PESQUISA VETERINÁRIA BRASILEIRA

Abstract in English:

ABSTRACT.- Santos A.S., Antoniass, N.A.B., Boabaid F.M., Bitencourt A.P.G., Almeida L.L., Canal C.W., Flores E.F. & Driemeier D. 2011. [Clinical, pathological, immunohistochemical and viral aspects of five calves persistently infected with bovine viral diarrhea virus in a farm of Rio Grande do Sul, Brazil.] Aspectos clínicos, patológicos, imuno-histoquímicos e virológicos em cinco bezerros persistentemente infectados com o vírus da diarreia viral bovina em uma propriedade do Rio Grande do Sul. Pesquisa Veterinária Brasileira 31(10):885-892. Setor de Patologia Veterinária, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9090, Porto Alegre, RS 91540-000, Brazil. E-mail: davetpat@ufrgs.br Bovine viral diarrhea virus (BVDV) is responsible for different syndromes that affect cattle worldwide causing important economic losses. This study analyzed the clinical, pathological, immunohistochemical and viral aspects of persistent infection by BVDV in five animals of a farm located in the county of Viamão, Rio Grande do Sul, southern Brazil. The clinical signs included growth impairment, nasal and ocular discharge and, in two animals, congenital cataract. The main gross lesions observed at the necropsy were enlargement of mesenteric lymph nodes and Peyer’s patches, and in one case, pododermatitis and crusted lesions on nasal planum and periocular region. Microscopic findings were characterized mostly by mononuclear infiltrate in the lamina propria, primarily in the small intestine and lymphoid depletion with histiocytic infiltrate in follicular centers of lymph nodes and Peyer’s patches. Viral antigens were more frequently demonstrated in epidermal keratinocytes, epithelium of hair follicles and dendritic cells of the dermis of the ears and skin, histiocytes and lymphocytes in lymph nodes, thyroid follicular cells, in the cytoplasm of neurons and to a lesser extent, in glial cells in the cerebral cortex and hippocampus. Viral isolation from blood samples and organs confirmed the presence of non-cytopathic BVDV. Moreover, viral RNA was detected by RT-PCR in serum samples. Phylogenetic analysis of a partial fragment of the5’ non-translated region of the viral genome allowed the classification of the sample as BVDV type 2b. The present study strengthens the need to investigate and to characterize BVD outbreaks and to describe its different clinic-pathological presentations.

• BVDV bovine viral diarrhea virus vírus da diarreia viral bovina

Abstract in Portuguese:

RESUMO.- Santos A.S., Antoniass, N.A.B., Boabaid F.M., Bitencourt A.P.G., Almeida L.L., Canal C.W., Flores E.F. & Driemeier D. 2011. [Clinical, pathological, immunohistochemical and viral aspects of five calves persistently infected with bovine viral diarrhea virus in a farm of Rio Grande do Sul, Brazil.] Aspectos clínicos, patológicos, imuno-histoquímicos e virológicos em cinco bezerros persistentemente infectados com o vírus da diarreia viral bovina em uma propriedade do Rio Grande do Sul. Pesquisa Veterinária Brasileira 31(10):885-892. Setor de Patologia Veterinária, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9090, Porto Alegre, RS 91540-000, Brazil. E-mail: davetpat@ufrgs.br O vírus da diarreia viral bovina (BVDV) é responsável por diferentes síndromes que afetam bovinos em todo o mundo, causando grandes perdas econômicas. O presente trabalho analisou as características clínicas, patológicas e imuno-histoquímicas e virais de cinco bovinos persistentemente infectados pelo BVDV de uma mesma propriedade, localizada no Município de Viamão, Rio Grande do Sul. Dentre os sinais clínicos verificados destacaram-se subdesenvolvimento, secreções nasais e oculares, além de catarata congênita unilateral em dois bovinos. As principais lesões observadas durante a necropsia consistiram de aumento dos linfonodos mesentéricos, evidenciação das placas de Peyer e pododermatite e lesões crostosas no plano nasal e na região periocular em um animal. Os achados microscópicos caracterizavam-se, principalmente, por infiltrado mononuclear na lâmina do intestino delgado e rarefação linfoide com infiltrado histiocitário nos centrofoliculares de linfonodos e nas placas de Peyer. Antígenos virais foram detectados por imuno-histoquímica principalmente em queratinócitos da epiderme, no epitélio de folículos pilosos e células mononucleares da derme de orelhas e pele; histiócitos e em linfócitos dos linfonodos; células foliculares da tireoide; no citoplasma de neurônios e, em menor escala, em células da micróglia no córtex cerebral e no hipocampo. O isolamento viral de amostras de sangue e órgãos dos animais confirmou a presença de BVDV não citopático. Também foi possível detectar a presença do genoma viral por RT-PCR no soro dos animais. A análise filogenética do fragmento parcial da região 5’ não traduzida do genoma viral permitiu a classificação da amostra viral como BVDV tipo 2b. O presente estudo reforça a necessidade de investigar e caracterizar surtos de BVD e descrever suas diferentes formas de apresentação.

