Co-infection by Neopora caninum and bovine viral diarrhea virus in cattle from Rio Grande do Sul, Brazil, destined to exportation / Coinfecção por Neospora caninum e vírus da diarreia viral bovina em bovinos do Rio Grande do Sul, Brasil, destinados à exportação

Reproductive tests in cattle are of great economic importance, given the impact it can have on the production system and may be caused by agents. Neospora caninum and Bovine Viral Diarrhea virus (BVDV) are considered of great importance as reproductive and should be considered responsible for keeping animals persistently infected. The present study included 479 calf serum samples for export in the state of Rio Grande do Sul (RS). All samples were screened for BVDV by an ELISA antigen. BVDV antigen-positive ELISA samples were isolated from BVDV in cell culture. An indirect immunofluorescence (IFT) technique was used to detect anti-N. caninum antibodies. Of the 479 export-treated serum samples, 361 were positive for BVDV antigens by ELISA and/or viral isolation test (361/479-75.36%), and 109 IFT-positive samples for N. caninum (109/479-22.75%). Despite detection of antibodies anti-N. caninum did not differ statistically between naturally infected BVDV and non-BVDV infected animals suggesting that there is no interference of BVDV infection on infection or detection rate of animals with N. caninum, positive animals in viral isolation and high DO in BVDV-Ag ELISA. may present active disease and consequent immunosuppression, contributing to a potential reactivation of N. caninum.(AU)

Testes reprodutivos em bovinos são de grande importância econômica, dado o impacto que podem ter no sistema de produção e podem ser causados por agentes. O Neospora caninum e o vírus da Diarreia Viral Bovina (BVDV) são considerados de grande importância como reprodutivos e devem ser considerados responsáveis por manter os animais persistentemente infectados. O presente estudo incluiu 479 amostras de soro de bezerro para exportação no estado do Rio Grande do Sul (RS). Todas as amostras foram rastreadas para BVDV por um antígeno ELISA. As amostras de ELISA positivas para o antigénio BVDV foram isoladas a partir de BVDV em cultura de células. Uma técnica de imunofluorescência indireta (IFT) foi utilizada para detectar anticorpos anti-N caninum. Das 479 amostras de soro tratadas para exportação, 361 foram positivas para antígenos de BVDV por ELISA e/ou teste de isolamento viral (361/479-75,36%) e 109 amostras positivas para IFT para N. caninum (109/479-22,75%). Apesar da detecção de anticorpos anti-N. caninum não diferiu estatisticamente entre animais infectados naturalmente BVDV e não BVDV sugerindo que não há interferência da infecção pelo BVDV na infecção ou taxa de detecção de animais com N. caninum, animais positivos em isolamento viral e alta DO em BVDV-Ag ELISA, pode apresentar doença ativa e consequente imunossupressão, contribuindo para uma potencial reativação de N. caninum.(AU)

Detección molecular de coinfección con los virus de la diarrea viral bovina (BVDV) en búfalos de agua (Bubalus bubalis) serológicamente negativos / Molecular analyses detect natural coinfection of water buffaloes (Bubalus bubalis) with bovine viral diarrhea viruses (BVDV) in serologically negative animals

La infección de los búfalos de agua (Bubalus bubalis) con los virus de la diarrea viral bovina (BVDV) ha sido confirmada mediante técnicas serológicas y moleculares en trabajos anteriores. Con el fin de determinar la presencia de animales persistentemente infectados y las especies y subtipos circulantes de BVDV en esta especie animal se realizó un estudio sobre una manada de búfalos de producción mixta con ganado bovino (Bossp.). Nuestros resultados serológicos mostraron un alto nivel de positividad frente a BVDV-1 y BVDV-2 dentro de la manada de búfalos. El análisis molecular sobre muestras de sangre de los animales serológicamente negativos reveló la presencia de ácido nucleico viral, lo que confirma la existencia de búfalos persistentemente infectados. El clonado y la secuenciación de la región 5 'UTR de algunas de las muestras obtenidas de búfalo reveló la presencia de coinfección natural con al menos dos subtipos diferentes de BVDV (1a y 1b) y con las especies virales BVDV-1 y BVDV-2

