Coxiella burnetii associated with BVDV (Bovine Viral Diarrhea Virus), BoHV (Bovine Herpesvirus), Leptospira spp, Neospora caninum, Toxoplasma gondii and Trypanosoma vivax in reproductive disorders in cattle / Coxiella burnetii associada ao BVDV (Vírus da Diarreia Viral Bovina), BoHV (Vírus do Herpes Bovino), Leptospira spp, Neospora caninum, Toxoplasma gondii e Trypanosoma vivax em distúrbios reprodutivos em bovinos

Abstract This is a cross-sectional study to assess the presence of antibodies in ruminants against selected pathogens associated with reproductive disorders in cattle in four Brazilian states, including the zoonotic agent Coxiella burnetii. The used tests were Virus Neutralization Assay for IBR and BVD, Microscopic Agglutination Test for Leptospira spp., Indirect Fluorescent Antibody Test (IFAT) for C. burnetii and Toxoplasma gondii, and Enzyme-Linked Immunosorbent Assay for Neospora caninum and Trypanosoma vivax. Seropositivity for C. burnetii was 13.7% with titers from 128 to 131,072; 57.8% for BoHV-1, with titers between 2 and 1,024; 47.1% for BVDV-1a, with titers from 10 to 5,120; 89.2% for N. caninum; 50% for T. vivax; and 52.0% for Leptospira spp., with titers between 100 to 800 (the following serovars were found: Tarassovi, Grippotyphosa, Canicola, Copenhageni, Wolffi, Hardjo, Pomona and Icterohaemorrhagiae); 19.6% for T. gondii with titer of 40. This is the first study that has identified C. burnetii in cattle associated with BoHV and BVDV, N. caninum, Leptospira spp., T. gondii and T. vivax. Thus, future studies should be conducted to investigate how widespread this pathogen is in Brazilian cattle herds.

Resumo Este é um estudo transversal para avaliar a presença de anticorpos em ruminantes contra patógenos selecionados e associados a distúrbios reprodutivos em bovinos de quatro estados brasileiros, incluindo o agente zoonótico Coxiella burnetii. Os testes utilizados foram Teste de Vírus-Neutralização para BoHV e BVDV, teste de Aglutinação Microscópica para Leptospira spp., Reação de Imunofluorescência Indireta for C. burnetii e Toxoplasma gondii, e Ensaio de Imunoabsorção Enzimática para Neospora caninum e Trypanosoma vivax. A soropositividade para C. burnetii foi de 13,7% com títulos de 128 a 131.072; 57,8% para BoHV-1, com títulos entre 2 a 1.024; 47,1% para BVDV-1a, com títulos de 10 a 5.120; 89,2% para N. caninum; 50% para T. vivax; e 52,0% para Leptospira spp., com títulos entre 100 a 800 (sorovares encontrados: Tarassovi, Grippotyphosa, Canicola, Copenhageni, Wolffi, Hardjo, Pomona e Icterohaemorrhagiae) 19,6% para T. gondii com título de 40. Este é o primeiro estudo que evidencia a participação de C. burnetii em bovinos associada ao Vírus da Rinotraqueíte bovina infecciosa e da diarreia viral bovina, N. caninum, Leptospira spp., T. gondii e T. vivax em bovinos. Desta forma, futuros estudos devem ser conduzidos a fim de investigar o quão disseminado se encontra este patógeno em rebanhos bovinos brasileiros.

Prostaglandin A1 inhibits the replication of bovine viral diarrhea virus

ABSTRACT Bovine viral diarrhea virus can cause acute disease in livestock, leading to economic losses. We show that Prostaglandin A1 inhibits bovine viral diarrhea virus replication in Madin-Darby bovine kidney cells (94% inhibition using 5 µg/mL). Light and electron microscopy of infected cells shows that Prostaglandin A1 also prevents virus-induced vacuolization, but at higher concentrations (10 µg/mL).