Abstract in English:

ABSTRACT.- Bianchi E., Martins M., Weiblen R. & Flores E.F. 2011. [Genotypic and antigenic profile of bovine viral diarrhea virus isolates from Rio Grande do Sul, Brazil (2000-2010).] Perfil genotípico e antigênico de amostras do vírus da diarréia viral bovina isoladas no Rio Grande do Sul (2000-2010). Pesquisa Veterinária Brasileira 31(8):649-655. Setor de Virologia, Departamento de Medicina Veterinária Preventiva, Centro de Ciências Rurais, Universidade Federal de Santa Maria, Av. Roraima 1000, Camobi, Santa Maria, RS 97105-900, Brazil. E-mail: eduardofurtadoflores@gmail.com Field isolates of bovine viral diarrhea virus (BVDV) display a high genetic and antigenic diversity that may difficult diagnosis and vaccine formulation. The present study presents a genetic and antigenic characterization of 20 BVDV isolates from Rio Grande do Sul, Brazil (2000-2010). The isolates were associated with a variety of clinical conditions that included respiratory or gastroenteric disease, skin lesions, abortions, animals with retarded growth, and persistently infected (PI) animals. Most isolates (19 or 95%) belong to the non-cytopathic biotype (NCP), one isolate (5%) had a mixture of viruses NCP and cytopathic (CP). Nucleotide sequencing of a region of 270 nucleotides of the 5’ untranslated region of the viral genome followed by phylogenetic analysis identified nine isolates of BVDV-2 (45%), eight BVDV-1 (40%). Three isolates were not classified in any genotype and were classified as atypical pestiviruses. It was not possible to associate genotypes or subgenotypes with clinical conditions: both BVDV-1 and BVDV-2 were involved in different clinico - pathological syndromes. Analysis of reactivity with a panel of 19 monoclonal antibodies (mAbs) revealed a marked variability in the major envelope glycoprotein (E2/gp53) among viruses of the same genotype, but especially among viruses from different genotypes. Virus neutralization assays (VN) with antisera of BVDV-1 and BVDV-2 reference strains against the isolates revealed variable levels of cross-reactivity between viruses of the same genotype, and lack or very low reactivity between viruses of different genotypes. These results indicate a similar frequency of BVDV-1 and BVDV-2 in the cattle population of Rio Grande do Sul, confirm the remarkable antigenic diversity of BVDV and reinforce the need to include viruses of genotypes BVDV-1 and BVDV-2 in vaccines. In addition, these results indicate the circulation of atypical pestiviruses in the cattle population of RS.

• Bovine viral diarrhea virus BVDV pestivirus diarréia viral bovina

Abstract in Portuguese:

RESUMO.- Bianchi E., Martins M., Weiblen R. & Flores E.F. 2011. [Genotypic and antigenic profile of bovine viral diarrhea virus isolates from Rio Grande do Sul, Brazil (2000-2010).] Perfil genotípico e antigênico de amostras do vírus da diarréia viral bovina isoladas no Rio Grande do Sul (2000-2010). Pesquisa Veterinária Brasileira 31(8):649-655. Setor de Virologia, Departamento de Medicina Veterinária Preventiva, Centro de Ciências Rurais, Universidade Federal de Santa Maria, Av. Roraima 1000, Camobi, Santa Maria, RS 97105-900, Brazil. E-mail: eduardofurtadoflores@gmail.com Isolados do vírus da diarréia viral bovina (BVDV) apresentam grande diversidade genética e antigênica, o que pode dificultar o diagnóstico e a formulação de vacinas. O presente trabalho apresenta um perfil genotípico e antigênico de 20 amostras do BVDV isoladas no Estado do Rio Grande do Sul entre 2000 e 2010. As amostras foram oriundas de uma variedade de condições clínicas, que incluíam doença respiratória ou gastroentérica aguda ou crônica, lesões cutâneas, abortos, animais com crescimento retardado, além de animais persistentemente infectados (PI). A maioria das amostras (19 ou 95%) pertence ao biótipo não-citopático (NCP); enquanto um isolado apresentou uma mistura de vírus NCP e citopático (CP). O sequenciamento e análise filogenética de uma região de 270 nucleotídeos da região 5’ não-traduzida do genoma viral permitiu identificar 9 isolados de BVDV-2 (45%) e 8 isolados de BVDV-2 (40%). Três amostras não agruparam filogeneticamente com nenhum dos genótipos, sendo classificados como pestivírus atípicos. Não foi possível associar os genótipos ou subgenótipos com as condições clínicas e, tanto os BVDV-1 quanto os BVDV-2 estavam envolvidos em diferentes síndromes clínico-patológicas. Análise de reatividade com um painel de 19 anticorpos monoclonais (AcMs) revelou uma variabilidade marcante na glicoproteína principal do envelope (E2) entre vírus do mesmo genótipo, e sobretudo, entre vírus de genótipos diferentes. Testes de neutralização viral (SN) com anti-soro de cepas de referência de BVDV-1 e BVDV-2 frente às amostras isoladas revelaram níveis variáveis de reatividade cruzada entre vírus do mesmo genótipo, e reatividade muito baixa ou ausente entre vírus de genótipos diferentes. Esses resultados indicam uma frequência semelhante de BVDV-1 e BVDV-2 na população estudada, confirmam a marcante variabilidade antigênica e reforçam a necessidade de se incluir vírus dos dois genótipos nas vacinas. Finalmente, indicam a presença de pestivírus atípicos circulantes na população bovina do RS.

Abstract in English:

ABSTRACT.- Arenhart S., Bauermann F.V., Vogel F.S.F., Weiblen R. & Flores E.F. 2010. Evidence of mixed persistent infections in calves born to cows challenged with a pool of bovine viral diarrhea virus isolates. Pesquisa Veterinária Brasileira 30(12):1053-1057. Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, Camobi, Santa Maria, RS 97105-900, Brazil. E-mail: eduardofurtadoflores@gmail.com Pregnant cows infected with noncytopathic (NCP) isolates of bovine viral diarrhea virus (BVDV) between days 40 and 120 days of gestation frequently deliver immunotolerant, persistently infected (PI) calves. We herein report the characterization of PI calves produced experimentally through inoculation of pregnant cows with a pool of Brazilian BVDV-1 (n=2) and BVDV-2 isolates (n=2) between days 60 and 90 of gestation. Two calves were born virus positive, lacked BVDV antibodies, but died 7 and 15 days after birth, respectively. Six other calves were born healthy, seronegative to BVDV, harbored and shed virus in secretions for up to 210 days. Analysis of the antigenic profile of viruses infecting these calves at birth and 30 days later with a panel of monoclonal antibodies indicated two patterns of infection. Whereas three calves apparently harbored only one isolate (either a BVDV-1 or BVDV-2), co-infection by two antigenically distinct challenge viruses was demonstrated in three PI calves. Moreover, testing the viruses obtained from the blood of PI calves by an RT-PCR able to differentiate between BVDV-1 and BVDV-2 confirmed the presence/persistence of two co-infecting viruses of different genotypes (BVDV-1 and BVDV-2) in these animals. These findings indicate that persistent infection of fetuses/calves - a well characterized consequence of fetal infection by BVDV – may be established concomitantly by more than one isolate, upon experimental inoculation. In this sense, mixed persistent infections with antigenically distinct isolates may help in understanding the immunological and molecular basis of BVDV immunotolerance and persistence.

