A blocking ELISA for the detection of specific antibodies to bovine respiratory syncytial virus

A blocking enzyme-linked immunosorbent assay (ELISA) has been adapted to detect specific antibodies in bovine sera to respiratory syncytial virus using a horseradish peroxidase-labeled monoclonal antibody to the fusion protein of the virus. This assay plus an indirect blocking ELISA and indirect ELISA were used to detect antibodies to the bovine respiratory syncytial virus (BRSV) in 159 field-origin bovine sera. Results of these assays were compared with serum antibody titers measured by the serum neutralization (SN) test. Over a 56-day period, the mean neutralization titers and the mean delta absorbance values for the blocking ELISA, on the same sera, showed similar declines. However, the calculated correlation coefficients between mean SN titer and mean absorbance value for the blocking ELISA of the individual sera ranged from -0.2 to -0.5 depending on the source of sera. Similar values were obtained whether using crude or purified viral antigen in the assays. Corresponding calculated correlation coefficients were generally higher for the indirect blocking ELISA or indirect ELISA than for the blocking ELISA. The blocking ELISA was between 70 and 64% as sensitive as the serum neutralization test with a specificity of 100 or 90% using the crude and purified viral antigen, respectively. The indirect blocking ELISA and indirect ELISA had similar calculated sensitivities and specificities. The blocking ELISA was faster to run than either of the other ELISA's or the neutralization test. Further, nonspecific background absorbance was obviated because the blocking ELISA detects antibodies to 1 specific viral protein, the fusion protein. These studies suggest that the blocking ELISA should be useful as a serological test for BRSV antibodies.     

Dot-enzyme linked immunosorbent assay as an alternative technique for the detection of bovine respiratory syncytial virus (BRSV) antibodies

A dot-ELISA test for the detection of anti-BRSV antibodies is described. The objective of this study was the standardisation of a test as a fast, inexpensive and effective alternative to detect anti-BRSV antibodies. Its sensitivity, specificity and usefulness were compared to a commercial ELISA-kit and to the standard serum neutralisation (SN) test. The standardisation of the technique was done using nitrocellulose disks soaked with a viral sample isolated in Brazil, BRSV-25-BR. The best results were obtained when the disks were sensitised with a purified antigen at a concentration of 0.7 microg/disk and the bovine serum was diluted 1: 200. The experiment used 423 samples of bovine serum collected in the main cattle breeding centres in Brazil. The standard SN, dot-ELISA technique and commercial ELISA kits scored 67.8%, 71.8% and 72.3% of the samples as positive, respectively. When compared to the SN test, the standardised dot-ELISA and the commercial ELISA tests presented relative sensitivities of 92.3% and 91.6% and relative specificities of 71.3% and 68.4% respectively. The results demonstrated that the dot-ELISA test is adequate for the objectives proposed by this study, being easy to use and economically viable. Thus, this test represents an alternative for BRSV serological diagnosis in the substitution of SN and commercial ELISA tests, recommendable for utilisation in laboratories with few resources.     

Prevalence of and risk factors for bovine respiratory syncytial virus (BRSV) infection in non-vaccinated dairy and dual-purpose cattle herds in Ecuador

A cross-sectional study was carried out to determine the seroprevalence and risk factors associated with bovine respiratory syncytial virus (BRSV) infection in non-vaccinated dairy and dual-purpose cattle herds from Ecuador. A total of 2,367 serum samples from 346 herds were collected from June 2008 to February 2009. A questionnaire, which included variables related to cattle, health, management measures, and the environment, was filled out in each herd. Presence of antibodies against BRSV was analyzed using a commercial indirect ELISA test. A logistic regression model was used to determine risk factors associated with BRSV at herd level. The individual seroprevalence against BRSV in non-vaccinated herds in Ecuador was 80.48% [1,905/2,367; 95% confidence interval (CI)?=?78.9-82.1]. The herd prevalence was 91.3% (316/346; 95% CI?=?88.3-94.3), and the intra-herd prevalence ranged between 25% and 100% (mean, 90.47%). The logistic regression model showed that the existence of bordering cattle farms, the dual-purpose farms, and the altitude of the farm (more than 2,338?m above sea level) were risk factors associated with BRSV infection. This is the first study about BRSV prevalence in Ecuador. It shows the wide spread of the BRSV infection in the country. The risk factors found will help to design effective control strategies.     