Detecção do virus 'HoBi'-like (BVDV-3) em bovino no semiárido do Estado da Paraíba / Detection of 'HoBi'-like (BVDV-3) vírus in a cow in the semiarid of Paraíba, Northeastern Brazil

Objetivou-se descrever os aspectos clínicos e anatomopatológicos, e a identificação viral de um caso de infecção pelo vírus 'Hobi'-like (BVDV-3) em bovino do semiárido paraibano, Nordeste do Brasil. Um bovino, fêmea, três meses de idade, foi levado ao Hospital Veterinário da UFCG apresentando salivação, dificuldade de apreensão do teto, falta de apetite, fezes escuras e em pouca quantidade. Diante da piora do quadro clínico optou-se por sua eutanásia in extremis, seguida da realização da necropsia e coleta de material para histopatologia. Histologicamente, nas mucosas do trato digestivo, havia edema, degeneração balonosa, necrose e infiltrado inflamatório, que foi observado na face dorsal da língua e no seu epitélio mais profundo. A imunohistoquímica de amostras de extremidade de pavilhão auricular demonstrou marcação antigênica positiva e pela RT-PCR foi possível detectar RNA viral do BVDV no soro sanguíneo, cujo efeito citopático em células epiteliais de rim bovino da linhagem "Madin Darby bovine kidney" (MDBK) não foi observado. O sequenciamento do gene 5'NCR demonstrou que o vírus isolado estaria mais relacionado ao 'Hobi'-like (BVDV-3). Após a confirmação do diagnóstico foram coletadas amostras de soro dos 23 animais do rebanho para sorologia por ELISA indireto, sendo constatada 69,6% (16/23) de soropositividade. A identificação deste novo caso de infecção por 'Hobi'-like na Paraíba reafirma a necessidade de um monitoramento regular para BVDV na região para detecção precoce da infecção dos rebanhos e adoção de medidas eficazes de prevenção e controle.(AU)

The aim of this study was to describe the clinical and anatomopathological aspects, and the viral identification of a case of 'Hobi'-like (BVDV-3) vírus infection in cattle from the semiarid of Paraiba State, Northeastern Brazil. A female bovine, three months old, was sent to the UFCG's Veterinary Hospital presenting salivation, difficulty of ceiling seizure, lack of appetite, dark feces and in small amounts. Due to worsening of symptoms it was decided to in extremis euthanasia, followed by the necropsy and collection of material for histopathology. Histologically, in the mucous membranes of the digestive tract there were edema, ballooning degeneration, necrosis and inflammatory infiltrate, which was observed on the dorsal surface of the tongue and in its deepest epithelium. The immunohistochemical of skin biopsies of the extremity of the ear (ear notches) showed positive antigenic marking and by RT-PCR it was possible to detect viral RNA of BVDV in the serum, whose cytopathic effect in epithelial cells of bovine kidney lineage "Madin Darby bovine kidney" (MDBK)was not observed. The sequencing of the 5'NCR gene showed that the virus isolated was more related to 'Hobi'-like (BVDV-3). After confirming the diagnosis serum samples were collected from 23 animals for serology by indirect ELISA, and a seropositivity of 69.6% (16/23) was found. The identification of this new case of 'Hobi'-like infection in Paraiba reaffirms the need for regular BVDV monitoring in the region to early detection of infection in the herds and adoption of effective preventive and control measures.(AU)

Factores inmunológicos que pueden influir en la presencia de síntomas clínicos en niños con giardiasis / Immunological factors influencing the presence of clinical symptoms in children with giardiasis