• BVDV bovine viral diarrhea virus diarréia viral bovina

Abstract in Portuguese:

RESUMO.- Arenhart S., Bauermann F.V., Vogel F.S.F., Weiblen R. & Flores E.F. 2010. Evidence of mixed persistent infections in calves born to cows challenged with a pool of bovine viral diarrhea virus isolates. [Evidências de infecção persistente mista em bezerros nascidos de vacas inoculadas com um pool de isolados do vírus da diarréia viral bovina.] Pesquisa Veterinária Brasileira 30(12):1053-1057. Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, Camobi, Santa Maria, RS 97105-900, Brazil. E-mail: eduardofurtadoflores@gmail.com Vacas prenhes infectadas com isolados não-citopáticos (NCP) do vírus da diarréia viral bovina (BVDV) entre os dias 40 e 120 de gestação frequentemente produzem bezerros imunotolerantes, persistentemente infectados (PI). Este artigo relata a caracterização de bezerros PI produzidos experimentalmente, pela inoculação de vacas prenhes com um pool de isolados brasileiros de BVDV-1 (n=2) e BVDV-2 (n=2) entre os dias 60 e 90 de gestação. Dois bezerros nasceram positivos para vírus, negativos para anticorpos, mas morreram aos dias 7 e 15 pós-nascimento, respectivamente. Outros seis bezerros nasceram saudáveis, soronegativos para o BVDV, replicaram e excretaram o vírus em secreções por até 210 dias. Análise antigênica dos vírus presentes nos bezerros PI com anticorpos monoclonais, ao nascimento e 30 dias após, revelou dois padrões de infecção. Três bezerros apresentavam apenas um dos vírus utilizados no desafio (BVDV-1 ou BVDV-2), enquanto outros três bezerros apresentavam co-infecção com mais de um vírus. Além disso, testando-se o sangue dos animais PI com um PCR capaz de diferenciar entre BVDV-1 e BVDV-2 observou-se a presença concomitante de vírus dos dois genótipos (BVDV-1 e BVDV-2) nestes três animais. Esses resultados indicam que infecção persistente - uma consequência bem conhecida da infecção fetal pelo BVDV - pode ser estabelecida concomitantemente por mais de um vírus, após infecção experimental. Nesse sentido, infecções persistentes mistas com isolados antigenicamente distintos podem ser úteis para o estudo das bases imunológicas e moleculares da imunotolerância e infecção persistente pelo BVDV.

Abstract in English:

ABSTRACT.- Arenhart S., Bauermann F.V., Oliveira S.A.M., Weiblen R. & Flores E.F. 2009. [Shedding and transmission of bovine viral diarrhea virus by persistently infected calves.] Excreção e transmissão do vírus da diarréia viral bovina por bezerros persistentemente infectados. Pesquisa Veterinária Brasileira 29(9):736-742. Departamento de Medicina Veterinária Preventiva, Centro de Ciências Rurais, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. E-mail: eduardofurtadoflores@gmail.com Persistently infected (PI) calves born to cows infected with non-cytopathic bovine virus diarrhea virus (BVDV) represent the main reservoir of the virus in nature. We herein report an investigation on the patterns of virus shedding and transmission by five PI calves produced experimentally through inoculation of pregnant cows with Brazilian BVDV isolates. Five calves that survived intrauterine infection were born healthy, lacking neutralizing antibodies to BVDV and harboring virus in the blood. After weaning - and following the disappearance of colostral antibodies - PI calves were monitored for virus in serum and body secretions (ocular, oral, nasal and genital) at weekly intervals for up to 150 days. For each animal, the virus titers in serum showed minor variations throughout the collections (with one exception that presented an increase late in infection), yet the titers varied widely among animals (from 102 to 106TCID50/mL). Virus shedding in secretions was detected steadily during all the observation period with minor titer variations for each particular animal. The highest titers were generally detected in nasal and ocular secretions (titers 104 to 106TCID50mL) whereas genital and oral secretions usually contained low amount of virus (102 to 103TCID50mL). To evaluate the kinetics of virus transmission by these animals, one PI was introduced on a group of 10 seronegative calves maintained with a high animal density simulating the conditions of an intensive management. All 10 contact calves seroconverted to BVDV by day 30. Another PI calf was introduced into a 48-head herd kept under a low animal density, extensive grass management. Among these animals, 8/48 (16.6%) seroconverted by day 10, 26/48 (54.1%) by day 40 and 37/48 (77%) were seropositive at day 100, when the monitoring was discontinued. These results show that continuous viremia and virus shedding in high titers in secretions by PI animals assure an efficient and rapid virus transmission to contact animals, being the kinetics of transmission much faster under intensive conditions.

