Differences in fluorescent antibody staining of bovine respiratory syncytial virus-infected cells by ovine and bovine sera

In indirect fluorescent antibody tests in which sera from cattle and sheep with respiratory disease problems were used to stain foetal bovine lung cells infected with a bovine respiratory syncytial virus strain, differences were noted in the pattern of fluorescence produced by some sheep sera and that produced by positive bovine sera. In serum neutralisation tests, also using a bovine respiratory syncytial virus strain, 4 of 7 sera giving this atypical pattern of fluorescence had very low neutralising antibody titres (highest 1/4), and 3 were negative. It is suggested that two related but antigenically distinguishable respiratory syncytial virus types are present in sheep, one of which is similar to bovine strains.     

Serology for diagnosis and epizootiological studies of bovine respiratory syncytial virus infections

The complement fixation test (CFT) and the virus neutralisation test (VNT), performed as a plaque reduction test, were employed to measure antibodies to bovine respiratory syncytial virus. The CFT with bovine sera was performed with supplementation of the complement factors in fresh guinea pig serum by an adequate amount of Clq-factor of the bovine species. Kinetics of maternally derived antibodies and the antibody response after spontaneous and experimental infections and after intramuscular vaccination were studied by both tests. Patterns of development of complement fixing and virus neutralising antibodies were generally similar and titres equalled each other in the test systems that are described. However a VNT detected antibodies a few days earlier after an infection than a CFT and peak-levels reached after a naturally acquired infection decreased faster in a CFT than in a VNT: a mean decrease of 3.1 and 1.4 log2 units was found in 13 weeks respectively. Mean half-life of passive antibodies was 25 days in a VNT. An infection with bovine respiratory syncytial virus could be diagnosed by serology, using a CFT on acute and convalescent serum samples of a number of animals in a group. Serology is preferable to virus isolation for routine diagnosis of bovine respiratory syncytial virus infections. Paired sera, collected at 14-day intervals and examined by CFT, are recommended for the diagnosis of the cause of respiratory disease. A VNT is preferable if low antibody levels are to be detected because non-specific reactions occur in a CFT at low serum dilutions.     

Comparison of a newly developed enzyme-linked immunosorbent assay with complement fixation and neutralisation tests for serology of bovine respiratory syncytial virus infections

An indirect double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection and titration of serum antibodies to bovine respiratory syncytial virus (BRSV). The ELISA was compared with a complement fixation (CF) test and a test for virus neutralising antibody in serum (virus neutralisation [VN] test). Testing sera collected in dairy herds revealed the closest correlation between the results of the ELISA and the CF test with respect to BRSV antibody titres. The VN test detected BRSV antibodies in a higher percentage of acute phase sera compared to the other two tests in field samples and in early bleedings of experimentally infected calves. However, the VN test was less effective in making a diagnosis of BRSV infections on the basis of a significant titre increase in paired sera. For this purpose the ELISA was found to be the most sensitive test.     

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.     

Serological study of a population of alpine chamois (Rupicapra r rupicapra) affected by an outbreak of respiratory disease

A serological survey of respiratory virus infections was carried out from 1998 to 2001 in Lecco province, Italy, as part of a health monitoring programme in a population of alpine chamois, many of which died of pneumonia in autumn and winter 2000 to 2001; 194 carcases of all age classes were found over a short period and in a small area. Eighteen of them, which were examined postmortem, consistently showed signs of severe fibrinous lobar pneumonia or catarrhal bronchopneumonia. Samples of serum from 145 chamois collected from hunted animals and carcases were tested by a virus neutralisation test against bovine respiratory syncytial virus (BRSV), bovine viral diarrhoea virus, bovine herpesvirus type 1 and parainfluenzavirus type 3. Positive results were detected only for BRSV. The area was divided into two subunits on the basis of the distribution of deaths; in the areas where fatalities were observed there was a significant increase of BRSV titres at the beginning of the outbreak. Furthermore, during the 2000 and 2001 hunting seasons antibody titres to BRSV were significantly higher in the areas where mortality occurred. The roe deer living in the same area were not affected by pneumonia and had a low prevalence of titres to BRSV which did not vary during the period of the study.     