El objetivo de este trabajo fue evaluar el perfil de la respuesta inmune según sintomatología intestinal en niños con giardiasis. Se realizó un estudio transversal clínico, inmunológico y parasitológico en 182 escolares (6-7 años) de la región de Barlovento, Estado Miranda. La evaluación clínica fue realizada haciendo énfasis en los síntomas intestinales y evaluación antropométrica. Se realizaron exámenes de heces seriadas. Los niveles de IgA secretora (total y específicos a G. duodenalis) así como los niveles de citocinas fueron determinados mediante ELISA. Los niveles de óxido nítrico usando el método colorimétrico de Griess. Análisis estadístico mediante Med calc Software, Versión 12.4.0. Encontramos que Los niveles plasmáticos de TNF-α y de óxido nítrico (en saliva) fueron más elevados en los niños con síntomas intestinales y giardiasis (P<0.001) mientras que los niveles de TGF-β, IL-10 y los niveles de IgA secretora total y especifica a G. duodenalis fueron más elevados (P<0.0001) en el grupo de asintomáticos. Los niveles de IL-6 fueron más elevados en los niños con giardiasis independientemente de la sintomatología. La co-infeccion con A. lumbricoides se asoció negativamente a la presencia de síntomas intestinales. Los indicadores antropométricos (Talla/edad, peso/Edad y Peso/ Talla) se asociaron positivamente a la presencia de citocinas reguladoras (P<0.005) y niveles de IgA secretora (P<0.0001). Así, Diferencias en el perfil de la respuesta inmune podrían asociarse a síntomas intestinales inducidos por giardiasis. Deficiencias en el estado nutricional se asocian a procesos inflamatorios y a la generación de síntomas mientras que la co-infección con helmintos intestinales favorece la reducción de la sintomatología.

We evaluated the immune profile associated to intestinal symptoms in children with giardiasis. A crosssectional study including clinical, immunological and parasitological aspects in 182 children (6-7 years) from the area of Barlovento, Miranda state, Venezuela was carried out. Clinical evaluation was performed with emphasis on intestinal symptoms and anthropometric parameters were also determined. Serial stool examinations were carried out. Secretory IgA levels (total and specific against G. duodenalis) and cytokine levels were determined by ELISA. Nitric oxide levels were determined using the Griess colorimetric method. Statistical analysis was performed by Med Calc Software, Version 12.4.0. We found that plasma levels of TNF-α and nitric oxide (salivary) were higher in children with intestinal symptoms and giardiasis (P<0.001) while the levels of TGF-β, IL-10 and IgA levels (total and anti-G. duodenalis) were higher (P<0.0001) in the asymptomatic group. The levels of IL-6 were higher in children with giardiasis regardless of symptoms. Coinfection with A. lumbricoides was associated negatively with the presence of intestinal symptoms. Anthropometric indicators (height / age, weight / age and weight / height) were positively associated with the presence of regulatory cytokines (P<0.005) and secretory IgA levels (P<0.0001). Therefore, differences in the immune profile may be associated with intestinal symptoms induced by giardiasis. Co-infection with intestinal helminths helps to reduce symptoms while nutritional deficiencies are associated with inflammatory processes and the generation of symptoms.

Development of a novel diagnostic test for detection of bovine viral diarrhea persistently infected animals using hair

The purpose of this study was to determine whether manually plucked hairs might serve as an alternative sample for a quantitative real time polymerase chain reaction (qRT-PCR) testing. Twenty three, 1~3 week old, non-bovine viral diarrhea virus (BVDV) vaccinated calves, found to be positive for BVDV by immunohistochemical staining, were selected and hairs were manually plucked from the ear. qRT-PCR was performed on samples consisting of more than 30 hairs (30~100) and whole blood. All 23 animals were positive for the virus by qRT-PCR performed on the whole blood and when samples of more than 30 hairs were assayed. Additionally, qRT-PCR was performed on groups of 10 and 20 hairs harvested from 7 out of 23 immunohistochemical staining-positive calves. When groups of 20 and 10 hairs were tested, 6 and 4 animals, respectively, were positive for the virus.

[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

Prevalência da infecção pelo vírus da diarréia viral bovina (BVDV) no estado de Goiás, Brasil / Bovine viral diarrhea virus (BVDV) infection in Goiás, Brazil