• Bovine viral diarrhea virus BVDV vírua da diarréia viral bovina

Abstract in Portuguese:

RESUMO.- Arenhart S., Bauermann F.V., Oliveira S.A.M., Weiblen R. & Flores E.F. 2009. [Shedding and transmission of bovine viral diarrhea virus by persistently infected calves.] Excreção e transmissão do vírus da diarréia viral bovina por bezerros persistentemente infectados. Pesquisa Veterinária Brasileira 29(9):736-742. Departamento de Medicina Veterinária Preventiva, Centro de Ciências Rurais, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. E-mail: eduardofurtadoflores@gmail.com Bezerros persistentemente infectados (PI) nascidos de vacas infectadas com amostras não-citopáticas do vírus da diarréia viral bovina (BVDV) se constituem nos principais reservatórios do vírus na natureza. Este trabalho relata uma investigação do padrão de excreção e transmissão viral por cinco bezerros PI produzidos experimentalmente pela inoculação de vacas prenhes com isolados brasileiros do BVDV. Cinco bezerros que sobreviveram a infecção intrauterina nasceram saudáveis, soronegativos e com a presença de vírus no sangue. Após o desmame - e desaparecimento dos anticorpos colostrais - os bezerros PI foram monitorados semanalmente durante 150 dias para a presença de vírus e títulos virais no soro e em secreções (ocular, oral, nasal e genital). Os títulos virais no soro de cada animal apresentaram pequenas variações durante o período (com exceção de um animal que apresentou um aumento de título tardiamente), mas os títulos variaram amplamente entre os animais (entre 102 e 106TCID50/ml). O vírus também foi excretado continuamente nas secreções de todos os animais, com pequenas variações de título entre as coletas. Os maiores títulos virais foram geralmente detectados nas secreções nasais e oculares (títulos de 104 a 106TCID50/mL), enquanto as secreções orais e genitais usualmente continham títulos virais baixos (102 a 103TCID50/mL). Com o objetivo de avaliar a dinâmica de transmissão viral, um bezerro PI foi introduzido em um grupo de 10 bezerros soronegativos, mantido com uma alta densidade animal e submetido a manejo diário para simular as condições de manejo semi-intensivo. Após 30 dias de convívio com o bezerro PI, todos os demais animais haviam soroconvertido ao BVDV. Para investigar a transmissão viral sob condições extensivas, outro bezerro PI foi incorporado a um rebanho de 48 animais mantido a campo, com baixa densidade animal e submetido a manejo extensivo. Dentre estes animais, 8/48 (16,6%) foram soropositivos para anticorpos no dia 10, 26/48 (54,1%) no dia 40 e 37/48 (77%) haviam soroconvertido no dia 100, quando encerrou-se o monitoramento. Estes resultados demonstram que a viremia e excreção viral contínua em altos títulos por animais PI assegura a transmissão rápida do BVDV a animais mantidos em contato, sendo a transmissão notadamente mais rápida em condições intensivas e de alta densidade animal.

Abstract in English:

ABSTRACT.- Diéguez F.J., Yus E., Sanjuán M.L., Vilar M.J. & Arnaiz I. 2008. Monitoring bovine viral diarrhea virus (BVDV) infection status in dairy herds. Pesquisa Veterinária Brasileira 28(12):588-592. Unidad de Epidemiología y Sanidad Animal, Instituto de Investigación y Análisis Alimentarios, Facultad de Veterinaria s/n, Lugo 27002, Spain. E-mail: franciscojavier.dieguez@usc.es This study was designed to assess the relationship between antibodies against bovine viral diarrhea virus (BVDV) determined in the bulk tank milk (BTM) and the within-herd seroprevalence. We also assessed the efficiency of measuring antibody levels in BTM samples to monitor BVDV infection status in a herd. In the 81 farms included in the study, BTM samples were obtained and blood samples withdrawn from all cattle older than one year. The infection status was then determined in serum and milk using a commercial blocking ELISA based on the detection of anti-p80 antibodies. Apart from these baseline serum and milk samples, another BTM sample was collected from each herd 9 months later, and a third BTM sample obtained 9 months after this. In these second and third milk samples, anti-BVDV antibodies were determined using the same ELISA kit. Statistical tests revealed good agreement between herd seroprevalences (% seropositive animals in the herd) and the antibody levels detected in the BTM samples. During the 18 months of follow-up, the farms with persistently infected cattle at the study outset (14.8% of the herds) showed a significant decrease in BTM antibody titers after virus clearance. Conversely, a significant increase in BTM antibody levels was observed in the herds infected with BVDV during the follow-up period. Our findings indicate that monitoring antibody levels in the BTM is a useful method of identifying changes in the BVDV infection status of a herd.

• Bovine viral diarrhea vírus BVDV antibodies diagnostic milk

Abstract in Portuguese:

ABSTRACT.- Diéguez F.J., Yus E., Sanjuán M.L., Vilar M.J. & Arnaiz I. 2008. Monitoring bovine viral diarrhea virus (BVDV) infection status in dairy herds. Pesquisa Veterinária Brasileira 28(12):588-592. Unidad de Epidemiología y Sanidad Animal, Instituto de Investigación y Análisis Alimentarios, Facultad de Veterinaria s/n, Lugo 27002, Spain. E-mail: franciscojavier.dieguez@usc.es This study was designed to assess the relationship between antibodies against bovine viral diarrhea virus (BVDV) determined in the bulk tank milk (BTM) and the within-herd seroprevalence. We also assessed the efficiency of measuring antibody levels in BTM samples to monitor BVDV infection status in a herd. In the 81 farms included in the study, BTM samples were obtained and blood samples withdrawn from all cattle older than one year. The infection status was then determined in serum and milk using a commercial blocking ELISA based on the detection of anti-p80 antibodies. Apart from these baseline serum and milk samples, another BTM sample was collected from each herd 9 months later, and a third BTM sample obtained 9 months after this. In these second and third milk samples, anti-BVDV antibodies were determined using the same ELISA kit. Statistical tests revealed good agreement between herd seroprevalences (% seropositive animals in the herd) and the antibody levels detected in the BTM samples. During the 18 months of follow-up, the farms with persistently infected cattle at the study outset (14.8% of the herds) showed a significant decrease in BTM antibody titers after virus clearance. Conversely, a significant increase in BTM antibody levels was observed in the herds infected with BVDV during the follow-up period. Our findings indicate that monitoring antibody levels in the BTM is a useful method of identifying changes in the BVDV infection status of a herd.