Infection of water buffaloes (Bubalus bubalis) with bovine viral diarrhea viruses (BVDV) has been confirmed in several studies by serological and molecular techniques. In order to determine the presence of persistently infected animals and circulating species and subtypes of BVDV we conducted this study on a buffalo herd, whose habitat was shared with bovine cattle (Bossp.). Our serological results showed a high level of positivity for BVDV-1 and BVDV-2 within the buffalo herd. The molecular analyses of blood samples in serologically negative animals revealed the presence of viral nucleic acid, confirming the existence of persistent infection in the buffaloes. Cloning and sequencing of the 5' UTR of some of these samples revealed the presence of naturally mix-infected buffaloes with at least two different subtypes (1a and 1b), and also with both BVDV species (BVDV-1 and BVDV-2)

[Bovine viral diarrhea in Qazvin province [Iran]: a seroprevalence study]

BVDV is one of the commonest infectious agents causing significant economical losses in the worldwide dairy cow industry. Serious control measures have been adopted against the infection in many countries, particularly in the developed ones. Investigation of the presence and prevalence of the infection is the first step, and an important one, for efficient control. This survey is aimed at these aspects of BVD in Qazvin Province. A total of 2,205 serum samples were gathered, almost randomly, from cows over 1-year old residing in 59 industrial dairy farms of Qazvin Province. The samples were taken from the cities of Abyek, Alborz and Buinzahra in the autumn of 2007 and the summer of 2008. A standard ELISA kit was applied for detecting antibodies against BVDV, using SPSS software [version 17] for corresponding analyses. The results indicated that 1,644 serum samples had positive reaction. Consequently, overall seroprevalence of infection in animals during the period surveyed was estimated at about 74.5%. Prevalence of the infection at herd level was 100%. Alborz with 83.2%, Abyek with 76.2%, and Buinzahra with 59.6% stood in order of seroprevalence. In the cases with an exact recorded history of parity and abortion, statistically significant relations were found between both variables and the test results [chi-square test with p=0.0005 and p=0.03, respectively]. This high seroprevalence of BVD infection in Qazvin Province, as in other parts of Iran, suggests that more and more emphasizes needs to be placed on the application of emergency measures for controlling the problem as soon as possible. In this regard, the detecting and elimination of PI calves must be considered as the first and essential action

In vitro amplification of BVDV field strains isolated in Argentina: effect of cell line and culture conditions / Amplificación in vitro de cepas de campo de virus de la diarrea viral bovina (VDVB) aisladas en Argentina: efecto de la línea celular y las condicioes de cultivo

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.

Monitoring bovine viral diarrhea virus (BVDV) infection status in dairy herds / Monitoramento do estado de infecção pelo vírus da diarréia viral bovina (BVDV) em rebanhos bovinos leiteiros

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.(AU)

Os objetivos do presente estudo foram avaliar a relação entre os níveis de anticorpos frente ao vírus da diarréia viral bovina (BVDV) no tanque de leite e a prevalência de animais seropositivos em cada rebanho; e também avaliar a eficiência da medição dos níveis de anticorpos no tanque de leite como método de monitoramento do status de infecção frente ao BVDV. Nos rebanhos estudados, obtiveram-se amostras de soro de todos os animais com idade superior a um ano, assim como uma amostra de tanque coletivo de leite. As amostras de soro e leite foram analisados por um teste ELISA de bloqueio baseado na detecção de anticorpos anti-p80. Posteriormente coletaram-se mais duas amostras do tanque de leite em cada exploração com intervalos de nove meses entre as coletas. Estas amostras foram analisadas com o mesmo ELISA. A análise estatística mostrou uma boa relação entre a soroprevalência dos rebanhos e a percentagem de inibição na amostra de tanque de leite. No decorrer do procedimento, aquelas explorações que possuíam animais PI no início do estudo (que representavam 14.8% dos rebanhos estudados) mostraram um decréscimo estatísticamente significativo dos níveis de anticorpos após a eliminação dos animais persistentemente infectados. Ao contrário, as explorações que sofreram a introdução da infecção durante o estudo mostram um incremento significativo nos níveis de anticorpos no leite. Nossas conclusões indicam que a avaliação de níveis de anticorpos no tanque de leite é um método útil de identificar mudanças do estado sorológico da infecção BVDV de rebanhos leiteiros.(AU)