O presente estudo objetivou estimar a prevalência de anticorpos contra o vírus da diarreia viral bovina (BVDV) em animais não vacinados e determinar os potenciais fatores de risco para a infecção em rebanhos bovinos no estado de Goiás, Brasil. Foram coletadas, entre março e setembro de 2002, amostras de soro de 3.533 animais em 888 propriedades localizadas em 232 municípios de Goiás. Essas amostras foram submetidas ao teste de soroneutralização e, em cada propriedade avaliada, aplicou-se um questionário epidemiológico para investigar possíveis fatores associados à infecção. Os dados obtidos foram analisados por meio do programa Epi Info versão 6.04. Adicionalmente, em 960 amostras soronegativas para anticorpos contra o BVDV, foi realizada pesquisa do antígeno viral, visando estimar a frequência de animais persistentemente infectados (PI). A soroprevalência para o BVDV foi de 64,0por cento em 784 amostras de soro e de 88,3por cento nas propriedades. Dos municípios estudados, 226 (97,4por cento) apresentaram, pelo menos, um animal/rebanho soropositivo. Em relação aos fatores de risco, apenas a idade mostrou-se associada à soropositividade nos animais, nas propriedades, nenhuma das variáveis analisadas foi considerada como fator de risco. A presença do antígeno viral foi detectada em quatro (0,4por cento) das amostras analisadas. Os resultados obtidos demonstram a expressiva disseminação do BVDV no rebanho bovino desta região.

This study was performed to estimate the seroprevalence of bovine viral diarrhea virus (BVDV) in a population of non-vaccinated cattle in Goiás State, Brazil, and to determine the potential risk factors related to the seropositivity. Serum samples were collected from 3,533 animals of 888 herds from 232 municipalities in Goiás between March and September, 2002. These samples were submitted to the serumneutralization test and an epidemiological questionnaire was filled out for each herd to investigate variables that could be associated with this infection. The data were analyzed using Epi Info 6.04. In addition, 960 BVDV antibody-negative sera were further tested by antigen-ELISA in order to identify persistently infectedanimals (PI). The seroprevalence was 64.0% and 784 (88.3%) herds had at least one seropositive animal. Two hundred and twenty six (97.4%) municipalities showed at least one positive herd/animal. Only age influenced the presence ofneutralizing antibodies to this virus in animals. None of the exposure variablesanalyzed was considered as a risk factor for the infection with BVDV in cattleherds. Four (0.4%) of 960 seronegative samples were BVDV antigen positive. Based on these results, we concluded that BVDV infection is spread among cattle herds in Goiás State, Brazil.

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.

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...

Recombinant E2 glycoprotein of bovine viral diarrhea virus induces a solid humoral neutralizing immune response but fails to confer total protection in cattle

Two recombinant baculoviruses were produced in order to obtain a bovine viral diarrhea virus (BVDV) immunogen: AcNPV/E2 expressing E2 glycoprotein, and AcNPV/E0E1E2 expressing the polyprotein region coding for the three structural proteins of BVDV (E0, E1, and E2). Mice were immunized with Sf9 cells infected with the recombinant baculoviruses in a water in oil formulation and the production of neutralizing antibodies was evaluated. Since E2 elicited higher neutralizing antibody titers than E0-E1-E2 polyprotein, it was selected to immunize cattle. Calves received two doses of recombinant E2 vaccine and were challenged with homologous BVDV 37 days later. The recombinant immunogen induced neutralizing titers which showed a mean value of 1.5 ± 0.27 on the day of challenge and reached a top value of 3.36 ± 0.36, 47 days later (84 days post-vaccination). On the other hand, sera from animals which received mock-infected Sf9 cells did not show neutralizing activity until 25 days post-challenge (62 days post-vaccination), suggesting that these antibodies were produced as a consequence of BVDV challenge. Even when no total protection was observed in cattle, in vitro viral neutralization assays revealed that the recombinant immunogen was able to induce neutralizing antibody synthesis against the homologous strain as well as against heterologous strains in a very efficient way.

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.

Coeficientes de correlación dentro de hatos y efectos de diseño para seropositividad a brucelosis, diarrea voral y rinotraqueitis infecciosa bovina de hatos lecheros / Intra-cluster correlation coeficients and desing effects for seropositivity to brucelosis, viral bovine diarrhea and infectious bovine rhinotracheitis