Abstract in English:

ABSTRACT.- Arenhart S., Silva L.F., Henzel A., Ferreira R., Weiblen R. & Flores E.F. 2008. [Fetal protection against bovine viral diarrhea virus (BVDV) in pregnant cows previously immunized with an experimental attenuated vaccine.] Proteção fetal contra o vírus da diarréia viral bovina (BVDV) em vacas prenhes previamente imunizadas com uma vacina experimental atenuada. Pesquisa Veterinária Brasileira 28(10):461-470. Departamento de Medicina Veterinária, Centro de Ciências Rurais, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil. E-mail: flores@ccr.ufsm.br This paper reports the antibody response and fetal protection in pregnant cows conferred by an experimental vaccine containing two attenuated strains of bovine viral diarrhea virus (BVDV-1 and BVDV-2). Cows (n=19) were vaccinated twice, with a 34-days-interval, with the experimental vaccine and together with non-vaccinated controls (n=18), were mated and challenged between days 60 and 90 of gestation by intranasal inoculation of four heterologous BVDV-1 and BVDV-2 isolates. The antibody response was evaluated by serum-neutralization tests performed at different intervals after vaccination (days 34, 78 and 138 post-vaccination [pv]). Fetal protection was monitored by ultrassonographic and clinical examination of the dams and fetuses during the rest of gestation; and through virological and serological examination of pre-colostral blood obtained from aborted and/or recently born fetuses/calves. At the day of challenge (day 138 pv), all vaccinated cows had neutralizing antibodies in high titers against BVDV-1 (1,280- ³10,240), and with one exception (titer 20), presented moderate to high titers to BVDV-2 (80-1,280). At the end of the monitoring, only three out of 18 control cows (16.6%) delivered healthy, virus-free calves. Fifteen non-vaccinated cows (83.3%) presented signs of fetal infection and/or had reproductive losses. Seven of these cows (38.8%) delivered virus-positive calves; five were healthy and survived (27.7%); two were premature or weak and lasted three and 15 days, respectively. The other eight cows (44.4%) aborted between day 30 post-challenge and the parturition; or delivered premature or stillbirth calves. In contrast, 17 out of 19 (89.4%) vaccinated cows delivery virus-free, healthy calves. One vaccinated cow aborted around day 130 post-challenge, yet this fetus could not be examined for the presence of virus. Another cow delivered a virus-positive calf (5.2%). In summary, the experimental vaccine induced adequate antibody titers in most animals and the immunological response induced by vaccination was able to prevent fetal infection and reproductive losses upon challenge with a pool of heterologous BVDV isolates. Hence, this experimental vaccine may be an attractive alternative for the prevention of reproductive losses associated with BVDV infection.

• Bovine viral diarrhea virus BVDV experimental vaccine fetal protection

Abstract in Portuguese:

ABSTRACT.- Arenhart S., Silva L.F., Henzel A., Ferreira R., Weiblen R. & Flores E.F. 2008. [Fetal protection against bovine viral diarrhea virus (BVDV) in pregnant cows previously immunized with an experimental attenuated vaccine.] Proteção fetal contra o vírus da diarréia viral bovina (BVDV) em vacas prenhes previamente imunizadas com uma vacina experimental atenuada. Pesquisa Veterinária Brasileira 28(10):461-470. Departamento de Medicina Veterinária, Centro de Ciências Rurais, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil. E-mail: flores@ccr.ufsm.br This paper reports the antibody response and fetal protection in pregnant cows conferred by an experimental vaccine containing two attenuated strains of bovine viral diarrhea virus (BVDV-1 and BVDV-2). Cows (n=19) were vaccinated twice, with a 34-days-interval, with the experimental vaccine and together with non-vaccinated controls (n=18), were mated and challenged between days 60 and 90 of gestation by intranasal inoculation of four heterologous BVDV-1 and BVDV-2 isolates. The antibody response was evaluated by serum-neutralization tests performed at different intervals after vaccination (days 34, 78 and 138 post-vaccination [pv]). Fetal protection was monitored by ultrassonographic and clinical examination of the dams and fetuses during the rest of gestation; and through virological and serological examination of pre-colostral blood obtained from aborted and/or recently born fetuses/calves. At the day of challenge (day 138 pv), all vaccinated cows had neutralizing antibodies in high titers against BVDV-1 (1,280- ³10,240), and with one exception (titer 20), presented moderate to high titers to BVDV-2 (80-1,280). At the end of the monitoring, only three out of 18 control cows (16.6%) delivered healthy, virus-free calves. Fifteen non-vaccinated cows (83.3%) presented signs of fetal infection and/or had reproductive losses. Seven of these cows (38.8%) delivered virus-positive calves; five were healthy and survived (27.7%); two were premature or weak and lasted three and 15 days, respectively. The other eight cows (44.4%) aborted between day 30 post-challenge and the parturition; or delivered premature or stillbirth calves. In contrast, 17 out of 19 (89.4%) vaccinated cows delivery virus-free, healthy calves. One vaccinated cow aborted around day 130 post-challenge, yet this fetus could not be examined for the presence of virus. Another cow delivered a virus-positive calf (5.2%). In summary, the experimental vaccine induced adequate antibody titers in most animals and the immunological response induced by vaccination was able to prevent fetal infection and reproductive losses upon challenge with a pool of heterologous BVDV isolates. Hence, this experimental vaccine may be an attractive alternative for the prevention of reproductive losses associated with BVDV infection.

