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.     

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.     

Properties of a respiratory syncytial virus isolated from a sheep with rhinitis

A virus isolated from a yearling cross-bred ewe was identified as respiratory syncytial virus (RSV) by indirect immunofluorescence and by virus neutralization with bovine RSV antisera. The virus caused a mild conjunctivitis in 3-month-old lambs when inoculated alone. Although clinical signs of pneumonia were not observed, there was gross and microscopic evidence of pulmonary inflammation in the lungs of lambs inoculated with either the sheep RSV isolate alone or in conjunction with Pasteurella haemolytica. Lung lesions in the dual infection were more severe, with approximately 10% of the total lung mass affected. Lavage fluids from lambs inoculated with virus and bacteria contained approximately 3 times more inflammatory cells than from control lambs or lambs inoculated with virus only. The sheep RSV isolate was classified as a mild respiratory pathogen in lambs of this age. Speculations on the potential importance of this virus in interspecies transmission to cattle and goats were discussed.     

A preliminary serological survey of viral antibodies in Peruvian sheep

This study reports the sero-prevalence of viral infections in sheep in Peru. Serum samples were collected from 34 mature healthy rams located in 3 different geographic regions of the country (north, central and south). The sera were tested for antibodies to the following viruses: respiratory syncytial virus (RSV); parainfluenza 3 (PI-3) virus; bovine viral diarrhea/border disease (BVD/BD) virus; bovine herpesvirus 1 (BHV-1); bluetongue (BT) virus; ovine progressive pneumoniae (OPP) virus; bovine leukosis virus (BLV). The serological studies showed that 47% were positive for RSV; 82% for PI-3; 3% for BVD/BD virus; 49% for BT virus; 13% for OPP virus. Antibodies were not detected to bovine herpesvirus 1 or to bovine leukosis virus.     

Analysis of ruminant respiratory syncytial virus isolates by RNAse protection of the G glycoprotein transcripts.

Two different respiratory syncytial virus (RSV) radiolabeled probes were used to characterize the genetic heterogeneity of 25 ruminant RSV isolates by the ribonuclease protection assay. A 32P-radiolabeled antisense RNA probe was transcribed from cloned ovine and bovine RSV G glycoprotein genes and then hybridized with total RNA isolated from infected cells with various ruminant RSV isolates. The results of this study, along with previously published nucleotide sequence data of the ovine RSV G glycoprotein gene, suggest the presence of at least 2 ruminant RSV subgroups. One subgroup is represented by RSV isolated from respiratory disease outbreaks from calves and goats, and the other is represented by RSV isolated from sheep.     

An immunoenzyme test used for the detection of antibodies to rinderpest: the advantage of using a purified virus cultivated in a cell line

Two types of in vitro assays (enzyme immunoassay and sero-neutralization test) were compared for their ability to detect antibodies to rinderpest virus in field sera from West African bovines. Purified virus grown on Vero cells (Ag/Vero) or bovine kidney cells (Ag/BK), were tested as antigen in enzyme-linked immunoassays (EIA). The results of the comparative evaluation of the two antigens by EIA, prove that Ag/Vero did enhance the sensitivity and the specificity of the test in comparison to Ag/BK and offers a 94% correspondence with seroneutralization.     

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.     

The frequency, distribution and effects of antibodies, to seven putative respiratory pathogens, on respiratory disease and weight gain in feedlot calves in Ontario.

During 1983-85, 279 calves requiring treatment for bovine respiratory disease and 290 comparison (control) animals from 15 different groups of feedlot calves were bled on arrival and again at 28 days postarrival. Their sera were then analyzed for antibodies to seven putative respiratory pathogens. On arrival, the prevalences of indirect agglutination titers to Pasteurella haemolytica, P. haemolytica cytotoxin, Mycoplasma bovis and M. dispar were greater than 50%, the prevalence of titers to bovine virus diarrhea virus (BVDV) was approximately 40%, and the prevalences of titers to infectious bovine rhinotracheitis virus (IBRV), bovine respiratory syncytial virus (RSV) and parainfluenza virus type 3 (PIV3) were all below 25%. Seroconversion during the first month after arrival occurred in more than half the calves to P. haemolytica cytotoxin, PIV3 and RSV. Seroconversion of agglutination titers to P. haemolytica, Mycoplasma and BVDV occurred in about 40% of calves, and seroconversion to IBRV was infrequent (less than 5%). Initial titers were negatively correlated to subsequent titer changes within organism. Initial titers, and titer changes between organisms were essentially independent. Light calves had an increased risk of being selected for treatment for respiratory disease. Seroconversion to P. haemolytica cytotoxin, RSV and BVDV were predictive of respiratory disease cases, explaining approximately 69% of all respiratory disease cases in the feedlots. It was not possible to accurately predict weight gain or relapse from the serological data.     