La estimación de los coeficientes de correlación dentro de hatos (CCI) o el efecto de diseño (D) para enfermedades infecciosas puede ser de interés en el diseño de encuestas y para calcular factores de inflación de varianza para estimadores de prevalencia en muestreos por conglomerados. Los valores de CCI y D para brucelosis, diarrea viral bovina (DVB) y rinotraqueitis infecciosa bovina (RIB) se calcularon utilizando los métodos de Bennett y análisis de varianza (VARCOMP). Los valores de CCI para brucelosis, DVB y RIB utilizando dichos métodos fueron 0,162 y 0,153; 0,044 y 0,015; y 0,110 y 0,128; y los valores de D fueron 3,33 y 3,09; 1,63 y 1,21; y 2,58 y 3,02; respectivamente. En conclusión, los valores de CCI y D para brucelosis, DVB y RIB fueron diferentes, según el método utilizado y para cada una de estas infecciones, por lo que se requieren tamaños de muestra diferentes para obtener la misma precisión en la estimación de sus prevalencias. El reporte de los valores de CCI y D sería de gran ayuda para la planeación y diseño de estudios epidemiológicos

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.

Detección de biotipos del virus de la Diarrea Viral Bovina (VDVB) a través de RT-PCR / Detection of Bovine Viral Diarrhea virus biotypes through RT-PCR

En el presente artículo se describe la normalización de la prueba de RT-PCR para ser empleada como herramienta en la detección del Virus de la Diarrea Viral Bovina (VDVB). Los iniciadores utilizados amplificaron un sector de 280 pb que se encuentra dentro del extremo 5’ UTR del genoma viral. Para el proceso de normalización, se usó como control positivo, cepas de referencia de VDVB (NADL, Osloss). Para evaluar reacción cruzada se usó una cepa de virus de Enfermedad de las Fronteras (BD). La obtención del cDNA se realizó por el método de ®random primers¼ (iniciadores aleatorios). La prueba detectó tanto cepas citopáticas, como no citopáticas del VDVB. El protocolo establecido demostró un buen funcionamiento in vitro y es la base para posteriores pruebas de validación y evaluación de la prueba diagnóstica.

The effect of infection of hatched blastocyst bovine embryos with border disease and bovine viral diarrhea viruses.

The effect of infection with teratogenic viruses at early stages of pregnancy is not fully understood. This study aimed to look at the effect of infection with teratogenic viruses such as bovine viral diarrhea virus (BVDV) and border disease virus (BDV), on early stage embryos at the hatched blastocyst stage. BVDV and BDV are known to cross the placenta of infected mothers and lead to congenital defects and death of developing fetuses. This study can be a good model for better understanding the effects of other teratogenic viruses such as Rubella virus in humans.

Magnitude, duraçäo e especificidade da resposta sorológica em bovinos vacinados contra o vírus da diarréia viral bovina (BVDV) / Magnitude duration and specicicity of the serological response in cattle vaccinated against bovine viral diarrhea virus (BVDV)

A resposta sorológica induzida por três vacinas comerciais inativadas contra o vírus da Diarréia Viral Bovina (BVDV) foi avaliada em bovinos imunizados três vezes (dias 0, 30 e 180) e testados a diferentes intervalos após a vacinaçäo. Trinta dias após a segunda vacinaçäo, 74,5 por cento (70/94) dos animais apresentavam anticorpos neutralizantes contra o BVDV-1 e 52,1 por cento (49/94) contra o BVDV-2. Os títulos médios (GMT) e o número de animais reagentes contra o BVDV-1 eram de 109,3 (32/36); 54,6 (22/28) e 25,5 (16/30) para as vacinas A, B e C, respectivamente; e de 19 (27/36), 42,3 (12/28) e 18,4 (10/30) contra o BVDV-2. Os títulos reduziram-se aos 180 dias, sendo que 31,9 por cento (30/94) dos animais já näo apresentavam atividade neutralizante frente ao BVDV-1 e 63,8 por cento (60/94) frente ao BVDV-2. Nesta data, os títulos médios e o número de animais positivos frente ao BVDV-1 eram de 28,3 (30/36), 28,3 (20/28) e 16,1 (14/30) e frente ao BVDV-2 de 16,8 (18/36), 21,6 (10/28) e 28,3 (6/30) para as vacinas A, B e C, respectivamente. Após o reforço (dia 180), os títulos médios contra o BVDV-1 aumentaram significativamente nos três grupos vacinais e contra o BVDV-2 apenas no grupo A. Trinta dias após, os títulos médios e o número de reagentes contra o BVDV-1 eram de 104,8 (23/24), 50,3 (24/26) e 43,7 (24/28) e contra o BVDV-2 de 33,4 (23/24), 23,3 (22/26) e 15,7 (22/28) para as vacinas A, B e C. Os títulos contra o BVDV-1 no dia 210 foram estatisticamente superiores aos títulos contra o BVDV-2 nos três grupos vacinais. O soro de alguns animais positivos de cada grupo foi testado frente a quatro amostras brasileiras de BVDV-1 e duas de BVDV-2. Além dos títulos baixos a moderados, os testes de neutralizaçäo cruzada revelaram variaçöes marcantes na atividade neutralizante frente a isolados de campo antigenicamente diferentes. Esses resultados demonstram que a vacinaçäo näo induziu uma resposta sorológica de magnitude e duraçäo adequadas na maioria dos animais, principalmente frente à grande diversidade antigênica das amostras de BVDV.