Abstract in English:

ABSTRACT.- Ferreira L.C.L., Flores E.F., Driemeier D., Melo O. & Lemos R.A.A. 2008. [Mucosal disease associated with generalized dermatitis in cattle, Mato Grosso do Sul, Brazil.] Doença das mucosas associada à dermatite generalizada em bovinos, Mato Grosso do Sul. Pesquisa Veterinária Brasileira 28(6):285-292. Departamento de Medicina Veterinária, Faculdade de Medicina Veterinária e Zootecnia, Universidade Federal de Mato Grosso do Sul, Av. Senador Filinto Müller 2443, Campo Grande, MS 79070-900, Brazil. E-mail: eqrural@nin.ufms.br This paper reports epidemiological, clinical, pathological and laboratory diagnostic aspects of a form of dermatitis associated with mucosal disease (MD) in cattle. It also focuses on the methods used for identifying persistently infected (PI) animals and on the impact of the disease on fertility and weaning rates in the affected herd. Cases of dermatitis associated with MD were diagnosed in two 12 and 24-month-old Nelore calves belonging to a beef cattle farm that operates the full cycle of production (calving, rearing, finishing) in Mato Grosso do Sul, Midwest Brazil. The clinical signs exhibited by affected cattle 0included slow, progressive weight loss; formation of diffuse skin crusts in multiple body areas; skin dryness; multiple ulcerations on the gums and dorsal surface of the tongue which evolved to longitudinal fissures; formation of keratinized projections; and detachment of hoof horn. In addition, diarrhea affected one animal in the late stage of the disease. Necropsies also revealed longitudinal erosions in the esophagus. Histological examination showed coagulation necrosis foci in esophageal and lingual mucosae, with neutrophil and lymphocyte infiltration. Skin lesions consisted of epidermal coagulation necrosis associated with neutrophil infiltration and hyperkeratosis. In both cases, clinical diagnosis was confirmed by the isolation and identification of cytopathic and noncytopathic biotypes of the bovine viral diarrhea virus (BVDV) and immunohistochemical detection of viral antigens in formalin fixed tissues. Out of 300 cattle that had contact with the affected animals, 38 were found to be seropositive - in high neutralizing titers - to BVDV. Blood samples from 1,025 young animals and 40 bulls from the farm were examined for the presence of BVDV to identify potential PI animals. The virus was isolated from blood of three calves in the initial test and, 12 months later, from two of them which had remained on the farm. Only one of these calves was found to be BVDV-positive by immuno-histochemical testing performed on ear-tissue samples. In the year following the birth of PI calves, the herd underwent decreases in fertility and weaning rates, which later returned to their previous levels. The resulting data demonstrate the presence of BVDV infection in beef herds in Mato Grosso do Sul and provide evidence as to include the disease in the differential diagnosis of causes of generalized dermatitis in cattle.

• Bovine viral diarrhea virus mucosal disease cattle diseases viral diseases dermatitis

Abstract in Portuguese:

ABSTRACT.- Ferreira L.C.L., Flores E.F., Driemeier D., Melo O. & Lemos R.A.A. 2008. [Mucosal disease associated with generalized dermatitis in cattle, Mato Grosso do Sul, Brazil.] Doença das mucosas associada à dermatite generalizada em bovinos, Mato Grosso do Sul. Pesquisa Veterinária Brasileira 28(6):285-292. Departamento de Medicina Veterinária, Faculdade de Medicina Veterinária e Zootecnia, Universidade Federal de Mato Grosso do Sul, Av. Senador Filinto Müller 2443, Campo Grande, MS 79070-900, Brazil. E-mail: eqrural@nin.ufms.br This paper reports epidemiological, clinical, pathological and laboratory diagnostic aspects of a form of dermatitis associated with mucosal disease (MD) in cattle. It also focuses on the methods used for identifying persistently infected (PI) animals and on the impact of the disease on fertility and weaning rates in the affected herd. Cases of dermatitis associated with MD were diagnosed in two 12 and 24-month-old Nelore calves belonging to a beef cattle farm that operates the full cycle of production (calving, rearing, finishing) in Mato Grosso do Sul, Midwest Brazil. The clinical signs exhibited by affected cattle 0included slow, progressive weight loss; formation of diffuse skin crusts in multiple body areas; skin dryness; multiple ulcerations on the gums and dorsal surface of the tongue which evolved to longitudinal fissures; formation of keratinized projections; and detachment of hoof horn. In addition, diarrhea affected one animal in the late stage of the disease. Necropsies also revealed longitudinal erosions in the esophagus. Histological examination showed coagulation necrosis foci in esophageal and lingual mucosae, with neutrophil and lymphocyte infiltration. Skin lesions consisted of epidermal coagulation necrosis associated with neutrophil infiltration and hyperkeratosis. In both cases, clinical diagnosis was confirmed by the isolation and identification of cytopathic and noncytopathic biotypes of the bovine viral diarrhea virus (BVDV) and immunohistochemical detection of viral antigens in formalin fixed tissues. Out of 300 cattle that had contact with the affected animals, 38 were found to be seropositive - in high neutralizing titers - to BVDV. Blood samples from 1,025 young animals and 40 bulls from the farm were examined for the presence of BVDV to identify potential PI animals. The virus was isolated from blood of three calves in the initial test and, 12 months later, from two of them which had remained on the farm. Only one of these calves was found to be BVDV-positive by immuno-histochemical testing performed on ear-tissue samples. In the year following the birth of PI calves, the herd underwent decreases in fertility and weaning rates, which later returned to their previous levels. The resulting data demonstrate the presence of BVDV infection in beef herds in Mato Grosso do Sul and provide evidence as to include the disease in the differential diagnosis of causes of generalized dermatitis in cattle.

Abstract in English:

ABSTRACT.- Cortez A., Heinemann M.B., Castro A.M.M.G., Soares M.S, Pinto A.M.V., Alfieri A.A., Flores E.F., Leite R.C. & Richtzenhain L.J. 2006. Genetic characterization of Brazilian bovine viral diarrhea virus isolates by partial nucleotide sequencing of the 5’-UTR region. Pesquisa Veterinária Brasileira 26(4):211-216. Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, Cidade Universitária, São Paulo, SP 05508-000, Brazil. E-mail: leonardo@usp.br Nineteen isolates of bovine viral diarrhea virus (BVDV) from Brazil were genetically characterized through partial nucleotide sequencing and analysis of the 5’UTR region. The isolates were grouped as BVDV-1 (11/19), BVDV-2 (6/19) or “atypical” pestivirus (2/19). Among the BVDV-1, eight isolates were classified as subgenotype BVDV-1a, whereas most (4 out of 6) BVDV-2 belonged to subgenotype 2b. Two isolates from aborted fetuses were not classified into any genetic group, being considered atypical BVDVs. Genetic diversity among Brazilian BVDV isolates may be responsible for vaccination and diag-nostic failure and therefore may influence the control strategies for BVDV infection in the country.

