial virus (BRSV) immunization was investigated using 4 different commercially available mixed vaccines. Forty, 5-6 month old, beef calves, randomly assigned to groups of 10, were vaccinated on day 0 and 21 with 1 of 3 inactivated vaccines, (3 groups), or a modified live virus (MLV) vaccine. BRSV-specific antibody responses were measured prior to vaccination and on day 35 by using an enzyme linked immunosorbent assay (ELISA), virus neutralization assay (VN), a fusion inhibition assay (FI); and responses were also measured for their ability to facilitate antibody dependent, complement mediated cytotoxicity (ADCMC) of BRSV infected cells. Sera from day 35 were, in addition, analyzed by use of an IgG1, IgG2 isotype specific ELISA. All vaccines induced significant increases in BRSV specific IgG antibody as measured by ELISA, but only one inactivated and the MLV vaccine induced significant increases in VN titers. Fusion inhibiting antibody titers were low or undetected in calves vaccinated with the inactivated vaccines. Vaccination with modified live virus induced significantly higher titers of fusion inhibiting antibodies, which are considered to be most highly correlated with protection. The VN to ELISA and FI to ELISA ratio of the calves that received MLV vaccine were significantly greater than the calves receiving the 3 inactivated vaccines. Vaccination with MLV induced the highest IgG2/IgG1 ratio. This difference was small, and only significant relative to 2 of the inactivated vaccine groups, which were not significantly different from each other. The higher proportion of IgG2 isotype in the MLV sera was not associated with lower ADCMC, a function not attributed to this isotype. The VN and FI titers, but not the ELISA value of the sera, were most predictive of ADCMC. The inactivation processes apparently alter epitopes and affect the induction of functional antibodies.     

Field application of the combination of neutralizing test and virus isolation, so-called spot test, to detect herds including cattle persistently infected with bovine viral diarrhea virus

To detect herds including cattle persistently infected (PI) with bovine viral diarrhea virus (BVDV), application of the combination of neutralizing antibody detection and virus isolation, so-called spot test, were performed on sera of 3 calves selected from each of 26 farms. Nine farms were judged as positive because 64 or more antibody titers were detected from 2 or more calves or BVDV was isolated from one or more calves. PI cattle were detected from 8 of the 9 farms. The positive judgment on one farm was obtained only when the indicator virus used on the neutralizing test was genotypically identical with the isolate from the farm. These results suggest that the spot test can be effective in detecting herds with PI cattle and that the accuracy may be influenced by the genotypes of the indicator viruses.     

Bovine viral diarrhea virus (BVDV) 1b: predominant BVDV subtype in calves with respiratory disease

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.     

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。     

Protection against respiratory infection with bovine virus diarrhoea virus by passively acquired antibody

Susceptibility to infection with bovine virus diarrhoea virus (BVDV) was compared for calves with varying amounts of specific antibody in their sera passively acquired from the ingestion of colostrum. Challenge consisted of intranasal exposure to a strain of BVDV isolated from an outbreak of respiratory disease. Resistance to infection, as judged by nasopharyngeal shedding of virus, was directly related to the titre of neutralizing antibodies in sera. Besides protecting against infection of the upper respiratory tract, passive antibody, which was mainly IgG1, also protected against viraemia and, to a lesser extent, leukopenia. In the presence of colostral antibody, neutralizing and IgG1 antibody responses were apparently inhibited, but a specific IgG2 response occurred.     

Comparison of the agar gel immunodiffusion test and ELISA in the detection of bovine leukosis virus antibody in cattle persistently infected with bovine virus diarrhoea virus

When six cattle persistently infected with bovine virus diarrhoea virus (BVDV) were inoculated with lymphocytes infected with bovine leukosis virus (BLV), a depressed antibody response to BLV was observed by ELISA which was due to a decrease in IgG1 synthesis. The ELISA was more sensitive and more reliable than the agar gel immunodiffusion (AGID) test in detecting BLV infection in cattle persistently infected with BVDV. Decreased antibody responses were manifested in the AGID test by negative, inconclusive or weakly positive reactions: only two of the six cattle developed antibodies that generated positive AGID reactions.     

Variation in E(rns) viral glycoprotein associated with failure of immunohistochemistry and commercial antigen capture ELISA to detect a field strain of bovine viral diarrhea virus

Bovine viral diarrhea virus (BVDV) affects cattle populations causing clinical signs that range from subclinical immunosuppression to severe reproductive and respiratory problems. Detection and removal of persistently infected (PI) calves is the single most important factor for control and eradication of BVDV. Current testing strategies to detect PI calves rely heavily on immunohistochemistry (IHC) and a commercially available antigen capture ELISA (ACE) assay. These viral assays depend on 1 or 2 monoclonal antibodies which target the E(rns) glycoprotein of BVDV. The sensitivity and specificity of these two tests have been reported previously. The purpose of this research was to characterize a strain of BVDV (AU501) that was undetectable using IHC and ACE based on a single monoclonal antibody, but was consistently detected in samples from a Holstein steer using virus isolation and PCR testing. Sequencing of this AU501 viral isolate revealed a unique mutation in the portion of the genome coding for the E(rns) glycoprotein. This unique field strain of BVDV demonstrates the risk of relying on a single monoclonal antibody for detection of BVDV. Multiple testing strategies, including polyclonal or pooled monoclonal antibodies that detect more than one viral glycoprotein may be necessary to detect all PI calves and facilitate eradication of BVDV.     