Tissue distribution of bovine viral diarrhea virus antigens in persistently infected cattle

The tissue distribution and cellular localization of viral antigens in three cattle with persistent bovine viral diarrhea virus (BVDV) infection was studied. In three cases, necropsy findings of oral ulcers, abmasal ulcers and necrosis of Peyer's patches were suspected have been caused by BVDV infection. Non-cytopathic BVDV was isolated from a tissue pool of liver, kidneys and spleen. Immunohistochemical detection of BVDV showed that BVDV antigens were detected in both epithelial and nonepithelial cells in all examined organs, including the gastrointestinal tract, liver, pancreas, lung, lymphatic organs (spleen, lymph nodes), adrenal gland, ovary, uterus, and the mammary gland. These findings support the hypothesis that animals with persistent BVDV infection spread BVDV through all routes, and that infertility in BVDV infection is associated with the infection of BVDV in the ovaries and uteri.

Identificaçäo do vírus da Diarréia Viral Bovina tipo 2 (BVDV-2) no Sul do Brasil / Identification of bovine virus diarrhea virus type-2(BVDV-2) in Southern Brazil

Amostras do vírus da Diarréia Viral Bovina (BVDV), denominadas de BVDV tipo 2 (BVDV-2), foram inicialmente identificadas em surtos de BVD aguda e enfermidade hemorrágica e têm sido isoladas predominantemente na América do Norte. O presente artigo descreve dois casos de enfermidade gastroentérica/respiratória seguidos de isolamento e identificaçäo de amostras de BVDV tipo 2 no sul do Brasil. Os vírus foram isolados de duas novilhas de diferentes rebanhos. Um dos animais apresentou enfermidade aguda, cursando com anorexia, atonia ruminal, diarréia escura ou muco-sanguinolenta, tenesmo e descarga nasal muco-purulenta. O outro animal desenvolveu enfermidade de longa duraçäo (7 meses), caracterizada por crescimento retardado, anorexia, quadros recorrentes de diarréia, dermatite interdigital, hemorragias digestivas e genitais ocasionais, conjuntivite, artrite e pneumonia crônica. Congestäo disseminada das mucosas, ulcerações extensivas e profundas na língua, palato e esôfago, áreas necróticas na mucosa do rúmen, áreas de congestäo e ulcerações cobertas com fibrina no intestino delgado foram os achados mais proeminentes. Antígenos do BVDV foram demonstrados por imunohistoquí-mica no epitélio da língua, nos pulmões e em linfonodos mesentéricos. Amostras näo-citopáticas do BVDV foram isoladas em cultivo celular a partir de leucócitos e do baço dos animais afetados e identificadas por imunofluorescência. Caracterizaçäo antigênica e análise filogenética desses isolados, e de outras duas amostras de BVDV isoladas de fetos coletados em matadouros, revelou tratar-se de BVDV tipo 2. A presença do BVDV tipo 2 na populaçäo bovina do Brasil possui um significado epidemiológico importante e pode ter conseqüências para o diagnóstico, estratégias de imunizaçäo e produçäo de vacinas(au)