• BVDV-1 BVDV-2 Pestivirus phylogenetic analysis

Abstract in Portuguese:

ABSTRACT.- Cortez A., Heinemann M.B., Castro A.M.M.G., Soares M.S, Pinto A.M.V., Alfieri A.A., Flores E.F., Leite R.C. & Richtzenhain L.J. 2006. Genetic characterization of Brazilian bovine viral diarrhea virus isolates by partial nucleotide sequencing of the 5’-UTR region. Pesquisa Veterinária Brasileira 26(4):211-216. Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, Cidade Universitária, São Paulo, SP 05508-000, Brazil. E-mail: leonardo@usp.br Nineteen isolates of bovine viral diarrhea virus (BVDV) from Brazil were genetically characterized through partial nucleotide sequencing and analysis of the 5’UTR region. The isolates were grouped as BVDV-1 (11/19), BVDV-2 (6/19) or “atypical” pestivirus (2/19). Among the BVDV-1, eight isolates were classified as subgenotype BVDV-1a, whereas most (4 out of 6) BVDV-2 belonged to subgenotype 2b. Two isolates from aborted fetuses were not classified into any genetic group, being considered atypical BVDVs. Genetic diversity among Brazilian BVDV isolates may be responsible for vaccination and diag-nostic failure and therefore may influence the control strategies for BVDV infection in the country.

Abstract in English:

Flores E.F., Weiblen R., Vogel F.S.F., Roehe P.M., Alfieri A.A. & Pituco E.M. 2005. [Bovine viral diarrhea virus (BVDV) infection in Brazil: history, current situation and perspectives.] A infecção pelo vírus da Diarréia Viral Bovina (BVDV) no Brasil - histórico, situação atual e perspectivas. Pesquisa Veterinária Brasileira 25(3):125-134. Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. E-mail: flores@ccr.ufsm.br Bovine viral diarrhea virus (BVDV) is one of the most important pathogens of cattle worldwide. BVDV infection and associated diseases have been reported in Brazil since the late 1960ties. Several serological, virological, clinical and pathological reports demonstrate the widespread distribution of BVDV infection among Brazilian cattle. In addition to variable levels of positive serology in beef and dairy cattle, BVDV antibodies have been occasionally detected in swine, wild boars, goats, cervids and water buffaloes. BVDV infection has been diagnosed in aborted fetuses, buffy coats of persistently infected (PI) animals, clinical specimens from animals suffering from different clinical syndromes, semen of bulls of artificial insemination (AI) centers, in healthy fetuses and in commercial fetal bovine serum and/or cultured cells. About 50 isolates have been genetically and/or antigenically characterized up to date, whilst roughly an equivalent number of isolates awaits characterization. Most of the characterized isolates belong to BVDV-1 genotype, non-cytopathic (NCP) biotype, yet some BVDV-2 (and some CP BVDV) have been identified as well. Brazilian BVDV isolates display a high antigenic variability and are markedly different from North American vaccine strains. A few inactivated, polyvalent vaccines are currently licensed in the country, yet vaccination is still incipient in many regions: only about 2.5 million doses were marketed in 2003. The low serological cross-reactivity between vaccine strains and field isolates has recently stimulated national industries to develop vaccines containing Brazilian BVDV-1 and BVDV-2 strains. The overall knowledge about BVDV infection in Brazil has grown considerably in the last years, due to an increasing number of laboratories performing diagnosis and research. Studies on the pathogenesis, serological and molecular epidemiology and production of reagents for diagnosis have contributed decisively for the recent growing knowledge on BVDV infections in the country.

• Bovine viral diarrhea virus BVDV epidemiology diagnostic.

Abstract in Portuguese:

Flores E.F., Weiblen R., Vogel F.S.F., Roehe P.M., Alfieri A.A. & Pituco E.M. 2005. [Bovine viral diarrhea virus (BVDV) infection in Brazil: history, current situation and perspectives.] A infecção pelo vírus da Diarréia Viral Bovina (BVDV) no Brasil - histórico, situação atual e perspectivas. Pesquisa Veterinária Brasileira 25(3):125-134. Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. E-mail: flores@ccr.ufsm.br Bovine viral diarrhea virus (BVDV) is one of the most important pathogens of cattle worldwide. BVDV infection and associated diseases have been reported in Brazil since the late 1960ties. Several serological, virological, clinical and pathological reports demonstrate the widespread distribution of BVDV infection among Brazilian cattle. In addition to variable levels of positive serology in beef and dairy cattle, BVDV antibodies have been occasionally detected in swine, wild boars, goats, cervids and water buffaloes. BVDV infection has been diagnosed in aborted fetuses, buffy coats of persistently infected (PI) animals, clinical specimens from animals suffering from different clinical syndromes, semen of bulls of artificial insemination (AI) centers, in healthy fetuses and in commercial fetal bovine serum and/or cultured cells. About 50 isolates have been genetically and/or antigenically characterized up to date, whilst roughly an equivalent number of isolates awaits characterization. Most of the characterized isolates belong to BVDV-1 genotype, non-cytopathic (NCP) biotype, yet some BVDV-2 (and some CP BVDV) have been identified as well. Brazilian BVDV isolates display a high antigenic variability and are markedly different from North American vaccine strains. A few inactivated, polyvalent vaccines are currently licensed in the country, yet vaccination is still incipient in many regions: only about 2.5 million doses were marketed in 2003. The low serological cross-reactivity between vaccine strains and field isolates has recently stimulated national industries to develop vaccines containing Brazilian BVDV-1 and BVDV-2 strains. The overall knowledge about BVDV infection in Brazil has grown considerably in the last years, due to an increasing number of laboratories performing diagnosis and research. Studies on the pathogenesis, serological and molecular epidemiology and production of reagents for diagnosis have contributed decisively for the recent growing knowledge on BVDV infections in the country.