Doença das mucosas associada à dermatite generalizada em bovinos, Mato Grosso do Sul / Mucosal disease associated with generalized dermatitis in cattle, Mato Grosso do Sul, Brazil

São descritos os aspectos epidemiológicos, clínicos, patológicos e diagnósticos de uma forma de dermatite associada à doença das mucosas (DM) em bovinos. Também são abordadas metodologias para a identificação de animais persistentemente infectados (PI) e o impacto nos índices zootécnicos no rebanho afetado. Os casos de dermatite associados com DM ocorreram em dois bovinos Nelore, de 12 e 24 meses de idade, pertencentes a uma fazenda de ciclo completo de bovinos de corte no estado de Mato Grosso do Sul. Os sinais clínicos nesses animais consistiam de emagrecimento lento e progressivo, formação de crostas difusas na pele de todo o corpo, pele ressecada, múltiplas ulcerações nas gengivas e face dorsal da língua, que evoluíram para fendas longitudinais, formação de projeções cornificadas e desprendimento dos cascos. Em um caso, também ocorreu diarréia no estágio final da doença. Na necropsia observaram-se ainda erosões longitudinais no esôfago. O exame histológico revelou focos de necrose de coagulação na mucosa do esôfago e língua, com infiltrado de neutrófilos e linfócitos. As lesões da pele consistiam de necrose de coagulação da epiderme associada com infiltrado de neutrófilos e hiperqueratose. Nos dois casos, a suspeita clínica foi confirmada pelo isolamento viral e identificação dos biótipos citopático e não-citopático do vírus da diarréia viral bovina (BVDV), além da detecção de antígenos virais em tecidos por imunoistoquímica. De um lote de 300 bovinos que tiveram contato com animais afetados, 38 foram testados e apresentaram altos títulos de anticorpos para o BVDV. Amostras de sangue coletadas de 1.025 animais jovens e 40 touros da propriedade foram submetidas a pesquisa de vírus para se identificarem possíveis animais persistentemente infectados (PI). O vírus foi isolado do sangue de três bezerros no teste inicial e, 12 meses depois, em dois deles que permaneceram na propriedade. Imunoistoquímica realizada em biópsia de orelhas identificou...

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...

RT-PCR em pools de soros sangüíneos para o diagnóstico da infecção aguda e de animais persistentemente infectados pelo vírus da diarréia viral bovina / RT-PCR in pools of bovine blood serum to detect acute infection and persistently infected animals with bovine viral diarrhea virus

Utilizou-se a técnica da RT-PCR para a detecção da região 5' UTR do genoma do vírus da diarréia viral bovina (BVDV) em pools de soros sangüíneos provenientes de um rebanho, constituído por 226 animais, que apresentava distúrbios da reprodução. A partir das amostras individuais de soro e de acordo com a categoria dos animais e o número de animais por categoria foram formados 10 pools (A a J) de soros. A primeira avaliação revelou a amplificação de um produto com 290pb nas reações referentes aos grupos D (35 vacas) e H (25 bezerros lactentes) que, após o desmembramento em amostras individuais, resultou na identificação de 11 vacas lactantes e 12 bezerros em amamentação positivos. Para a identificação de animais persistentemente infectados (PI) entre os 23 positivos na primeira avaliação, realizou-se a segunda colheita de soros sangüíneos, três meses após. A RT-PCR das amostras individuais de soro revelou resultado positivo em cinco bezerros. Em dois, foi possível isolar o BVDV em cultivo de células MDBK. A especificidade das reações da RT-PCR foi confirmada pelo seqüenciamento dos produtos amplificados a partir do soro de uma vaca com infecção aguda, de um bezerro PI e das duas amostras do BVDV isoladas em cultivo celular. A utilização da RT-PCR em pools de soros sangüíneos demonstrou ser uma estratégia rápida de diagnóstico etiológico e de baixo custo tanto para a detecção de infecção aguda quanto de animais PI.