An enzyme-linked immunosorbent assay for detection of antibodies against bovine pestivirus

A competitive enzyme-linked immunosorbent assay was developed and compared with the serum neutralisation test for bovine pestivirus using 508 cattle sera and serial serum samples from a goat hyperimmunized with five bovine pestivirus isolates. There was 96.7% agreement between the two tests. The relative sensitivity of the enzyme-linked immunosorbent assay compared to the serum neutralisation test was 95.2% and the relative specificity was 99.4%. The titres of individual animals in the assay did not show a close correlation with serum neutralisation test titres. This may be because the antibodies measured in the two tests are directed against different viral proteins. The enzyme-linked immunosorbent assay has the advantage of being quicker and cheaper than the serum neutralisation test. The configuration used in the ELISA means sera from all species can be tested for pestivirus antibody using the same set of reagents.     

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.     

Detection of bovine respiratory syncytial virus using a heterologous antigen-capture enzyme immunoassay

Based on the marked antigenic similarities that exist between antigens of the human and bovine strains of respiratory syncytial virus (RSV), an enzyme immunoassay (EIA) designed to detect human RSV was used to detect bovine RSV. The commercial test kit (RSV EIA) consists of a solid phase (beads) coated with a capture antiserum prepared against the Long strain of human RSV. The RSV EIA test was compared with the method of inoculation of cell cultures and fluorescent antibody (FA) staining of lung tissue for the detection of bovine RSV. Using a cell culture-propagated stock of strain 375 of bovine RSV, the threshold of sensitivity of the EIA test for the cattle strain of RSV was determined to be less than or equal to 10(2.3) CCID50/ml. In addition, RSV EIA detected the bovine RSV in nasal samples obtained from 3 experimentally inoculated cattle. The RSV EIA exhibited a sensitivity of greater than or equal to 80% during the period that shedding of infectious virus took place. All of the bovine RSV FA-positive lung samples (n = 37) were positive by the RSV EIA. Twenty-six of the remaining 214 bovine RSV FA-negative lung samples were positive by the RSV EIA. The RSV EIA was also used to test 137 nasal swabs obtained from cases of bovine respiratory disease. Of these, 38 tested positive by RSV EIA. All samples that tested positive by EIA were confirmed by blocking assays using hyperimmune serum anti-bovine RSV and a pool of monoclonal antibodies specific for that virus.     

Studies on the occurrence and significance of bovine respiratory syncytial virus in Saskatchewan.

Seventy-six percent of 49 blood samples from Saskatchewan cattle had serum antibodies against bovine respiratory syncytial virus. Experimental infection of one week and seven month old calves with bovine respiratory syncytial virus (Iowa strain) caused transient fever, mucopurulent nasal discharge and coughing but no macroscopic or microscopic lesions attributable to bovine respiratory syncytial virus.     

Prevalence of ovine and bovine respiratory syncytial virus infections in cattle determined with a synthetic peptide-based immunoassay.

Subgroup-specific peptide-based enzyme-linked immunosorbent assays from the G-protein of the ovine and bovine respiratory syncytial virus (RSV), respectively, were used to determine the prevalence of the ovine and bovine subgroup strains of RSV infections in cattle. A total of 1,102 bovine serum samples were obtained from 6 diagnostic laboratories located in the northwestern and the southeastern USA and were tested for antibody to either the bovine or ovine subgroups of RSV. Antibody to viruses from each subgroup was present in samples from each region and all states tested. The Southeast had a higher prevalence of the bovine subgroup strains (69.5%). Then did the Northwest (40.9%). The prevalence of the ovine strain was similar for the two regions (16.7% in the southeast, 14.9% in the northwest). The overall prevalence was 56.6% for the bovine strain and 15.9% for the ovine strain. These results suggest members of the ovine subgroup of RSV circulate in the cattle population but with less frequency than those viruses of the bovine subgroup.     