Prevalence of bovine respiratory syncytial virus (BRSV) and bovine herpesvirus-4 (BHV-4) in cattle from Ethiopia

A serological study was done to establish the occurrence and determine the prevalence of two important respiratory tract pathogens, bovine respiratory syncytial virus (BRSV) and bovine herpesvirus-4 (BHV-4), in cattle in Ethiopia. Prevalence rates for specific antibodies of 92.5% and 22.3% were recorded for BRSV and BHV-4, respectively. The presence of antibodies against these viruses in cattle from Ethiopia is recorded for the first time in this report. Our data suggests diseases caused by these viruses occur in Ethiopia but, perhaps because disease signs are not specific, they have not been recognized in the past.     

A seroepidemiologic study of bovine respiratory syncytial virus (BRSV) in Turkey

In this study, a survey of cattle for neutralizing antibodies to respiratory syncytial virus (RSV) in Turkey is presented. 490 serum samples were collected from several state farms and some private flocks in different regions of Turkey. The serologic examination was done by the microtiter serum-neutralization technique. In this report, a positive serum antibody titer at dilutions of 1:2 was demonstrated in 226 (46.12%) of the 490 sera tested.     

Application of a modified indirect fluorescent antibody test to the detection of antibodies to bovine respiratory syncytial virus in Ontario cattle.

A modified indirect fluorescent antibody test for the detection of serum antibodies to bovine respiratory syncytial virus was developed. The test made use of Terasaki plastic microtiter plates in which bovine respiratory syncytial virus (Saskatchewan strain) infected Georgia bovine kidney cells were grown and fixed in situ by a modified acetone fixation procedure. Evans blue dye was used as a counterstain to reduce nonspecific fluorescence. In a study of 986 field sera from a geographically broad cross-section of mature Ontario cattle, 95% of the samples were found to be positive at or above a 1:2 dilution. No seronegative regions, counties or herds were identified. When representative samples covering a range of indirect fluorescent antibody titers were further examined by a microtiter virus neutralization assay, a significant agreement was found between the two tests. Up to a fourfold decrease in titer was observed when antigen coated plates were stored at -70 degrees C for four months. The modified indirect fluorescent antibody test for bovine respiratory syncytial virus antibody detection proved to be a rapid, practical procedure for use in the diagnostic laboratory. This study confirms that bovine respiratory syncytial virus is widespread in the Ontario cattle population and that most mature cattle can be assumed to have been exposed to this virus.     

Isotype-specific ELISAs for the detection of antibodies to bovine respiratory syncytial virus

Isotype-specific ELISAs for the detection of antibodies to bovine respiratory syncytial virus (BRSV) are described. BRSV-specific IgG1 and IgG2 were determined in indirect double antibody sandwich assays. For IgA and IgM antibody capture assays were used. The isotype specificity of the assays was confirmed by the observation that samples with a high titre of BRSV-specific antibodies of particular isotype were negative in the assays for the other isotypes and vice versa. Comparison of the results obtained in the ELISAs and in the virus neutralisation test showed that acute phase antibodies were more efficiently detected in the latter. It also showed that the presence of BRSV-specific IgA was not correlated with neutralising activity in vitro. The serum antibody response of BRSV-infected seronegative calves from the field consisted of a nearly simultaneous increase of IgM, IgA and IgG1-antibodies in the acute phase of the disease, while the IgG2-response followed at various intervals thereafter. In young animals with maternal antibodies a different pattern was found. There was no increase in IgG1 and IgG2, but six of eight animals showed a weak IgM response and two of these six calves also showed a weak and short lasting IgA response. Because maternal antibodies are insufficiently effective in protecting calves against BRSV, the presence of such antibodies at mucosal surfaces was investigated. Maternal immunity was found to be restricted to IgG1 antibodies in serum. This agrees with the failure of maternal antibodies to protect mucosal surfaces against BRSV infection.     