A versatile flow cytometry-based immunofluorescence inhibition assay for the detection of bovine viral diarrhea virus-specific antibodies

A fast, sensitive and reliable flow cytometry-based (FACS = fluorescence activated cell sorting) immunofluorescence inhibition assay (FACS-IFI) for the detection of virus-specific antibodies in sera is described. The method was evaluated using sera from cattle experimentally infected with bovine viral diarrhea virus (BVDV). Virus-infected cells, which were fixed and permeabilized, were incubated with diluted sera from immunized or control animals. Monoclonal antibodies (mabs) against different viral proteins were added, and detected with ALEXA488-conjugated goat-antimouse antibodies. The fluorescence signals were detected by flow cytometry and determined as mean channel values. Results were expressed as percent fluorescence inhibition compared to standardized negative sera. The FACS-IFI test with sera from experimentally infected animals was highly sensitive and specific. Comparison of the FACS-IFI results with a commercially available blocking ELISA, an indirect ELISA and the standard serum neutralization test showed a strong correlation. Furthermore, the detection of protein-specific antibodies was possible using the FACS-IFI test.     

A single dilution enzyme-linked immunosorbent assay for the quantitative detection of antibodies to bovine herpesvirus type 1

Three serological assays were compared for detection of antibodies to bovine herpes-virus type 1. These were virus neutralization (VN), enhanced complement fixation (CF) and enzyme-linked immunosorbent assay (ELISA). The ELISA was developed using an infected cell lysate antigen and purified virus and was optimized in relation to antigen and antisera dilutions. The CF assay was enhanced by the addition of bovine complement. These 3 assays were compared for detection of: specific virus antibody titers; sero-conversions; early antibody response in experimentally-infected cattle. Both ELISA end-point titers and single dilution values were found to be more sensitive than the CF or VN assays for specific antibody level quantitation. With a single dilution ELISA test procedure a correlation was obtained between ELISA values and VN titers. Using the single dilution ELISA test the assay also detected antibodies in experimentally-infected cattle before either the VN or CF assays, and agreed with the VN test in 35/38 seroconversions found by 4-fold or more VN changes between acute and convalescent paired sera from naturally-infected animals. The single dilution ELISA was a rapid and sensitive test for routine antibody detection in bovine sera.     

Evaluation of two commercial enzyme-linked immunosorbent assays for detection of bovine viral diarrhoea virus in serum and skin biopsies of cattle

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.     

Bovine herpesvirus-1 (BHV-1), bovine leukemia virus (BLV) and bovine viral diarrhea virus (BVDV) infections in Algerian dromedary camels (<Emphasis Type="Italic">Camelus dromaderius</Emphasis>)

This study was performed to investigate the presence of bovine herpesvirus-1 (BHV-1), bovine leukemia virus (BLV) and bovine viral diarrhea virus (BVDV) infections in dromedary camels (Camelus dromaderius) kept in mixed herds with sheep and goats in Algeria, since the prevalence of BHV-1, BVDV, and BLV infections among dromedary camels in Algeria is unknown. Totally, 111 camel sera were collected from two provinces (Laghouat and Ghardaia) in Algeria. The sera were analyzed for BHV-1 specific antibodies, BVDV specific antibodies and BVDV antigen using the ELISA, and BLV nucleic acid using PCR. The seropositivity rate was 9.0% for BVDV-specific antibody, although 41.4% of camels tested were positive for BVDV antigen. Moreover, there was no evidence of BHV-1 and BLV infections. The results indicated that camels might represent an important source for BVDV infection in all ruminants, including cattle, sheep, and goats bred in mixed herds in Algeria, since they had a higher BVDV prevalence rates. Therefore, the prevention and control measures for BVDV infection should put in place in camel populations to limit the spread of BVDV infection to ruminant populations in Algeria.     

Development of an enzyme linked immunosorbent assay (ELISA) for the detection of bovine serum antibody to bovine viral diarrhoea virus

An enzyme linked immunosorbent assay (ELISA) was developed to detect antibody to bovine viral diarrhoea virus (BVDV) in bovine serum. The ELISA results were compared with those of the serum neutralisation test (SNT) using serums from 6 experimentally infected calves bled at intervals from 0 to 154 days postinfection and 886 field samples. The optical density (OD) produced by a single dilution of test serum was compared with a standard curve and the result expressed in ELISA units. Despite wide variation between absolute ELISA and SNT results, an agreement of 97% was obtained when reciprocal SNT titres greater than or equal to 8 and ELISA units greater than or equal to 10 were taken as indicative of a specific reaction. The ELISA was shown to be an efficient method of measuring antibody in bovine serum samples and would assist in any large scale screening of cattle herds for BVDV antibody.     