Serological studies of parainfluenza type 3 virus, bovine adenovirus type 3 and bovine respiratory syncytial virus infection in beef calves

Six serum samples were taken at monthly intervals from birth to weaning from each of 41 newborn calves in the autumn and spring calf crops of a beef cow--calf herd. The serum hemagglutination-inhibition (HI) antibody titres to parainfluenza type 3 virus (PIV-3), virus-neutralization (VN) antibody titres to bovine adenovirus type 3 (BAV-3) and bovine respiratory syncytial virus (BRSV) were determined using microtitration techniques. There was serological evidence of a significantly higher incidence of infection with BAV-3 in the fall calves than in the spring calves. Serological responses to BAV-3 were not detected in calves with VN titres of greater than 1/256. Serological evidence of subclinical infection with PIV-3 occurred mainly in late February or early March during a period of marked environmental temperature fluctuations. Serological evidence of a high incidence of infection with BRSV was obtained for both the fall and spring calf crops. Serum antibody appeared to be unable to prevent infection with BRSV. An association between infection with BRSV and clinical pneumonia was found in 3 out of 9 calves. BAV-3 infection was related to pneumonia in only 1 instance; however, there was simultaneous evidence of BRSV infection in this calf. PIV-3 infection was found to be related to pneumonia in only 1 instance. There was serological evidence of infection with BAV-3 in association with the occurrence of diarrhea in 3 calves.     

Significant rising antibody titres to influenza A are associated with an acute reduction in milk yield in cattle

Sporadic cases of an acute fall in milk production, "milk drop", were investigated in a Holstein Friesian dairy herd in Devon. The investigation was a case control study with two controls per case. Paired blood samples demonstrated that rising antibody titres to human influenza A/England/333/80 (H1N1) and human influenza A/Eng/427/88 (H3N2) were associated with an acute fall in milk production. Rising titres to bovine respiratory syncytial virus (BRSV), bovine virus diarrhoea virus (BVD), infectious bovine rhinotracheitis (IBR) and parainfluenza virus 3 (PI3) were not associated with an acute fall in milk production. Cases with rises in antibody to influenza A had significantly higher respiratory scores and rectal temperatures than their controls. The mean loss of milk production for the cases with rises in antibody to influenza A compared to their controls was 159.9L. This study provides further evidence that influenza A persists in cattle and causes clinical disease.     

Failure to detect antibody to bovine respiratory syncytial virus in bovine fetal serum.

Sera obtained from 147 bovine fetuses estimated to be between 120 and 270 days of gestation at an abattoir were tested for antibody to bovine respiratory syncytial virus. Antibody to bovine respiratory syncytial virus was not detected in any of the sera examined. Based on the results of this study and a review of the literature, it appears that transplacental infection by bovine respiratory syncytial virus does not occur, or is uncommon.     

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.     

Superneutralization test--a new virus serology examination procedure]

The serum neutralisation test, based on the antigen-antibody reaction and cytopathic action of a given virus upon certain in-vitro cultured cell systems, was expanded to the dimensions of a super-neutralisation test by including specific control sera with known titre and applying then to all samples positive to serum neutralisation through repeated addition of virus to tubular cell cultures with no CPE in the serum neutralisation test. Another antigen-antibody reaction was introduced by repeated addition of virus. In samples with less antibody (lower titre) due to lacking or partial neutralisation of added virus such reaction gave a CPE of with the intensity was inversely proportional to the titre. The CPE (cytopathic effect) will disappear beyond a certain titre limit owing to complete neutralisation of added virus. The point of disappearance can be controlled to the purpose of the given test by appropriate choice of the virus dose. This approach will entail negligible extra expenses but enable, in addition to qualitative assessment of reagents, quantitative identification of their antibody titres, and this will, no doubt, add to the informative weight of the test result. The advent of the super-neutralisation test has added to laboratory diagnosis in the field of virus serology a conventiently applicable, uninvolved, and highly effective technique.     