Bovine respiratory syncytial virus (BRSV): a review

Bovine respiratory syncytial virus (BRSV) infection is the major cause of respiratory disease in calves during the first year of life. The study of the virus has been difficult because of its lability and very poor growth in cell culture. However, during the last decade, the introduction of new immunological and biotechnological techniques has facilitated a more extensive study of BRSV as illustrated by the increasing number of papers published. Despite this growing focus, many aspects of the pathogenesis, epidemiology, immunology etc. remain obscure. The course and outcome of the infection is very complex and unpredictable which makes the diagnosis and subsequent therapy very difficult. BRSV is closely related to human respiratory syncytial virus (HRSV) which is an important cause of respiratory disease in young children. In contrast to BRSV, the recent knowledge of HRSV is regularly extensively reviewed in several books and journals. The present paper contains an updated review on BRSV covering most aspects of the structure, molecular biology, pathogenesis, pathology, clinical features, epidemiology, diagnosis and immunology based on approximately 140 references from international research journals.     

Use of the indirect fluorescent antibody test in the detection of bovine respiratory syncytial virus antibodies in bovine serum.

The indirect fluorescent antibody test was adapted for identifying bovine respiratory syncytial virus and its specific antibody, using goat turbinate (GTU) cells. The virus caused maximal cytopathic effects in GTU cells 4 to 8 days postinfection, but fluorescence was not readily detected during this period. Fluorescence was maximal in infected GTU cells at 24 to 36 hours postinfection, but could be detected 48 hours postinfection. Bovine serums (331) which had been submitted to the Oklahoma Animal Disease Diagnostic Laboratory were tested for antibodies to this virus, and 73.6% were found to be positive.     

Cell-mediated immune response in cattle to bovine respiratory syncytial virus

Cattle inoculated with bovine respiratory syncytial virus (BRSV) were evaluated for the development of a cell-mediated immune response. Results of the leukocyte migration-inhibition test under agarose and the delayed hypersensitivity test indicated that a cell-mediated immune response was elicited after intranasal inoculation of calves with BRSV. Migration inhibition in the leukocyte migration-inhibition test was detected by postinoculation day (PID) 5 and reached maximum inhibition on PID 21. Inhibition of leukocyte migration was still evident by PID 42 when values were still appreciably greater than preinoculation values. All of the calves inoculated with BRSV developed a delayed hypersensitivity skin response when challenge exposed intradermally with BRSV antigen.     

Atypical interstitial pneumonia (AIP) in calves and young cattle in Schleswig-Holstein in conjunction with an infection by the bovine respiratory syncytial virus (BRSV)

It is reported on atypical interstitial pneumonia (AIP) in 16 mostly Holstein-Frisian calves and feeders from 13 different farms in Schleswig-Holstein in association with an infection by the respiratory syncytial virus (BRSV). All animals were submitted with identical clinical histories. Macroscopically the lung lesions were characterized by alveolar and interstitial edema and emphysema. Microscopically there was a diffuse interstitial pneumonia with formation of hyaline membranes and multinucleated giant cells. From the investigation material of the 16 animals BRSV was confirmed by direct immunofluorescence with monoclonal antibodies in 4 animals from 4 different farms.     

Detection of IgE antibodies to bovine respiratory syncytial virus

The role of IgE antibodies against respiratory syncytial virus has attracted attention for both human and bovine disease. To detect such antibodies, we have developed an enzyme-linked immunosorbent assay (ELISA) specific for bovine respiratory syncytial virus (BRSV). Firstly, anti-serum strongly positive for BRSV-specific IgE was produced by immunizing a levamisole-treated calf with BRSV. The presence and specificity of BRSV-specific IgE in this animal was confirmed with the Praunitz-Kustner (PK) technique. Potential interference in an ELISA by other BRSV-specific immunoglobulin isotypes was eliminated by preferential precipitation of serum samples with 27.5% saturated ammonium sulfate. The correlation between the PK and the ELISA assay was greater than 93% and the ELISA was found to be more specific than the PK. Indeed, in a pilot experimental infection study, the serum levels of BRSV-specific IgE were found to correlate with the symptom expression following repetitive live virus aerosolization. This may prove to be a useful rapid test to study both herd immunity and the potential pathogenic influence of IgE.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to bovine viral diarrhoea virus (BVDV) in cattle sera

