RESUMO
In this communication, we report the presence of RNA of bovine viral diarrhea virus (BVDV) as a contaminant of different biological products used in Mexico for veterinary vaccine production. For this purpose, six batches of monovalent vaccines, eight cell line batches used for vaccine production, and 10 fetal bovine serum lots (FBS) commercially available in Mexico from different suppliers were tested by reverse transcription polymerase chain reaction (RT-PCR). Viral RNA was detected in 62.5% of the samples analyzed. Phylogenetic analysis revealed the presence of the subgenotypes BVDV-1a, 1b, and BVDV-2a in the tested samples. Collectively, these findings indicate that contamination by BVDV RNA occurs in commercial vaccines and reagents used in research and production of biological products. The ramifications of this contamination are discussed.
Assuntos
Vírus da Diarreia Viral Bovina Tipo 1/genética , Vírus da Diarreia Viral Bovina Tipo 2/genética , Vacinas Virais/genética , Animais , Doença das Mucosas por Vírus da Diarreia Viral Bovina/imunologia , Bovinos , Linhagem Celular , Vírus da Diarreia Viral Bovina Tipo 1/imunologia , Vírus da Diarreia Viral Bovina Tipo 2/imunologia , Genótipo , Síndrome Hemorrágica Bovina/microbiologia , México , Filogenia , RNA Viral/genética , Vacinas Virais/imunologiaRESUMO
The pestiviruses bovine viral diarrhea virus 1 and 2 (BVDV-1 and -2, respectively) and HoBi-like pestivirus (HoBiPeV) are important pathogens of cattle, and a number of reverse-transcription PCR (RT-PCR)-based assays have been developed for their detection in clinical specimens. We evaluated a newly designed set of pan-bovine pestivirus primers (BP189-389) in a gel-based RT-PCR screening test for pestiviruses in the sera of beef calves destined for export from southern Brazil. Serum samples positive for BVDV antigens by an antigen ELISA ( n = 135) were submitted to RT-PCR assays using different sets of primers, followed by nucleotide sequencing of the amplicons. RT-PCR with pestivirus primers 324-326 detected 110 positive samples: BVDV-1 ( n = 62), BVDV-2 ( n = 38), and HoBiPeV ( n = 10). A PCR using primers HCV90-368 detected 97 positive samples (64 BVDV-1; 33 BVDV-2). An additional RT-PCR round using BVDV-2-specific primers (2F-2R) detected 45 positive samples (including 38 detected by primers 324-326 and 33 by HCV90-368); whereas a RT-PCR using HoBiPeV-specific primers (N2-R5) detected 26 positive samples (including 10 detected by primers 324-326).The assay using the primers BP189-389 detected all 135 ELISA-positive samples, including the 26 HoBiPeV detected by primers N2-R5. Our results demonstrated that primers BP189-389 compare favorably against other primer sets in the detection of bovine pestiviruses, especially HoBiPeV. This conventional PCR may be useful for efficient detection of pestiviruses in bovine sera and other specimens as well, especially in laboratories without real-time PCR equipment.
