Detection of bovine viral diarrhea virus by amplification on polycation-treated cells followed by enzyme immunoassay / Detección del virus de la diarrea viral bovina por amplificación sobre células tratadas con policationes seguida de enzimoinmunoensayo

A bovine viral diarrhea virus (BVDV) amplification method combined with an enzyme immunoassay was developed to detect BVDV antigens in seropositive cattle. Reconstitution assays conducted by adding decreasing amounts of BVDV (Singer strain) to Madin-Darby bovine kidney (MDBK) cells showed that the sensitivity threshold of the combined assay was 10-7 TCID50. BVDV amplification was carried out in polycation (DEAE-Dextran and polybrene)- treated MDBK cells. Treated cells were able to replicate both ether-treated virus and neutralizing antibody-coated virus. Ammonium chloride decreased virus replication in polycation-treated cells, suggesting viral penetration by endocytosis. BVDV detection was tested in leukocytes from 104 seropositive cattle from 2 unvaccinated commercial closed dairy herds with high seroprevalence. Lysates and co-cultures of peripheral blood leukocytes (PBL) were tested, directly or after up to 6 blind passages in normal or polycation-treated cells. BVDV was detected in 10/104 cattle after only one co-culture of PBL in treated cells. No virus was detected in whole blood or plasma samples. BVDV positive and negative cattle were retested three times, achieving consistent results. The finding of immune carriers supports the possibility that these animals may constitute an epidemiological risk.

Se desarrolló un método de detección de antígenos del virus de la diarrea viral bovina (BVDV) combinando amplificación viral con enzimoinmunoensayo. El método combinado presentó una sensibilidad de 10-7 TCID50 en ensayos con diluciones decrecientes de BVDV cepa Singer sobre la línea celular MDBK. La amplificación del título viral se efectuó sobre células MDBK tratadas con policationes Estas células replicaron tanto el BVDV tratado con éter como el unido a anticuerpos. La replicación viral en las células tratadas disminuyó ante la presencia de cloruro de amonio, lo que sugiere la penetración viral por endocitosis. El BVDV se determinó en leucocitos de 104 bovinos seropositivos de dos rodeos en producción, cerrados y con alta seroprevalencia. Los leucocitos de sangre periférica (LSP) fueron lisados y analizados directamente o luego de hasta 6 pasajes ciegos sobre células normales o tratadas con policationes. El BVDV se detectó en 10 de los 104 animales después de solamente un cultivo de LSP en células tratadas. No se pudo detectar presencia viral en las muestras de sangre o plasma. Los estudios se repitieron tres veces en animales BVDV positivos y negativos, con resultados consistentes. El hallazgo de bovinos seropositivos portadores del virus indica la posibilidad de que estos animales puedan significar un riesgo epidemiológico.

Adjuvant effect of DEAE-dextran and tetanus toxoid on whole cell heat inactivated phenol preserved typhoid vaccine.

Active mouse protection test (AMPT) and enzyme linked immunosorbent assay (ELISA) were used to determine the immunogenicity of whole cell typhoid vaccine when administered in conjunction with either tetanus toxoid (TT) or DEAE-Dextran (DD). Immunization of mice with whole cell typhoid vaccine showed enhanced potency either when administered in conjunction with TT or DD and values were statistically significant (p < 0.05) in comparison to conventional or standard typhoid vaccines. For ELISA, the mice were immunized with 2 different schedules, one in which a single dose of 0.25 ml subcutaneously (s/c) was administered and in another two doses of 0.25 ml each s/c, 14 days apart. In case of single dose schedule of immunization D vaccine (Whole cell typhoid + 5 mg/ml DD) showed significant increase of immune response (3.201 log10) as compared to plain vaccine (2.550 log10). Two dose schedule further increased the titres to 3.856 log10. DD adjuvanted vaccine showed higher potency by AMPT as compared to the TT adjuvanted vaccine or plain vaccine. The present study clearly demonstrates that a single dose of 0.25 ml which is equivalent to half of the conventionally used single human dose of typhoid vaccine adjuvanted with DD can significantly improve the immunogenicity of the vaccine.