A microtitre ELISA has been established for the quantitation of antibodies to bovine viral diarrhoea virus (BVDV). Single dilutions of sera were assayed and units of antibody were calculated from a standard curve. In order to detect the maximum number of responding animals both IgG1 and IgG2 antibody should be assayed, although detection of IgG1 alone was nearly as effective. The ELISA was as sensitive as the virus neutralization test for detection of antibody; comparison of an ELISA that detected IgG1 plus IgG2 antibody to BVDV with the virus neutralization test gave a correlation coefficient (r) of 0.89 (P less than 0.001 for 95 compared sera). Although similar amounts of IgG1 and IgG2 antibodies were present in sera from both experimentally- and naturally-infected cattle, antibody to BVDV in colostrum and in the sera from young calves was predominantly IgG1. The number of adult cows with antibody was 40 out of 41 while 36 of 44 calves reared in a beef unit were found to have produced antibody by the time they were 31.5 weeks old, an indication of the high prevalence of BVDV in the cattle population.     

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to maedi-visna virus

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to maedi-visna virus (mvv) in sheep is described, in which microtitre plates are with a partly purified preparation of mvv. The antibodies bound are detected by a horseradish peroxidase conjugate.The results obtained with ELISA on a total of 493 serum samples from several commercial flocks were compared to those of a routine agar gel precipitation test (AGPT) and a complement fixation test (CFT).All samples which scored positive in AGPT, CFT or both (20.8%) were also found positive by ELISA. In addition, with ELISA a further 11.5% of the samples were positive. Serum samples from maedi-free flocks, from sheep suffering from sheep pulmonary adenomatosis and from lambs immunized against other viruses were all negative by ELISA. The assay has been used routinely for some years and proved to be specific, sensitive and suited for screening of large numbers of serum samples.     

Prevalence and specificity of antibodies to bovine respiratory syncytial virus in sera from feedlot and range cattle

The specificity of serum antibodies for the polypeptides of bovine respiratory syncytial virus (BRSV) was examined, using sera obtained from feedlot and range cattle. Test results in sera from feedlot cattle indicated a 60% rate of seroconversion and 95% seropositivity to BRSV, associated with lack of clinical signs indicative of respiratory tract disease. Exposure to other common respiratory tract viruses also was high (greater than or equal to 92% to bovine herpesvirus type 1, bovine viral diarrhea virus, and para-influenza virus type 3). Test results in sera from range cattle indicated BRSV seropositive rates of 28% in calves, 49% in yearling cattle, and 70% in mature cows; clinical signs of respiratory tract disease were not observed in these cattle. Antibodies to BRSV in sera from cattle in both environments reacted predominantly with polypeptides of molecular weight 80,000 through 85,000, 40,000, and 28,000. Reactivity to a glycoprotein of molecular weight between 43,000 and 44,000 and to several glycopolypeptides of smaller molecular weight increased in serum specimens obtained from feedlot cattle between time of entry into the feedlot and slaughter.     

Severe respiratory disease in dairy cattle in New York State associated with bovine respiratory syncytial virus infection

Severe respiratory disease associated with bovine respiratory syncytial virus (BRSV) infection has been identified in dairy cattle in New York State. The cases identified occurred in dairy calves and heifers. The disease was characterized in 4 animals by pathologic changes including interstitial pneumonia, necrotizing bronchiolitis with multinucleated syncytial epithelial cells and interstitial emphysema. BRSV antigen was demonstrated in lung samples or was isolated in tissue culture in all 4 cases. A retrospective survey of 6279 bovine diagnostic accessions between 1977 and 1982 revealed 66 cases of interstitial pneumonia, often with concurrent bronchiolitis. In this 5 year period, only 1 case in 1981 had interstitial pneumonia and bronchiolitis with pathologic features consistent with BRSV infection. It is concluded that pathogenic BRSV has entered New York State and that it is contributing to clinical respiratory disease in dairy cattle.