Abstract in English:

Lima M., Flores E.F., Weiblen R., Flores F.S.F. & Arenhart S. 2004. [Characterization of bovine viral diarrhea virus (BVDV) types 1 and 2 isolates for use in vaccines.] Caracterização de amostras atenuadas do vírus da Diarréia Viral Bovina (BVDV) tipos 1 e 2 para uso em vacinas. Pesquisa Veterinária Brasileira 24(1):35-42. Depto Medicina Veterinária Preventiva, Centro de Ciências Rurais, Universidade Federal de Santa Maria, Santa Maria, RS 97105-900, Brazil. E-mail: flores@ccr.ufsm.br This article reports the characterization of two cytopathic isolates of bovine viral diarrhea virus (BVDV-1: IBSP-2; BVDV-2:SV-253) submitted to multiple passages (n=30) in tissue culture associated with ultraviolet irradiation. The vaccine candidate strains were characterized in vitro (plaque size and morphology, growth kinetics and antigenic profile) and in vivo (attenuation and serological response in calves). In vitro characterization of biologically cloned viruses obtained at passages 0, 1, 10, 20 and 30 demonstrated that the attenuation process did not significantly affect the phenotypic and antigenic properties of the viruses. No major differences in plaque size and morphology and in the growth kinetics in tissue culture were observed among the viruses obtained at different passages. Likewise, the antigenic profile of these viruses did not change upon successive passages in tissue culture, as ascertained by the pattern of binding by 48 monoclonal antibodies (mAbs). Intramuscular inoculation of both viruses (IBSP-2: 107.3 TCID50; SV-253: 106.8 TCID50) at passage 30 (p30) in twelve 15 months old heifers did not produce clinical signs, demonstrating the attenuation of the viruses. Following inoculation, infectious virus was detected in leucocytes of most inoculated animals (10/12) between days 3 and 6 post-inoculation (pi) and in nasal secretions of three animals (days 4, 7 and 8pi). However, the vaccine viruses were not transmitted to three seronegative calves maintained as sentinels. All vaccinated calves seroconverted at day 14 post-vaccination. A moderate to high serum neutralizing response against five Brazilian BVDV-1 (titers from 80 to ³1,280) and four Brazilian BVDV-2 isolates (titers from 20 to 640) was observed at day 33 post-vaccination (pv). In general, the highest titers were observed against the Brazilian BVDV-1 isolates. At day 240 post-vaccination, the animals received a booster administration (IBSP-2: 107.3 TCID50 and SV-253: 106.8 TCID50). Revaccination resulted in a strong anamnestic response in most animals, with increasing antibody titers mainly to BVDV-2. These are promising results towards the future use of these strains in modified-live vaccines for the control of BVDV infection in Brazil.

• Bovine viral diarrhea virus (BVDV) modified-live vaccine attenuation.

Abstract in Portuguese:

Lima M., Flores E.F., Weiblen R., Flores F.S.F. & Arenhart S. 2004. [Characterization of bovine viral diarrhea virus (BVDV) types 1 and 2 isolates for use in vaccines.] Caracterização de amostras atenuadas do vírus da Diarréia Viral Bovina (BVDV) tipos 1 e 2 para uso em vacinas. Pesquisa Veterinária Brasileira 24(1):35-42. Depto Medicina Veterinária Preventiva, Centro de Ciências Rurais, Universidade Federal de Santa Maria, Santa Maria, RS 97105-900, Brazil. E-mail: flores@ccr.ufsm.br This article reports the characterization of two cytopathic isolates of bovine viral diarrhea virus (BVDV-1: IBSP-2; BVDV-2:SV-253) submitted to multiple passages (n=30) in tissue culture associated with ultraviolet irradiation. The vaccine candidate strains were characterized in vitro (plaque size and morphology, growth kinetics and antigenic profile) and in vivo (attenuation and serological response in calves). In vitro characterization of biologically cloned viruses obtained at passages 0, 1, 10, 20 and 30 demonstrated that the attenuation process did not significantly affect the phenotypic and antigenic properties of the viruses. No major differences in plaque size and morphology and in the growth kinetics in tissue culture were observed among the viruses obtained at different passages. Likewise, the antigenic profile of these viruses did not change upon successive passages in tissue culture, as ascertained by the pattern of binding by 48 monoclonal antibodies (mAbs). Intramuscular inoculation of both viruses (IBSP-2: 107.3 TCID50; SV-253: 106.8 TCID50) at passage 30 (p30) in twelve 15 months old heifers did not produce clinical signs, demonstrating the attenuation of the viruses. Following inoculation, infectious virus was detected in leucocytes of most inoculated animals (10/12) between days 3 and 6 post-inoculation (pi) and in nasal secretions of three animals (days 4, 7 and 8pi). However, the vaccine viruses were not transmitted to three seronegative calves maintained as sentinels. All vaccinated calves seroconverted at day 14 post-vaccination. A moderate to high serum neutralizing response against five Brazilian BVDV-1 (titers from 80 to ³1,280) and four Brazilian BVDV-2 isolates (titers from 20 to 640) was observed at day 33 post-vaccination (pv). In general, the highest titers were observed against the Brazilian BVDV-1 isolates. At day 240 post-vaccination, the animals received a booster administration (IBSP-2: 107.3 TCID50 and SV-253: 106.8 TCID50). Revaccination resulted in a strong anamnestic response in most animals, with increasing antibody titers mainly to BVDV-2. These are promising results towards the future use of these strains in modified-live vaccines for the control of BVDV infection in Brazil.