Assuntos
Doenças dos Bovinos/diagnóstico , Monitoramento Epidemiológico/veterinária , Infecções por Pestivirus/veterinária , Pestivirus/isolamento & purificação , Animais , Doença das Mucosas por Vírus da Diarreia Viral Bovina/diagnóstico , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Brasil , Bovinos , Doenças dos Bovinos/virologia , Vírus da Diarreia Viral Bovina/isolamento & purificação , Síndrome Hemorrágica Bovina/diagnóstico , Síndrome Hemorrágica Bovina/virologia , Infecções por Pestivirus/diagnóstico , Infecções por Pestivirus/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterináriaRESUMO
The detection of Bovine Viral Diarrhea virus (BVDV) infection, especially among persistently infected calves (PI), should be performed earlier in order to eliminate the source of the infection and to prevent the spread of the disease in the herd. However, colostrum intake can influence the results of some of the tests used to diagnose the BVDV infection. Therefore, this study evaluated the efficacy of serum neutralization (SN) test in conjunction with reverse transcription-polymerase chain reaction (RT-PCR) for the diagnosis of BVDV infection before colostrum intake. The deliveries of the animals were assisted to select 52 newborn Holstein calves for inclusion in the study. Initially, the whole blood and serum samples were collected from the calves before (T0) and after (T1) the colostrum intake. The calves that were RT-PCR positive at any of the time-points were retested on the 30th day post birth (T2). The presence of specific antibodies for BVDV was evaluated by SN, and that of viral RNA by the RT-PCR. The BVDV-specific antibodies were observed in the serum of 13.46% (7/52) of the calves at T0 because of fetal infection. At T1, seroconversion was observed in 100% (52/52) of the calves. The geometric mean titers (GMT) of the antibodies for BVDV increased significantly from T0 (14.52) to T1 (2490) (P = 0.0001). Of the four calves that were RT-PCR positive before colostrum...(AU)
Infecção causada pelo BVDV, especialmente bezerras persistentemente infectadas (PI), deve ser detectada precocemente para eliminação da fonte de infecção e disseminação da doença no rebanho. No entanto, a mamada de colostro interfere em alguns testes adotados para o diagnóstico da Diarréia Viral Bovina. Assim, esta pesquisa avaliou o uso da soroneutralização (SN) em associação com a reação em cadeia de polimerase precedida da transcrição reversa (RT-PCR) para diagnóstico da infecção pelo BVDV antes da mamada de colostro. Partos foram acompanhados para seleção de 52 bezerras da raça Holandesa. Inicialmente foram coletadas amostras de sangue total e soro de todos os animais antes (T0) e após a mamada do colostro (T1). Os animais positivos no RT-PCR em qualquer momento foram retestados aos 30 dias de vida (T2). A detecção de anticorpos específicos para o BVDV foi feita por meio da técnica de soroneutralização e a detecção do RNA viral pela técnica de RT-PCR. Foram observados anticorpos neutralizantes no soro sanguíneo em 13,46% (7/52) bezerras no T0, proveniente de infecção fetal; e no T1 observou-se soroconversão de 100% (52/52) das bezerras. Os títulos médios geométricos (GMT) de anticorpos para BVDV aumentaram significativamente do T0 (14,52) para o T1 (2.490) (P=0,0001). Quatro bezerros foram positivos no RT-PCR antes da mamada de colostro (T0), sendo que dois deles eram...(AU)
Assuntos
Animais , Bovinos , Vírus da Diarreia Viral Bovina/patogenicidade , Síndrome Hemorrágica Bovina/diagnóstico , Colostro/virologiaRESUMO
The detection of Bovine Viral Diarrhea virus (BVDV) infection, especially among persistently infected calves (PI), should be performed earlier in order to eliminate the source of the infection and to prevent the spread of the disease in the herd. However, colostrum intake can influence the results of some of the tests used to diagnose the BVDV infection. Therefore, this study evaluated the efficacy of serum neutralization (SN) test in conjunction with reverse transcription-polymerase chain reaction (RT-PCR) for the diagnosis of BVDV infection before colostrum intake. The deliveries of the animals were assisted to select 52 newborn Holstein calves for inclusion in the study. Initially, the whole blood and serum samples were collected from the calves before (T0) and after (T1) the colostrum intake. The calves that were RT-PCR positive at any of the time-points were retested on the 30th day post birth (T2). The presence of specific antibodies for BVDV was evaluated by SN, and that of viral RNA by the RT-PCR. The BVDV-specific antibodies were observed in the serum of 13.46% (7/52) of the calves at T0 because of fetal infection. At T1, seroconversion was observed in 100% (52/52) of the calves. The geometric mean titers (GMT) of the antibodies for BVDV increased significantly from T0 (14.52) to T1 (2490) (P = 0.0001). Of the four calves that were RT-PCR positive before colostrum...
Infecção causada pelo BVDV, especialmente bezerras persistentemente infectadas (PI), deve ser detectada precocemente para eliminação da fonte de infecção e disseminação da doença no rebanho. No entanto, a mamada de colostro interfere em alguns testes adotados para o diagnóstico da Diarréia Viral Bovina. Assim, esta pesquisa avaliou o uso da soroneutralização (SN) em associação com a reação em cadeia de polimerase precedida da transcrição reversa (RT-PCR) para diagnóstico da infecção pelo BVDV antes da mamada de colostro. Partos foram acompanhados para seleção de 52 bezerras da raça Holandesa. Inicialmente foram coletadas amostras de sangue total e soro de todos os animais antes (T0) e após a mamada do colostro (T1). Os animais positivos no RT-PCR em qualquer momento foram retestados aos 30 dias de vida (T2). A detecção de anticorpos específicos para o BVDV foi feita por meio da técnica de soroneutralização e a detecção do RNA viral pela técnica de RT-PCR. Foram observados anticorpos neutralizantes no soro sanguíneo em 13,46% (7/52) bezerras no T0, proveniente de infecção fetal; e no T1 observou-se soroconversão de 100% (52/52) das bezerras. Os títulos médios geométricos (GMT) de anticorpos para BVDV aumentaram significativamente do T0 (14,52) para o T1 (2.490) (P=0,0001). Quatro bezerros foram positivos no RT-PCR antes da mamada de colostro (T0), sendo que dois deles eram...
