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.

Differentiation of a porcine reproductive and respiratory syndrome virus vaccine strain from North American field strains by restriction fragment length polymorphism analysis of ORF 5.

The suitability of restriction fragment length polymorphism (RFLP) analysis for differentiating a porcine reproductive and respiratory syndrome virus (PRRSV) vaccine strain from other North American field strains was investigated. Open reading frame 5 nucleotide sequence data of the vaccine virus, its parent strain VR-2332, and 22 other strains of PRRSV included in this study indicated that 3 restriction enzyme gel patterns characterize the vaccine virus and the parent strain genotype. The combined 3 RFLP patterns differentiate the vaccine and parent virus from other PRRSV strains. This test will be a valuable tool in epidemiologic studies and will be useful in identifying individual strains in cases of multistrain PRRSV infections.

Characterization of the humoral immune response to porcine reproductive and respiratory syndrome (PRRS) virus infection.

The development of the humoral immune response against porcine reproductive and respiratory syndrome (PRRS) virus was monitored by an indirect fluorescent antibody (IFA) test, immunoperoxidase monolayer assay (IPMA), enzyme-linked immunosorbent assay (ELISA), and serum virus neutralization (SVN) test over a 105-day period in 8 pigs experimentally infected with ATCC strain VR-2402. Specific antibodies against PRRS virus were first detected by the IFA test, IPMA, ELISA, and the SVN test 9-11, 5-9, 9-13, and 9-28 days postinoculation (PI), respectively, and reached their maximum values by 4-5, 5-6, 4-6, and 10-11 weeks PI, respectively, thereafter. After reaching maximum value, all assays showed a decline in antibody levels. Assuming a constant rate of antibody decay, it was estimated by regression analysis that the ELISA, IFA, IPMA, and SVN antibody titers would approach the lower limits of detection by approximately days 137, 158, 324, and 356 PI, respectively. In this study, the immunoperoxidase monolayer assay appeared to offer slightly better performance relative to the IFA test, ELISA, and SVN test in terms of earlier detection and slower rate of decline in antibody titers. Western immunoblot analysis revealed that antibody specific for the 15-kD viral protein was present in all pigs by 7 days PI and persisted throughout the 105-day observation period. Initial detection of antibodies to the 19-, 23-, and 26-kD proteins varied among pigs, ranging from 9 to 35 days PI. Thereafter, the antibody responses to these 3 viral proteins of PRRS virus continued to be detected throughout the 105-day study period.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and characterization of a bovine rhinovirus isolated from Iowa cattle with acute respiratory tract disease.

Viral isolate FS1-43 isolated from a calf with acute respiratory tract disease was characterized as a bovine rhinovirus. This virus was antigenically indistinguishable from rhinovirus strains C-07 and VC-96. The virus replicated in low passage Madin-Darby and Georgia bovine kidney cells. A relative noncytopathogenic replication occurred in epithelial bovine turbinate, bovine fibroblastic turbinate, and bovine lung cells. Ciliary activity of tracheal explants was unaffected during eight serial passages. Viral replication was detected earlier and viral titers were higher at 33 C than at 37 C. Rotation of cultures did not affect viral titers but was necessary to produce a cytopathic effect in Madin-Darby bovine kidney cells.

Comparative pathogenicity of selected bovine viral diarrhea virus isolates in gnotobiotic lambs.

The infectivity and pathogenicity of selected bovine viral diarrhea virus (BVDV) isolates were determined in gnotobiotic, colostrum-deprived neonatal lambs. Five-day-old cesarean-derived gnotobiotic lambs were exposed to 1 of 10 BVDV isolates via aerosol suspension. These isolates were from tissues or secretions of calves or lambs affected with respiratory tract disease, weak neonatal calves, aborted bovine fetuses, or reference Singer or Draper BVDV. The pathogenicity of each isolate, relative to the others, was evaluated in lambs by measurement of the neutralizing antibody response, virus isolation from nasal secretions or tissues, and postmortem lesions. The BVDV isolates varied in their infectivity and pathogenicity. Singer, the cytopathic reference strain, was the most lymphotrophic isolate and stimulated the greatest neutralizing antibody response. Encephalitis was the most consistent lesion observed and was used as the final determinant of relative pathogenicity of the viruses. The most neuropathogenic isolates were the 2 viruses originating from lambs affected with respiratory tract disease, the 2 weak neonatal calf isolates, and 1 isolate from an aborted bovine fetus. The least pathogenic isolates were the 2 reference isolates, Draper and Singer; the 2 mucosal disease isolates; and 1 isolate originating from an aborted bovine fetus.

