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1.
Rev. argent. microbiol ; 38(4): 209-215, oct.-dic. 2006. ilus, graf, tab
Artigo em Inglês | LILACS | ID: lil-634529

RESUMO

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.


Assuntos
Animais , Bovinos , Feminino , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Vírus da Diarreia Viral Bovina/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Cultura de Vírus/métodos , Sangue/virologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/diagnóstico , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/virologia , DEAE-Dextrano/farmacologia , Brometo de Hexadimetrina/farmacologia , Rim , Plasma/virologia , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade
2.
Rev Argent Microbiol ; 38(4): 209-15, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17370574

RESUMO

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.


Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Vírus da Diarreia Viral Bovina/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Cultura de Vírus/métodos , Animais , Sangue/virologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/diagnóstico , Bovinos/microbiologia , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/virologia , DEAE-Dextrano/farmacologia , Feminino , Brometo de Hexadimetrina/farmacologia , Rim , Plasma/virologia , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade
3.
Prev Vet Med ; 72(1-2): 49-54; discussion 215-9, 2005 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-16253360

RESUMO

Detection of bovine virus diarrhoea virus (BVDV) in one vaccinated beef cattle and three non-vaccinated dairy herds was investigated on peripheral blood leukocytes (PBL) with or without previous treatment followed by a capture ELISA (cELISA). Using the combination of PHA and polycation treatment, PBL from 229 seropositive cattle were studied and could be classified in four different states of BVDV infection. Lysed PBL from four animals were directly positive in cELISA (Category I), PBL of 17 animals were positive after PHA stimulation (Category II), 15 animals were positive only after PHA stimulation plus polycation treatment (Category III), while virus could not be detected in 193 seropositive cattle. Wild-type BVDV strains were isolated by co-culture on polycation-treated MDBK cells from 11 of these seropositive animals. BVDV antibodies of these same animals were able to neutralize their own virus, indicating that virus persists in PBL in spite of strain-specific antibodies. No apparent change of leukocyte subpopulations could be detected in any category of virus-positive animals. Thus, BVDV may be present in the PBL of some cattle, even in the presence of a specific active immune response.


Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Portador Sadio/veterinária , Portador Sadio/virologia , Vírus da Diarreia Viral Bovina/isolamento & purificação , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/diagnóstico , Doença das Mucosas por Vírus da Diarreia Viral Bovina/epidemiologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/imunologia , Portador Sadio/diagnóstico , Portador Sadio/imunologia , Bovinos , Vírus da Diarreia Viral Bovina/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Leucócitos/imunologia
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