Assuntos
Animais , Bovinos , Colostro/virologia , Síndrome Hemorrágica Bovina/diagnóstico , Vírus da Diarreia Viral Bovina/patogenicidadeRESUMO
São descritos os aspectos epidemiológicos, clínicos e patológicos de um surto de intoxicação por Pteridium arachnoideum e Pteridium caudatum em bovinos no Estado de Mato Grosso. A distribuição dessas plantas no Estado, a intensidade de invasão de pastagens e alguns fatores associados à invasão das pastagens por Pteridium spp. são também descritos. Pteridium spp. foi observada em 83 propriedades de nove municípios de Mato Grosso e deste total, amostras de 22 propriedades foram coletadas para identificação taxonômica. Em 22 propriedades identifica-se P. arachnoideum e em duas dessas havia também P. caudatum. O desmatamento e a realização de queimadas parecem estar relacionados com a invasão de Pteridium spp. nas pastagens. Na propriedade em que ocorreu a doença, 306 bovinos foram introduzidos em uma pastagem formada por Brachiaria brizantha intensamente invadida por P. arachnoideum e P. caudatum e desses 22 bovinos adoeceram e morreram. Os principais sinais clínicos foram febre alta, apatia, fadiga, fraqueza e decúbito. Na necropsia havia graus variáveis de hemorragias em vários órgãos e cavidades e infartos ocasionais. Microscopicamente, a alteração mais importante consistiu em aplasia severa da medula óssea vermelha. Os achados epidemiológicos, clínicos e patológicos associados à identoificação taxonômica da planta fundamentam o diagnóstico de intoxicação aguda por P. arachnoideum e P. caudatum em bovinos. O crescente desmatamento e a frequente utilização de queimadas em Mato Grosso podem contribuir para que esta doença venha a ser uma importante fonte de prejuízos para a pecuária do Estado.
Epidemiological features, clinical signs and pathological findings of a spontaneous acute poisoning by Pteridium arachnoideum and Pteridium caudatum in cattle in the State of Mato Grosso, Brazil, are described. The plant distribution, magnitude and some aspects about the pasture invasion by P. arachnoideum and P. caudatum in the State are also described. Pteridium spp. were found in 83 farms from nine counties and 22 of them were submitted to taxonomic identification. P. arachnoideum was found in 22 farms and in two farms also P. caudatum. Deforestation and burning appear to be associated with pasture invasion by Pteridium spp. On the farm where the poisoning occurred 306 cattle were introduced into a pasture of Brachiaria brizantha intensely invaded by P. arachnoideum and P. caudatum. Twenty two cattle were poisoned by the plants and died. The main clinical signs were hemorrhages, high fever, apathy, fatigue, weakness and recumbency. Gross findings included variable degrees of hemorrhages in several organs and cavities with occasional infarcts. The most consistent histological finding was marked bone marrow aplasia. Epidemiological, clinical and pathological findings associated with the taxonomic identofication of the plant support the diagnosis of acute poisoning by P. arachnoideum and P. caudatum in cattle. The increasing rate of deforestation and frequent burning in Mato Grosso can contribute to this disease which becomes a major cause of economic losses to livestock production in the State.