Seroepidemiologic survey for antibodies to selected viruses in the respiratory tract of lambs

A serologic study was conducted to determine the prevalence of antibodies to, and infection rate of, Mastadenovirus ovi 5, M ovi 6, parainfluenza-3 (PI-3) virus, bovine herpesvirus-1 (BHV-1), respiratory syncytial virus (RSV), bovine viral diarrhea (BVD) virus, and ovine progressive pneumonia (OPP) virus in lambs at a ram lamb growth-rate test station. For 2 consecutive years, serum samples were prepared from blood collected from 1- to 2-month-old ram lambs as they entered the test station (1st sample) and again 2 months later (2nd sample). The 1st year, 59 producers submitted 237 lambs; the 2nd year, 65 producers submitted 253 lambs. Microtitration serum virus-neutralization tests were used to determine antibody titers for M ovi 5, M ovi 6, PI-3 virus, BHV-1, and BVD virus. Antibodies to RSV and OPP virus were determined, using indirect hemagglutination and agar-gel immunodiffusion, respectively. Based on results of the 1st blood samples collected, the mean prevalence for both years was as follows: 95% of the lambs were seropositive for M ovi 5; 87.2% for PI-3 virus; 84.5% for RSV; 41.7% for M ovi 6; 8.7% for BVD virus; 5.4% for BHV-1; and 3.3% for OPP virus. Based on the 2-year mean, M ovi 6 had the highest infection rate (207 of 484 [42.8%]) as determined by the number of lambs evaluated having a greater than or equal to 4-fold increase in serum antibody titer from the 1st to the 2nd sampling. Infection rates of the other viruses were: 31.0% for M ovi 5; 15.3% for PI-3 virus; 5.6% for RSV; 0.6% for BVD virus; and 0.4% for BHV-1. One lamb became seropositive for OPP virus the 2nd year.     

Expression and characterisation of the ovine respiratory syncytial virus (ORSV) G protein for use as a diagnostic reagent.

Respiratory syncytial virus (RSV) causes severe lower respiratory tract infection in children and calves. Antibodies to ovine RSV (ORSV) are common in sheep, but the clinical disease is not well defined. There is no report of ORSV infection in Australian sheep although respiratory distress syndrome has been described. This discrepancy may be due to the lack of a suitable diagnostic test. In this report, we have characterised the ORSV G protein in an attempt to study its relatedness to human and bovine RSV (HRSV, BRSV) and for use in the development of a suitable diagnostic assay. Full length and a truncated variant of ORSV G protein were expressed in mammalian cells and the expressed proteins characterised by indirect immunofluorescence and radioimmunoprecipitation assays. Our results indicate that like HRSV, the ORSV G protein is heavily glycosylated. The expressed protein was membrane bound as well as secreted and could be purified from culture supernatants and may be suitable for use in development of a diagnostic assay.     

Comparison of competitive ELISA, indirect ELISA and standard AGID tests for detecting blue-tongue virus antibodies in cattle and sheep

The performances of a competitive enzyme-linked immunosorbent assay (ELISA) using a group specific monoclonal antibody against bluetongue virus, an indirect ELISA and the standard agar gel immunodiffusion (AGID) test were compared in the detection of serum antibody against bluetongue virus. Test sera consisted of 1300 bovine, 530 ovine and 160 carpine samples from bluetongue-free areas of Canada, 605 bovine and ovine field samples from the USA and Barbados and 464 samples from 79 cattle and sheep experimentally infected with 19 South African and five USA serotypes of bluetongue virus. The diagnostic specificity of the competitive ELISA, as determined for the bluetongue virus-free cattle sera was superior (99.92 per cent) to that of the indirect ELISA (99.85 per cent) and the AGID (99.0 per cent). The specificities of the competitive ELISA for sheep (99.63 per cent) and goats (100.0 per cent) sera were also higher than those of the AGID test. The performance of the ELISA tests was similar whether a gamma-ray-irradiated (2.0 Mrad) or a non-irradiated bluetongue virus antigen preparation was used. The competitive ELISA results for bovine field sera from endemic areas demonstrated a relatively low level of agreement (92.04 per cent) with AGID test results, with 9.7 per cent false negatives. The possible presence in these sera of antibody to cross-reacting antigens or to other orbiviruses, eg, epizootic haemorrhagic disease virus, which react in the AGID but not in the competitive ELISA may account for this lack of agreement.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

A comparison of five serological tests for bovine brucellosis.