Serologic survey of selected pathogens in white-tailed and mule deer in western Nebraska.

Exposure of free-ranging white-tailed deer (Odocoileus virginianus) and mule deer (Odocoileus hemionus) in western Nebraska to selected livestock pathogens was determined by serology and attempted virus isolation. Antibodies to bluetongue virus, epizootic hemorrhagic disease virus, and bovine respiratory syncytial virus were present in both species of deer. No serologic reactors to Brucella or Anaplasma were found. Attempts to isolate bluetongue virus were negative.

Serosurvey for selected pathogens in hunter-killed pronghorns in western Nebraska.

Exposure of pronghorns (Antilocapra americana) in western Nebraska in 1983 to selected livestock pathogens was examined by serology and attempted virus isolation. Antibodies were present to the agents of bluetongue, epizootic hemorrhagic disease, and bovine respiratory syncytial virus. There were no serologic reactors to Brucella, and attempts to isolate the viruses of bluetongue and epizootic hemorrhagic disease were negative.

Conjunctivitis associated with a Mycoplasma-like organism in swine.

A midwestern producer reported high incidence of conjunctivitis and keratoconjunctivitis in a herd of crossbred finishing swine. Complete necropsy was performed on 3 pigs with bilateral mucopurulent conjunctivitis and chemosis; other gross lesions were not seen. Mycoplasma sp was isolated from conjunctival swab specimens obtained from 1 pig; small numbers of streptococci and coagulase-negative staphylococci were isolated from conjunctival swab specimens from all 3 pigs. Neither swine influenza virus nor pseudorabies virus was isolated from conjunctival swab specimens. Histologically, the 3 pigs had chronic lymphoplasmacytic conjunctivitis with lymphofollicular hyperplasia and foci of epithelial and goblet cell hyperplasia. Ultrastructural examination of conjunctival specimens from the 3 pigs revealed large numbers of Mycoplasma-like organisms adhered to superficial conjunctival cells. Mycoplasma-like organisms also were seen in membrane-bound vacuoles in superficial conjunctival cells. Bacteria (including chlamydiae) or viruses were not seen ultrastructurally. The lymphoproliferative nature of the conjunctival lesion and the evidence of adhered and intracellular organisms suggested an etiologic role for a Mycoplasma-like organism in the disease in these pigs.

Detection of a cell line contaminated with hog cholera virus.

Cell lines from the repository of the American Type Culture Collection were examined for possible contamination with bovine viral diarrhea virus. During testing, hog cholera virus (HCV) was detected in the IB-RS-2 D10 porcine kidney cell line. This variant of HCV was avirulent for pigs and seldom induced detectable concentrations of antibody against reference viruses (HCV-Ames or bovine viral diarrhea virus-NY1) in serum of inoculated pigs. Additionally, this variant of HCV did not confer protection to pigs against virulent HCV. The contaminated cell line had been distributed to > 20 laboratories in the United States. The cell line was not used in field studies and has been destroyed.

Bovine respiratory syncytial virus.

The current knowledge is reviewed in regards to the importance of bovine respiratory syncytial virus in the bovine respiratory disease complex. The epidemiology, clinical disease, pathologic findings, pathogenesis, diagnosis, treatment, and prevention of this viral disease are discussed.

Experimental transmission of an apparent viral pneumonia in conventional and gnotobiotic pigs.

Endemic pneumonia in five- to eight-week-old pigs induced microscopic lesions of proliferative interstitial pneumonia which were compatible with a viral aetiology. The disease was transmitted experimentally to conventional and gnotobiotic pigs by means of a lung homogenate filtered through a 0.22 micron filter. No common viral respiratory pathogens of pigs were isolated. Two types of virus particles were observed in cell culture by electron microscopy; one was about 70 nm in diameter and had an envelope and short surface spicules, the other also had an envelope, was elongated, pleomorphic, measured 80 x 320 nm and was coated by antibodies.