Assuntos
Animais , Bovinos , Avaliação de Sintomas/veterinária , Bovinos , Medula Óssea/fisiopatologia , Pastagens/efeitos adversos , Pteridium/intoxicação , Síndrome Hemorrágica Bovina , Intoxicação por Plantas/veterinária , Dispersão VegetalRESUMO
São descritos os aspectos epidemiológicos, clínicos e patológicos de um surto de intoxicação por Pteridium arachnoideum e Pteridium caudatum em bovinos no Estado de Mato Grosso. A distribuição dessas plantas no Estado, a intensidade de invasão de pastagens e alguns fatores associados à invasão das pastagens por Pteridium spp. são também descritos. Pteridium spp. foi observada em 83 propriedades de nove municípios de Mato Grosso e deste total, amostras de 22 propriedades foram coletadas para identificação taxonômica. Em 22 propriedades identifica-se P. arachnoideum e em duas dessas havia também P. caudatum. O desmatamento e a realização de queimadas parecem estar relacionados com a invasão de Pteridium spp. nas pastagens. Na propriedade em que ocorreu a doença, 306 bovinos foram introduzidos em uma pastagem formada por Brachiaria brizantha intensamente invadida por P. arachnoideum e P. caudatum e desses 22 bovinos adoeceram e morreram. Os principais sinais clínicos foram febre alta, apatia, fadiga, fraqueza e decúbito. Na necropsia havia graus variáveis de hemorragias em vários órgãos e cavidades e infartos ocasionais. Microscopicamente, a alteração mais importante consistiu em aplasia severa da medula óssea vermelha. Os achados epidemiológicos, clínicos e patológicos associados à identoificação taxonômica da planta fundamentam o diagnóstico de intoxicação aguda por P. arachnoideum e P. caudatum em bovinos. O crescente desmatamento e a frequente utilização de queimadas em Mato Grosso podem contribuir para que esta doença venha a ser uma importante fonte de prejuízos para a pecuária do Estado.(AU)
Epidemiological features, clinical signs and pathological findings of a spontaneous acute poisoning by Pteridium arachnoideum and Pteridium caudatum in cattle in the State of Mato Grosso, Brazil, are described. The plant distribution, magnitude and some aspects about the pasture invasion by P. arachnoideum and P. caudatum in the State are also described. Pteridium spp. were found in 83 farms from nine counties and 22 of them were submitted to taxonomic identification. P. arachnoideum was found in 22 farms and in two farms also P. caudatum. Deforestation and burning appear to be associated with pasture invasion by Pteridium spp. On the farm where the poisoning occurred 306 cattle were introduced into a pasture of Brachiaria brizantha intensely invaded by P. arachnoideum and P. caudatum. Twenty two cattle were poisoned by the plants and died. The main clinical signs were hemorrhages, high fever, apathy, fatigue, weakness and recumbency. Gross findings included variable degrees of hemorrhages in several organs and cavities with occasional infarcts. The most consistent histological finding was marked bone marrow aplasia. Epidemiological, clinical and pathological findings associated with the taxonomic identofication of the plant support the diagnosis of acute poisoning by P. arachnoideum and P. caudatum in cattle. The increasing rate of deforestation and frequent burning in Mato Grosso can contribute to this disease which becomes a major cause of economic losses to livestock production in the State.(AU)
Assuntos
Animais , Bovinos , Bovinos , Pastagens/efeitos adversos , Pteridium/intoxicação , Avaliação de Sintomas/veterinária , Medula Óssea/fisiopatologia , Intoxicação por Plantas/veterinária , Dispersão Vegetal , Síndrome Hemorrágica BovinaRESUMO
The aim of this work was to study the in vitro amplification of BVDV (Pestivirus, Flaviridae) field isolates from Argentina in MDBK, BoTur and BHK-21 continuous cell lines. Field isolates 99/134 (mucosal disease), 00/693 (mucosal disease), 04P7016 (respiratory disease) and 04/89 (mucosal disease), genotype 1b, were used and compared with the Singer and NADL reference strains, genotype 1a. Additionally, cell lines derived from explants of bovine testis (RD-420), bovine uterus (NCL-1) and porcine kidney (PKZ) were tested as alternative substrates for BVDV propagation in vitro. The effect of cell line, harvest time and infection protocol was evaluated. The viral titers observed depended on the virus and harvest time but not on the infection protocol. We found that MDBK and BoTur cell lines were susceptible to the infection whereas BHK-21 and PKZ were not. NADL