The 5' untranslated region of the bovine viral diarrhea virus (BVDV) genome was detected by RT-PCR assay in pools of blood sera samples collected from a cattle herd (n=226 animals) with reproductive failures. Based on the classes of animal and the number of animals per class, the individual blood serum samples were distributed in 10 sera pools (A to J). During the first evaluation a 290bp amplicon was amplified in reactions from groups D (35 cows) and H (25 sucking calves). The individual analysis of serum from groups D and H resulted in positive reactions in serum samples from 11 cows and 12 calves. For the identification of persistently infected (PI) animals, three months after the first examination, blood serum samples from 23 positive animals were reevaluated by RT-PCR, resulting in five positive calves. In two of these calves the BVDV was isolated in MDBK cell culture. The specificity of RT-PCR amplicons from one cow with acute infection, one PI calf, and two wild type BVDV strains isolated in cell culture were confirmed by nucleotide sequencing. The use of RT-PCR in pools of blood sera proved to be a quick and low cost strategy for the etiological diagnosis of the acute infection as well as to detect PI animals thereby favoring the implementation of control and prophylaxis measures.

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.

Detecção de ácidos nucléicos de Brucella spp., Leptospira spp., herpesvirus bovino e vírus da diarréia viral bovina, em fetos bovinos abortados e em animais mortos no perinatal / Detection of Brucella spp., Leptospira spp., bovine herpesvirus and bovine viral diarrhea virus nucleic acids in aborted fetuses and bovines dead perinatal

Samples of 114 bovine fetuses and 10 calves, which dead in perinatal period, were examined for detection of DNA. The most common detected agent was Brucella spp. in 17 samples (13.7 percent) followed by Leptospira spp. in 4 cases (3.2 percent),bovine herpesvirus (BHV) and bovine viral diarrhea (BVDV) in 3 animals (2.4 percent) each, and 1 for the association of BVDV and BHV. In 77.4 percent (96/124) of the samples it was not possible to detect any agent.

Co-infections with bovine herpesvirus type 5 and bovine viral diarrhoea virus

During a series of experiments attempting to reproduce clinically apparent bovine herpesvirus type 5 (BoHV-5) infections, a group of calves was inadvertently infected with bovine viral diarrhoea virus (BVDV). Another group of calves was infected with BoHV-5 only. This paper reports the outcome of such infections. Two out of six calves solely infected with BoHV-5 displayed moderate to severe clinical signs. Three out of four calves of the group co-infected with BoHV-5 and BVDV developed severe clinical signs, two of them died. BoHV-5 virus was isolated to higher titres and for a longer period from the group of calves infected with both viruses. These results suggest that BVDV may enhance clinical signs induced by BoHV-5 and may also play a role in extending the period of BoHV-5 shedding.

Durante a realização de experimentos envolvendo inoculações experimentais com herpesvírus bovino tipo 5 (BoHV-5), um grupo de bovinos foi acidentalmente também inoculado com vírus da diarréia viral bovina (BVDV). Os dados obtidos nesta co-infecção foram então comparados a aqueles observados em animais inoculados exclusivamente com BoHV-5. No grupo infectado com BoHV-5 somente, dois dos seis animais inoculados mostraram sinais clínicos de moderados a graves. No grupo co-infectado com BoHV-5 e BVDV, três dos quatro animais desenvolveram doença grave, e dois deles morreram. BoHV-5 foi isolado em títulos maiores e por um período de tempo mais longo no grupo co-infectado. Os resultados sugerem que o BVDV pode exacerbar os sinais clínicos induzidos pelo BoHV-5 e, ainda, aumentou os níveis de excreção deste último.

