Bluetongue virus: production and study of viral antigen for serological diagnosis.

A soluble antigen, produced from the culture supernatant of VERO cells infected with bluetongue virus serotype 4 (BTV-S4) and concentrated by sequential ultrafiltration with membranes with cut-off values 10(3) and 25 x 10(3) NMWP, showed complete identity to standard antigens when compared by agar gel immunodiffusion (AGID) and SDS-PAGE profiles, revealing that the main protein component responsible for the AGID reaction has a molecular weight of about 60 kDa corresponding probably to the NS1 protein.

Comparison of indirect immunofluorescence with agar gel immunodiffusion for the diagnosis of bluetongue virus infection.

1. Sera from 190 cows and from 72 sheep were examined to compare the results obtained with the agar gel immunodiffusion (AGID) and indirect immunofluorescence (IIF) tests for the diagnosis of bluetongue (BT) disease. 2. In the AGID test, 96 of 190 (50.5%) cattle serum samples and 38 of 72 (52.7%) sheep serum samples were positive, for a total of 134 out of 262 (51.1%) sera. In the IIF test, 98 of 190 (51.6%) cattle serum samples and 39 of 72 (54.2%) sheep serum samples were positive, for a total of 137 out of 262 (52.3%) sera. 3. The fluorescence of the IIF test presented a granular cytoplasmic aspect, which in some cells was observed only on the cell membranes. 4. Statistical analysis of the data showed close agreement between the two techniques, regardless of the kind of sera examined. The IIF test showed high sensitivity (93.8% and 92.1%), specificity (91.4% and 88.2%) and positive (91.8% and 89.7%) and negative (93.48% and 90.9%) predictive values for cattle serum and sheep serum, respectively. 5. The results obtained with IIF were comparable to those obtained with the AGID test, indicating that both techniques can be used routinely in epidemiologic studies of BT. However, the IIF offers the additional advantages that it can be used for antibody quantification and for the detection of viral antigens in BT-infected cell lines.

Identificación de anticuerpos de la lengua azul por la técnica de inmunodifusión en gel de agar

Se preparó un antígeno soluble del virus de la lengua azul (VLA) para ser utilizado en pruebas de inmunodifusión en gel de agar (IDGA). Dicho antígeno es grupo específico, y es capaz de detectar anticuerpos inducidos por cualquiera de los 24 serotipos del VLA. Fue producido a partir del VLA serotipo 4 y controlado en IDGA frente a antígeno y sueros de referencia (NVSL, Ames, EUA; LARA, Campinas, Brasil; Veterinary Diagnostic Technology, Inc., EUA) y por la técnica inmunoenzimática (ELISA) con anticuerpo monoclonal 3-17-A3 (IADR, Pirbright, Inglaterra). Todas las pruebas mostraron una reacción de total identidad con los reactivos controles.

A soluble antigen of the bluetongue virus (BTV) was prepared for use in immunodifusion in agar gel tests (IDAG). The antigen is group specific and is capable of detecting antibodies induced by any of the 24 BTV serotypes. It was produced from type 4 BTV serotype and controled in IDAG against reference antigens and sera (NVSL, Ames, USA; LARA, Campinas, Brazil; Veterinary Diagnostic Technology, Inc. USA) and by the enzyme-linked immunosorbent assay (ELISA) with 3-17-A3 monoclonal antibody (IADR, Pirbright, England). All tests yielded a reaction of total identify with the control reagents.

Comparison of indirect immunofluorescencee with agar gel immunodiffusion for the diagnosis of bluetongue virus infection

Sera from 190 cows and from 72 sheep were examined to compare the results obtained with the agar gel immundiffusion (AGIP) and indirect immunofluorescence (IIF) tests for the diagnosis of bluetongue (BT) disease. In the AGIP test, 96 of 190 (50.5%) cattle serum samples and 38 of 72 (52.7%) sheep serum samples were positive, for a total of 134 out of 262 (51.1%) sera. In the IIF test, 98 of 190 (51.6%) cattle serum samples and 39 of 72 (54.2%) sheep serum samples were positive, for a total of 137 out of 262 (52.3%) sera. The fluorescence of the IIF test presented a granular cytoplasmic aspect, which in some cells was observed only on the cell membranes. Statistical analysis of the data showed close agreement between the two techniques, regardless of the kind of sera examined. The IIF test showed high sensitivity (93.8% and 92.1%), specificity (91.4% and 88.2%) and positive (91.8% and 89.7%) and negative (93.48% and 90.9%) predictive values for cattle serum and sheep serum, respectively. The results obtained with obtained with IIF were comparable to those obtained with the AGIP test, indicating that both techniques can be used routinely in epidemiologic studies of BT. However, the IIF offers the additional advantages that it can be used for antibody quantification and for the detection of viral antigens in BT-infected cell lines