viral titers, 00/693 and 04/89, increased from 24 to 48 h p.i. in BoTur cells and then reached a plateau, whereas those of 99/134 and 04P7016 remained constant between 24 and 72 h p.i. BVDV Singer, on the other hand, presented a maximum titer at 24 h p.i. and then decreased. BVDV-NADL titers increased in MDBK and NCL-1 but not in RD-420 between 24 and 48 h p.i., and then decreased at 72 h p.i. These facts lead us to conclude that neither the subgenotypes (1a, 1b) nor the clinical symptoms of the animal from the virus had been isolated seem to affect the virus cell line kinetics of viral replication in vitro. On the other hand, the most homogenous behavior, the most similar replication curves, and highest titers observed in MDBK and NCL-1 seem to indicate that these lines are generally more susceptible to BVDV replication.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Vírus da Diarreia Viral Bovina/crescimento & desenvolvimento , Síndrome Hemorrágica Bovina/virologia , Cultura de Vírus/métodos , Replicação Viral , Animais , Argentina/epidemiologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/epidemiologia , Bovinos , Técnicas de Cultura de Células/métodos , Linhagem Celular/virologia , Cricetinae , Vírus da Diarreia Viral Bovina/isolamento & purificação , Cães , Feminino , Síndrome Hemorrágica Bovina/epidemiologia , Rim/citologia , Masculino , Mesocricetus , Especificidade de Órgãos , Suínos , Testículo/citologia , Útero/citologiaRESUMO
The aim of this work was to study the in vitro amplification of BVDV (Pestivirus, Flaviridae) field isolates from Argentina in MDBK, BoTur and BHK-21 continuous cell lines. Field isolates 99/134 (mucosal disease), 00/693 (mucosal disease), 04P7016 (respiratory disease) and 04/89 (mucosal disease), genotype 1b, were used and compared with the Singer and NADL reference strains, genotype 1a. Additionally, cell lines derived from explants of bovine testis (RD- 420), bovine uterus (NCL-1) and porcine kidney (PKZ) were tested as alternative substrates for BVDV propagation in vitro. The effect of cell line, harvest time and infection protocol was evaluated. The viral titers observed depended on the virus and harvest time but not on the infection protocol. We found that MDBK and BoTur cell lines were susceptible to the infection whereas BHK-21 and PKZ were not. NADL viral titers, 00/693 and 04/89, increased from 24 to 48 h p.i. in BoTur cells and then reached a plateau, whereas those of 99/134 and 04P7016 remained constant between 24 and 72 h p.i. BVDV Singer, on the other hand, presented a maximum titer at 24 h p.i. and then decreased. BVDV-NADL titers increased in MDBK and NCL-1 but not in RD-420 between 24 and 48 h p.i., and then decreased at 72 h p.i. These facts lead us to conclude that neither the subgenotypes (1a, 1b) nor the clinical symptoms of the animal from the virus had been isolated seem to affect the virus cell line kinetics of viral replication in vitro. On the other hand, the most homogenous behavior, the most similar replication curves, and highest titers observed in MDBK and NCL-1 seem to indicate that these lines are generally more susceptible to BVDV replication.
Se estudió la interacción de aislamientos de campo de Argentina del VDVB (Pestivirus, Flaviridae) en las líneas celulares continuas MDBK, BoTur y BHK-21. Se utilizaron los virus de campo genotipo 1b, 99/134, 00/693 (casos compatibles con enfermedad de las mucosas) y 04P7016 (cuadro respiratorio) y las cepas de referencia genotipo 1a Singer y NADL. Además se evaluó la interacción de VDVB-NADL con las líneas celulares experimentales de bovino RD-420 y NCL-1 y de riñón porcino (PKZ). Se usaron 2 protocolos de infección. Los títulos virales observados dependieron del virus y del tiempo de infección y no así del modo de infección. Mientras que MDBK y BoTur resultaron susceptibles a la infección, BHK-21 y PKZ no lo fueron. Los virus NADL, 00/693 y 04/89 incrementaron su título entre las 24 y las 48 h p.i. en BoTur para mantenerlo posteriormente; los virus 99/134 y 04P7016 no presentaron variaciones y la cepa Singer presentó título máximo a las 24 h p.i para luego descender. La cinética del virus NADL en las células MDBK, RD-420 y NCL-1 tuvo un incremento de título para MDBK y NCL-1 entre las 24 y 48 h p.i que descendió a las 72 h p.i. La interacción virus-línea celular no estaría relacionada con el sub-genotipo del virus (1a o 1b), ni con el cuadro clínico; las células MDBK y NCL-1 serían más susceptibles a la replicación del VDVB.