A search for RNA insertions and NS3 gene duplication in the genome of cytopathic isolates of bovine viral diarrhea virus

Calves born persistently infected with non-cytopathic bovine viral diarrhea virus (ncpBVDV) frequently develop a fatal gastroenteric illness called mucosal disease. Both the original virus (ncpBVDV) and an antigenically identical but cytopathic virus (cpBVDV) can be isolated from animals affected by mucosal disease. Cytopathic BVDVs originate from their ncp counterparts by diverse genetic mechanisms, all leading to the expression of the non-structural polypeptide NS3 as a discrete protein. In contrast, ncpBVDVs express only the large precursor polypeptide, NS2-3, which contains the NS3 sequence within its carboxy-terminal half. We report here the investigation of the mechanism leading to NS3 expression in 41 cpBVDV isolates. An RT-PCR strategy was employed to detect RNA insertions within the NS2-3 gene and/or duplication of the NS3 gene, two common mechanisms of NS3 expression. RT-PCR amplification revealed insertions in the NS2-3 gene of three cp isolates, with the inserts being similar in size to that present in the cpBVDV NADL strain. Sequencing of one such insert revealed a 296-nucleotide sequence with a central core of 270 nucleotides coding for an amino acid sequence highly homologous (98 percent) to the NADL insert, a sequence corresponding to part of the cellular J-Domain gene. One cpBVDV isolate contained a duplication of the NS3 gene downstream from the original locus. In contrast, no detectable NS2-3 insertions or NS3 gene duplications were observed in the genome of 37 cp isolates. These results demonstrate that processing of NS2-3 without bulk mRNA insertions or NS3 gene duplications seems to be a frequent mechanism leading to NS3 expression and BVDV cytopathology.

Enfermidade gastroentérica e respiratória em bezerros inoculados com amostras brasileiras do vírus da Diarréia Viral Bovina tipo 2 (BVDV-2) / Gastroenteric and respiratory disease in calves inoculated with brazilian isolates of Bovine Viral Diarrhea virus type 2 (BVDV - 2)

Duas amostras brasileiras do vírus da Diarréia Viral Bovina tipo 2 (BVDV-2) foram inoculadas em bezerros com o objetivo de avaliar a sua virulência e estudar a patogenia da infecçäo. Previamente à inoculaçäo, os animais foram imunodeprimidos com dexametasona. Quatro bezerros com idades entre 45 e 90 dias (grupo A) foram inoculados com a amostra SV-260 (n=2) ou LV-96 (n=2) e quatro bezerros com 6 a 8 meses de idade foram inoculados com a amostra SV-260 (grupo B). Após a inoculaçäo, os bezerros do grupo A apresentaram anorexia, depressäo, hipertermia, sinais de infecçäo respiratória e diarréia profusa, acompanhada de melena em dois animais. Os sinais respiratórios e digestivos progrediram, e os animais morreram ou foram sacrificados in extremis entre os dias 7 e 12 pós-inoculaçäo. Ulceras e erosöes no trato digestivo (língua, n=4; esôfago, n=1; rúmen, n=1 e abomasso, n=3), edema pulmonar (n=4) e na mucosa do abomasso (n=3); equimoses e sufusöes na serosa do baço (n=2), no rúmen, no intestino delgado e no ceco (n=1), no coraçäo (n=1) e na mucosa da bexiga (n=1) e intussuscepçäo intestinal (=1) foram os achados macroscópicos mais marcantes. Ulceras e erosöes, acompanhadas de infiltrado mononuclear na mucosa e submucosa do trato digestivo e depleçäo linfóide nos linfonodos e placas de Peyer, foram as alteraçöes microscópicas mais freqüentes. O vírus foi detectado em vários tecidos e órgäos. Antígenos virais foram demonstrados por imuno-histoquímica, principalmente em células epiteliais do trato digestivo; em células mononucleares nos espaços perivasculares e peribronquiais; na cápsula e septos de linfonodos; e em linfócitos e células mononucleares das placas de Peye e baço. Os animais do grupo B apresentaram depressäo, hipertermia, sinais moderados de infecçäo respiratória e digestiva, ulceraçöes na língua e bochecha, mas recuperaram-se após alguns dias. Esses resultados demonstram que as amostras de BVDV-2 foram capazes de reproduzir a enfermidade aguda quando inoculadas em bezerros e que as conseqüências clínico-patológicas da infecçäo foram mais severas nos animais mais jovens.