Toxoplasmosis and border disease in 54 Swedish sheep flocks. Seroprevalence and incidence during one gestation period.

Serum samples from 704 animals from 54 Swedish sheep flocks were analysed by ELISA twice during 1 breeding season for antibodies to Toxoplasma gondii and border disease virus (BDV). An ELISA, originally developed for the detection of antibodies to bovine viral diarrhoea virus (BVDV) in cattle, was assessed on sheep sera and the results were compared with those obtained in a virus neutralization test. The correlation between the 2 assays proved good. Before breeding, 132 (19%) sheep in 42 flocks had antibodies to T. gondii and 7 (1%) sheep in 5 flocks were seropositive to BDV. During the observation period 4 sheep seroconverted to T. gondii and 13 to BDV, giving an incidence rate of 0.7% and 1.9% respectively. No clinical signs due to the infections were observed. In 5 flocks the frequency of barrenness, abortion or stillbirths exceeded 5%, 5% and 8%, respectively, but there was no evidence that this was attributable to the agents studied. The proportion of BDV-positive flocks was significantly higher among flocks that had been in contact with cattle than among those that had not.     

Development and evaluation of an enzyme-linked immunosorbent assay for bovine antibody (IgG) to Pasteurella haemolytica

The sensitivity of an indirect enzyme-linked immunosorbent assay (ELISA) for bovine IgG serum antibody to Pasteurella haemolytica was compared with that of an indirect hemagglutination (IHA) test. Pasteurella haemolytica serotypes were grown in a chemically defined cell culture medium, and soluble antigens released into the growth medium were used in the ELISA and IHA test. An ELISA with serotype-1 antigen consistently detected antibody in sera that were positive by IHA test (correlation, 99%). Sera reacting with serotype-1 ELISA antigens also reacted with ELISA antigens prepared from other serotypes. Although ELISA titers averaged 5 log2 units higher than IHA titers, plots of titers determined by the 2 methods were approximately linear. Titer increases detected in paired serum samples by either test were similar. The ELISA was more sensitive than was the IHA in detecting colostral IgG antibody in serum of newborn calves. The ELISA uses a simple, stable antigen preparation and detects antibody to P haemolytica serotypes that commonly infect cattle.     

Spreading of West Nile virus infection in Croatia

West Nile virus (WNV) is an emerging zoonotic pathogen with rapid global expansion. The virus circulation is confirmed in many countries of Mediterranean Basin and Southern and Central Europe. In our study detection of specific WNV antibodies was performed in horses and cattle sera samples collected from October 2010 to April 2011. Serum samples were randomly taken from different parts of Croatia and tested by IgG and IgM ELISA. Positive serological results were confirmed by virus neutralization assay (VN-assay) and plaque reduction neutralization test (PRNT). Results showed that WNV antibodies were present in 72 out of 2098 horse sera (3.43%) and 3 of 2695 cattle sera (0.11%). The highest seroprevalence was found in Eastern Croatia in counties next to Hungarian, Serbian and Bosnia and Herzegovinian state borders. In Adriatic part of Croatia positive animals were found only in the westernmost county, near Slovenian and Italian borders. Geographic distribution and number of positive horses indicated that WNV is highly present in Croatia and spreading from East to West. However, positive horses in westernmost part of country indicate possible second origin of spreading. Location of serological positive cattle supports the hypothesis that seropositive cattle could be indicators of high WNV activity in the respective geographic regions.     

Immunoglobulin M-specific enzyme-linked immunosorbent assay for serodiagnosis of bovine respiratory syncytial virus infections

An antibody-capture enzyme-linked immunosorbent assay (ELISA) for detection of immunoglobulin (Ig) M antibodies to bovine respiratory syncytial virus (BRSV) in cattle was developed. Monoclonal antibody to bovine IgM was used as the catching antibody. The IgM-ELISA was used, as well as a BRSV-specific IgG ELISA to determine the kinetics of IgM and IgG antibody responses to BRSV infections in cattle. High IgM and IgG antibody titers developed after naturally occurring or induced BRSV infection of calves (6 to 7 months old). Induced infection resulted in an IgM response that was first detectable at postinoculation day (PID) 11 reached a maximum at PID 13, and became undetectable again about PID 28. An IgG response also was detected by PID 11. However, a maximum response was not reached before PID 23, and titers remained high (until PID 80). In naturally occurring infection, IgM and IgG responses in calves were observed in the acute phase of epizootics of respiratory tract disease. Patterns of IgM and IgG response curves were similar to those observed in experimentally infected calves. The involvement of BRSV in an epizootic of respiratory tract disease in 8 calves (2 to 3 weeks old) was demonstrated by the detection of BRSV in several lung lavage samples. All calves had existing IgG antibodies to BRSV which were interpreted to be maternally derived. None of the calves responded with an increase in IgG antibody titer. However, a weak but distinct BRSV IgM antibody response occurred in 6 calves.(ABSTRACT TRUNCATED AT 250 WORDS)