Five serological assays: the buffered plate antigen test, the standard tube agglutination test, the complement fixation test, the hemolysis-in-gel test and the indirect enzyme immunoassay were diagnostically evaluated. Test data consisted of results from 1208 cattle in brucellosis-free herds, 1578 cattle in reactor herds of unknown infection status and 174 cattle from which Brucella abortus had been cultured. The complement fixation test had the highest specificity in both nonvaccinated and vaccinated cattle. The indirect enzyme immunoassay, if interpreted at a high threshold, also exhibited a high specificity in both groups of cattle. The hemolysis-in-gel test had a very high specificity when used in nonvaccinated cattle but quite a low specificity among vaccinates. With the exception of the complement fixation test, all tests had high sensitivities if interpreted at the minimum threshold. However, the sensitivities of the standard tube agglutination test and indirect enzyme immunoassay, when interpreted at high thresholds were comparable to that of the complement fixation test. A kappa statistic was used to measure the agreement between the various tests. In general the kappa statistics were quite low, suggesting that the various tests may detect different antibody isotypes. There was however, good agreement between the buffered plate antigen test and standard tube agglutination test (the two agglutination tests evaluated) and between the complement fixation test and the indirect enzyme immunoassay when interpreted at a high threshold. With the exception of the buffered plate antigen test, all tests were evaluated as confirmatory tests by estimating their specificity and sensitivity on screening-test positive samples.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzyme-linked immunosorbent assay for differentiation of the antibody response of cattle naturally infected with Brucella abortus or vaccinated with strain 19

Purified O chain of Brucella abortus was passively attached to polystyrene to differentiate antibody responses of cattle vaccinated with B abortus strain 19 from those of naturally infected cattle. In the indirect assay, using O polysaccharide as antigen, a single serum dilution was used and mouse monoclonal antibody to bovine L chain conjugated with horseradish peroxidase was the detection reagent. Measurable antibody was not found in sera of vaccinated cattle, except for 3 sera from cattle that were persistently infected with strain 19. Sera from 25 cattle infected with pathogenic strains contained antibody on the basis of results of indirect enzyme immunoassay, using smooth lipopolysaccharide or O chain as antigens, or results of competitive enzyme immunoassay, using the O-chain antigen. Results in sera from calves with experimentally induced Yersinia enterocolitica serotype 0:9 infection or inoculated with a low dose of B abortus strain 2308 were comparable with those in sera of cattle that were vaccinated with strain 19. The data correlated with those from competitive enzyme immunoassay, using one serum dilution and horseradish peroxidase-conjugated mouse monoclonal antibody to smooth lipopolysaccharide. On the basis of results of the indirect enzyme immunoassay, all sera (except those samples obtained before inoculation) contained antibody to smooth lipopolysaccharide.     

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.     

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.     

Validation of a second generation competitive enzyme immunoassay (CELISA) for the diagnosis of brucellosis in various species of domestic animals

A second generation competitive enzyme immunoassay (CELISA) for detection of bovine antibody to Brucella abortus was developed to eliminate reagent variables in the assay. This assay was different from earlier CELISA formats in that it used recombinant protein A and protein G immunoglobulin receptors (PAG), labelled with horseradish peroxidase, thus eliminating the requirement for polyclonal anti-mouse-enzyme conjugate for detection. This allowed standardization of the assay. The CELISA uses a monoclonal antibody specific for a common epitope of the O-polysaccharide (OPS) of smooth lipopolysaccharide (SLPS) derived from B. abortus S1119.3. This antibody did not react with PAG. This monoclonal antibody was used to compete with antibody in the bovine test serum to the smooth lipopolysaccharide (SLPS) antigen. Reaction of bovine antibody was then measured directly with the PAG enzyme conjugate. In this case, development of colour in the reaction indicated a positive reaction. The performance characteristics of the new CELISA, sensitivity, specificity and exclusion of antibody of B. abortus S19 vaccinated animals, were very similar to those of the classical CELISA and to the indirect enzyme immunoassay (IELISA) when using sera deemed positive by isolation of the bacterium, either from individual animals or from some animals on the premises. All sera were tested by the buffered antigen plate agglutination test (BPAT) and the complement fixation test (CFT). Only samples positive on both BPAT and CFT were considered as positive and only samples negative on both tests were used considered negative. Sufficient samples from cattle, swine, sheep and goats to validate the test were included based on OIE guidelines suggesting inclusion of a minimum of 300 positive and 1000 negative samples.