Production and characterization of monoclonal antibodies to Brazilian isolates of bovine viral diarrhea virus

Three Brazilian isolates of bovine viral diarrhea virus (BVDV), antigenically distinct from the standard North American isolates, were selected to immunize BALB/c mice in order to obtain hybridoma cells secreting anti-BVDV monoclonal antibodies (mAbs). Two hybridoma clones secreting mAbs, reacting specifically with BVDV-infected cells (mAbs 3.1C4 and 6.F11), were selected after five fusions and screening of 1001 hypoxanthine-aminopterin-thymidine-resistant clones. These mAbs reacted in an indirect fluorescent antibody (IFA) assay with all 39 South and North American BVDV field isolates and reference strains available in our laboratory, yet failed to recognize other pestiviruses, namely the hog cholera virus. The mAbs reacted at dilutions up to 1:25,600 (ascitic fluid) and 1:100 (hybridoma culture supernatant) in IFA and immunoperoxidase (IPX) staining of BVDV-infected cells but only mAb 3.1C4 neutralized virus infectivity. Furthermore, both mAbs failed to recognize BVDV proteins by IPX in formalin-fixed paraffin-embedded tissues and following SDS-PAGE and immunoblot analysis of virus-infected cells, suggesting they are probably directed to conformational-type epitopes. The protein specificity of these mAbs was then determined by IFA staining of CV-1 cells transiently expressing each of the BVDV proteins: mAb 3.1C4 reacted with the structural protein E2/gp53 and mAb 6.F11 reacted with the structural protein E1/gp25. Both mAbs were shown to be of the IgG2a isotype. To our knowledge, these are the first mAbs produced against South American BVDV isolates and will certainly be useful for research and diagnostic purposes

Characterization of mixed infections with different strains of bovine rotavirus in an outbreak of diarrhea in dairy herds in Goiás, Brazil

Ten faecal samples of bovine rotavirus from calves less than 30 days old from an outbreak of diarrhea in Hidrolândia, Goiás, Brazil were submitted to serological and molecular characterization, using enzyme immonuassay for subgrouping and serotyping, PAGE for determination of electropherotypes and PCR for genome typing. Nine samples belonged to group A/subgroup I rotavirus one samples was group A / soubgroup non-I/non-II. Four samples were characterized as G10P[11] (B223-like), four samples showed a mixture of two rotavirus strains (G6G10 and P[5]P[11]), one sample was characterized as G6P[11] and one sample was characterized only by G serotyping/genotyping, and did not react with any primer used. Two electropherotypes were detected and both were present in the same animal. This study demonstrates that two different electropherotypes and/or serotypes of bovine rotavirus can circulate in the same outbreak.

Contaminación del suero fetal bovino con virus de la diarrea viral bovina / Contamination of bovine fetal serum with bovine viral diarrhea virus

Fetal bovine serum (FBS) used in cell culture may be contaminated with viruses, among them bovine viral diarrhea virus (BVDV) affecting the production of biological reagents and the results of diagnosis. The filtration process used in the preparation of commercial FBS abrogates most viral agents that may be present in raw FBS, but BVDV may pass through the filters because of its small size and its pleomorphism. While detection of bovine herpes virus-1 and parainfluenza-3 (PI-3) is determined by observation of the cytopathic effect, and also by hemadsorption in the case of PI-3, the most frequently isolated BVDV is non cytopathic, and infects cells without morphological alterations, inducing problems that arise after several cell generations. Batches of raw and processed FBS were analyzed. Frequencies of BVDV detection in raw serum in Argentina were similar to those published for USA. By conventional methods for BVDV detection, only 2 of 20 commercial batches of FBS had BVDV. Using cell cultures maintained with high concentrations of the serum under study for at least 2 weeks, with detection of viral antigen by indirect immunofluorescence, the percentage of BVDV detection was 80. This method shows that most lots of commercial FBS contain BVDV. RT-PCR allows faster detection of the viral genome, but it must be validated, as it does not show viral replication. To eliminate the problem of BVDV contamination in FBS, only gamma irradiated FBS is used in our laboratory.