Prevention of bovine respiratory syncytial virus infection and clinical disease by vaccination

A double blind field trial was carried out with a live attenuated bovine respiratory syncytial virus vaccine. The trial involved 530 calves, two to 10 months old, on 27 dairy farms, where respiratory problems due to bovine respiratory syncytial virus infections had been observed during the preceding year. In 17 herds either all calves were vaccinated (nine groups) or all calves received a placebo (eight groups). In 10 herds half the number of calves were vaccinated and the other half kept as non-vaccinated controls. Calves were vaccinated intramuscularly twice with an interval of four to five weeks. These groups were under regular clinical observation and animals were tested periodically for antibodies to bovine respiratory syncytial virus and parainfluenza type 3 virus. Serological examination indicated that no bovine respiratory syncytial virus infection had occurred prior to the first vaccination in August. Vaccination did not cause adverse reactions. Low concentrations of neutralising and complement fixing antibodies were induced by vaccination and a sharp increase of antibody titres was observed after natural infection of vaccinated animals. Infections with bovine respiratory syncytial virus occurred in six out of eight non-vaccinated groups, in nine out of 10 partly vaccinated groups and in only two out of nine completely vaccinated groups. Virus infection in completely vaccinated groups was significantly reduced compared with partly vaccinated and non-vaccinated groups. The incidence of bovine respiratory syncytial virus lower respiratory disease was significantly reduced in completely vaccinated groups compared to non-vaccinated groups. Generally only mild signs of upper respiratory disease were present in completely vaccinated groups after bovine respiratory syncytial virus infection.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Prevalence of antibodies to bovine respiratory syncytial virus, bovine viral diarrhea virus, bovine herpesvirus-1, and bovine parainfluenza-3 virus in sheep and goats in Quebec

Serum samples were collected from 1,075 clinically normal sheep and goats from 77 flocks in 7 agricultural regions of Quebec from June to August 1982. Sheep and goats were tested for antibodies to bovine respiratory syncytial virus, bovine viral diarrhea virus, and bovine herpes-virus-1 by the indirect fluorescent antibody technique and for parainfluenza-3 virus by the hemagglutination inhibition test. The prevalence of antibodies in animals to respiratory syncytial virus was 31%; to bovine viral diarrhea virus, 22.2%; to bovine herpesvirus-1, 10.8%; and to parainfluenza-3 virus, 23.2%. Antibodies prevailed in similar proportions in young (less than 1 year) and adult (greater than 1 year) animals.     

Serologic studies of bovine respiratory syncytial virus in Minnesota cattle

Serum antibody titers to bovine respiratory syncytial virus were determined for 559 cattle. Serum samples were obtained through the Minnesota State-Federal Brucellosis Laboratory and were collected over a 1-year period. Results of this study revealed an antibody prevalence of 65.5% to bovine respiratory syncytial virus. The distribution of antibody titers is presented, as well as analysis of titers based on breed, sex, and age of the cattle.     

Prevalence of antibodies to seven viruses in a flock of ewes in Minnesota

Blood samples were collected from a flock of healthy ewes at a University of Minnesota research station. Sera from these blood samples were tested for antibodies against 7 viruses, using 3 tests (eg, virus-neutralization test for bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, bovine adenovirus type 3, and bovine respiratory syncytial virus; hemagglutination inhibition test for parainfluenza virus type 3; and agar-gel immunodiffusion test for lentivirus of ovine progressive interstitial pneumonia and bluetongue virus). The number of seropositive ewes for each antibody type were 1 of 377 (0.3%) for bovine viral diarrhea virus, 2 of 377 (0.5%) for infectious bovine rhinotracheitis virus, 29 of 378 (7.6%) for bovine adenovirus type 3, 200 of 378 (52.5%) for bovine respiratory syncytial virus, 273 of 373 (71.7%) for parainfluenza virus type 3, and 210 of 379 (55%) for ovine progressive pneumonia virus. All ewes were seronegative for bluetongue virus antibodies.