Análise antigênica e molecular de amostras citopáticas do vírus da diarréia viral bovina / Antigenic and molecular analysis of cytopathic isolates of bovine viral diarrhea virus

Sete amostras citopáticas do vírus da Diarréia Viral Bovina (BVDV) isoladas de casos clínicos e do sangue de bezerros de rebanhos com problemas reprodutivos foram analisadas. Todas as amostras caracterizadas possuíam uma mistura de vírus citopáticos (cp) e näo-citopáticos (ncp), que foram clonados biologicamente, originando populaçöes puras de vírus de cada biotipo. Os clones cp e ncp obtidos foram caracterizados antigenicamente com um painel de anticorpos monoclonais (MAbs) e quanto à expressäo da proteína näo-estrutural NS3. A análise de reconhecimento pelos MAbs revelou dois padröes de reatividade: 1. Em cinco casos, os vírus cp e ncp de uma mesma amostra mostraram-se antigenicamente muito semelhantes entre si, indicando tratar-se de verdadeiros "pares" de vírus, nos quais o vírus cp origina-se do ncp através de mutaçöes ou recombinaçöes; 2. Duas amostras, no entanto, continham vírus cp e ncp com diferenças antigênicas consideráveis entre si. A análise de poliptídeos näo-estruturais das amostras ncp através de Western immunoblot revelou uma única banda de reatividade, de massa aproximada de 125kDa, correspondente à proteína näo-estrutural NS23. As amostras cp expressaram, além da NS23, um polipeptídeo de massa aproximada de 80kDa, correspondente à NS3. Duas amostras cp apresentaram diferenças na migraçäo da NS23. Uma amostra apresentou a NS23 com massa menor do que 125kDa, enquanto outra amostra apresentou duas bandas de reatividade, com massas menor e maior que a NS23 dos demais vírus, respectivamente. Esses resultados confirmam achados anteriores de que amostras de campo citopáticas do BVDV geralmente possuem vírus dos dois biotipos e que o fenótipo citopático está associado à expressäo da proteína NS3. O isolamento de amostras citopáticas do sangue de animais clinicamente normais de um feto, no entanto, demonstra que a ocorrência de vírus cp näo se restringe a casos da Doença das Mucosas.

Antigenic characterization of Brazilian bovine viral diarrhea virus isolates by monoclonal antibodies and cross-neutralization

Nineteen Brazilian isolates of bovine viral diarrhea virus (BVDV) were characterized antigenically with a panel of 19 monoclonal antibodies (mAbs) (Corapi WV, Donis RO and Dubovi EJ (1990) American Journal of Veterinary Research, 55: 1388-1394). Eight isolates were further characterized by cross-neutralization using sheep monospecific antisera. Analysis of mAb binding to viral antigens by indirect immunofluorescence revealed distinct patterns of reactivity among the native viruses. Local isolates differed from the prototype Singer strain in recognition by up to 14 mAbs. Only two mAbs - one to the non-structural protein NS23/p125 and another to the envelope glycoprotein E0/gp48 - recognized 100 per cent of the isolates. No isolate was recognized by more than 14 mAbs and twelve viruses reacted with 10 or less mAbs. mAbs to the major envelope glycoprotein E2/gp53 revealed a particularly high degree of antigenic variability in this glycoprotein. Nine isolates (47.3 per cent) reacted with three or less of 10 E2/gp53 mAbs, and one isolate was not recognized by any of these mAbs. Virus-specific antisera to eight isolates plus three standard BVDV strains raised in lambs had virus-neutralizing titers ranging from 400 to 3200 against the homologous virus. Nonetheless, many antisera showed significantly reduced neutralizing activity when tested against heterologous viruses. Up to 128-fold differences in cross-neutralization titers were observed for some pairs of viruses. When the coefficient of antigenic similarity (R) was calculated, 49 of 66 comparisons (74.24 per cent) between viruses resulted in R values that antigenically distinguish strains. Moreover, one isolate had R values suggesting that it belongs to a distinct serologic group. The marked antigenic diversity observed among Brazilian BVDV isolates should be considered when planning diagnostic and immunization strategies.

Caracterização preliminar de amostras do vírus da Diarréia Viral Bovina (BVDV) isoladas no Brasil / Preliminary characterization of brazilian isolates of bovine viral diarrhea virus (BVDV)

O presente artigo relata a caracterização inicial de 19 amostras do vírus da Diarréia Viral Bovina (BVDV) isoladas no Brasil, com relação a aspectos biológicos, antigênicos e moleculares. Onze amostras foram isoladas de fetos bovinos, seis foram obtidas do sangue de animais clinicamente saudáveis de rebanhos com problemas reprodutivos e duas amostras foram isoladas de casos clínicos de enfermidade gastrentérica. Os casos de doença entérica afetaram animais jovens e cursaram com diarréia, às vezes sanguinolenta, erosões e ulcerações na mucosa oronasal e do trato digestivo, e eventualmente hemorragias digestivas e petéquias na vulva. Dezesseis amostras (84,2%), incluindo aquelas isoladas de fetos e dos casos clínicos, pertencem ao biotipo não-citopático (ncp). A replicação de outras três amostras (15,8%), foi caracterizada pelo aparecimento de vacuolização e destruição progressiva do tapete celular. A análise das amostras que produziram citopatologia, após clonagem, revelou tratar-se de populações mistas composta de vírus citopáticos (cp) e não-citopáticos. A análise de polipeptídeos virais através de SDS-PAGE seguida de "Western-immunoblot" revelou a produção da proteína não-estrutural NS3/p80 em células infectadas com as amostras cp. Em contraste, não se evidenciou a geração da NS3/p80 em células infectadas com as amostras ncp que produziram apenas o polipeptídeo precursor NS23/p125. A subsequente análise de reatividade frente a um painel de 15 anticorpos monoclonais (AcMs) revelou uma diversidade antigênica marcante entre os isolados, sobretudo na glicoproteína E2/gp53. Embora um AcM contra essa glicoproteína reagiu com 18 isolados (94,7%), outros nove AcMs anti-E2/gp53 reconheceram entre zero e 57,9% das amostras brasileiras. A grande variabilidade antigênica detectada entre as amostras brasileiras do BVDV pode ter importantes implicações para o diagnóstico e estratégias de controle e imunização contra o vírus. TERMOS DE INDEXAÇÃO: Vírus da Diarréia Viral Bovina, BVDV, biotipos, diversidade antigênica

El virus de la DVB como agente contaminante en cultivo de tejidos animales / Bovine viral diarrhea virus as a contaminating agent in animal tissue cultures

El pestisvirus que produce la diarrea viral bovina (V.D.V.B), ocasiona porblemas reproductivos en el ganado vacuno y se encuentra ampliamente difundido en el país. La presencia de cepas no citopatogénicas del mismo, las cuales normalmente no se evidencian en los cultivos celulares contaminados con éste virus, han constituido un limitante para el establecimiento y mantenimiento de cultivos primarios y líneas celulares libres del V.D.V.B., para uso rutinario en actividades diagnósticas o investigativas. El suero fetal bovino (SFB) es la fuente usual de contaminación para los cultivos de tejidos animales, dadas las escasas previsiones para el control de su calidad, ya que la recolección y mercadeo del SFB se realiza con criterios artesanales y sin los debidos controles de calidad. Específicamente en el caso del V.D.V.B., se pueden presentar dos situaciones con el SFB: que contenga partículas virales o que posea niveles de anticuerpos contra el virus; en ambos casos se darán problemas en los procedimientos realizados con dichos sueros. Con el fin de controlar las contaminaciones de los cultivos celulares con el VDVB, en el Postgrado de la Facultad de Medicina Veterinaria de la Universidad Nacional, se ha empleado la técnica de inmunofluorescencia indirecta (IFI) para detectarlas tanto, en el SFB como en diferentes cultivos celulares, obteniéndose para el SFB el 25.8 por ciento de postivos de un total de 32 muestras de diferentes orígenes y para cultivos celulares el 42.8 por ciento de positivos de 28 lotes, procedentes de diferente origen animal y centros de investigación, resultados que son comparables con los obtenidos por otros laboratorios internacionales como el National Animal Diseases Laboratory (NADL) del Departamento de Agricultura de USA. Dentro de las recomendaciones para la prevención y control del problema en laboratorios de diagnóstico e investigación médica y veterinaria, que empleen tecnologías relacionadas con manejo y mantenimiento de cultivos celulares, están las de implementar técnicas para la detección del virus como la IFI, la peroxidasa o la inmunoprecipitación al igual que el manejo de un bajo número de fetos en la conformación de lotes de SFB