Development and Evaluation of an Indirect ELISA: Serological Survey to Detect Specific Antibodies to Bovine Herpesvirus 4

ABSTRACTThe aim of this work was to develop and evaluate an indirect enzyme-linked immunosorbent assay (ELISA) and made a serological screening for specific antibodies to BoHV-4. Bovine serum samples were collected from different Brazilian states and evaluated for the presence of antibodies for BoHV-4 and BoHV-1. The serological results obtained showed that the indirect ELISA assay could be applied for the detection of specific antibodies for BoHV-4. The ELISA test allowed concluding that BoHV-4 is present in bovines in every Brazilian state from which serum samples were collected. The ELISA assay here standardized proved to be useful for the epidemiological studies and showed a positivity range from 1.8 to 66% in Brazil.

Bovine Herpesvirus 4 in Parana State, Brazil: case report, viral isolation, and molecular identification

Bovine Herpesvirus 4 (BoHV-4) is a member of Gammaherpesvirinae sub-family and belongs to genus Rhadinovirus. This virus has been associated with different clinical manifestations and research activity has put forward a strong correlation among virus infection, postpartum metritis, and abortion. The goal of this work was to characterize a virus strain isolate from a cow’s uterine outflow. From swabs drawn of uterine secretion, a virus strain was isolated and characterized by its cytopathology, morphology, and molecular biology approaches. In culture there was CPE development, characterized mainly by long strands with several small balloons along them, radiated from infected cells. Electron microscopy analysis revealed virus particles that had icosahedrical capsid symmetry surrounded by a loose envelope, typical of a herpesvirus. A 2,571 bp PCR product after HindIII digestion generated four fragments, whose base pair composition were 403, 420, 535, and 1,125 bp. Restriction enzymes HindIII and BamHI generated the expected diagnostic bands as well as a 2,350 bp hypermolar fragment as a result of BamHI treatment to demonstrate that agent was a bovine herpesvirus 4, appertaining to DN-599 group.

Immune response after rabies oral immunization in mice

The objective of this study was to formulate an anti-rabies oral vaccine from the cell culture vaccine PV TECPAR to elicit the production of antibodies against the rabies in mice. A vaccine was developed using 10(7.5) DL50/0.03 ml viral antigens homogenised in lanovaseline to facilitate oral administration. Mice were vaccinated two times for seroconversion. Sera of the vaccinated mice showed a higher level of antibody production than the control group. These results could be used to direct the development of an anti-rabies oral vaccine.

Production and characterization of monoclonal antibodies anti fragment Fc of bovine IgG

The aim of this work was to produce and characterize monoclonal antibodies anti bovine immunoglobulin G (IgG). Out of seven hybridomas, two were chosen based on the ELISA'S absorbance values and were labeled B4F11 and B3H12. These monoclonals were analyzed through Western Blot for IgG fragments obtained by proteolysis with papain, separated by electrophoresis in polyacrylamide gel electrophoresis with â-mercaptoetanol as reducing agent. This revealed that, possibly, the B4F11 was directed to a conformational antigen, and that B3H12 reacted in a specific fashion with Fc (Bovine IgG crystallizable fragment). This antibody could be used in the development of reagents to immunoassays relevant for research and diagnosis.

No Brasil, anticorpos anti-espécie específica usados em métodos de diagnóstico geralmente são importados, aumentando o custo das análises. Visando produzir insumos para métodos diagnóstico por enzimaimunoensaio, o presente trabalho teve como objetivo produzir e caracterizar anticorpos monoclonais anti imunoglobulina G (IgG) bovina. De sete hibridomas obtidos e que apresentaram valores relevantes de absorbância em teste imunoenzimático (ELISA indireta), dois clones com melhor performance foram selecionados e designados B4F11 e B3H12. Estes anticorpos monoclonais foram analisados em Western Blot para reatividade com fragmentos de IgG bovina, obtidos por proteólise com papaína e separados por eletroforese em gel de poliacrilamida, com presença do agente redutor beta-mercaptoetanol. A eletroforese mostrou que o anticorpo B4F11, foi direcionado para um antígeno conformacional, e que o monoclonal B3H12 reagiu especificamente com a porção Fc da IgG bovina (fragmento cristalizável). Este anticorpo será utilizado no desenvolvimento de reagentes para imunoensaios de interesse à pesquisa e diagnósticos.

Megacólon tóxico em colite experimental induzida por enemas de ácido acético / Toxic megacolon in experimental colite induced by acethic acid enemas

O objetivo do estudo foi avaliar os aspectos morfológicos da colite induzida pelo ácido acético a 25 por cento, 35 por cento e 50 por cento, via retal. Foram utilizados 40 ratos Sprague-Dawley-Tecpar, divididos em quatro grupos: grupo A (n = 10) - enemas de ácido acético a 25 por cento; grupo B (n = 10) - enemas de ácido acético a 35 por cento; grupo C (n = 10) - enemas de ácido acético a 50 por cento; e grupo D (n = 10) - enemas de ácido acético a 10 por cento (controle). Os aspectos morfológicos foram graduados de acordo com os critérios propostos por Moraes. A intensidade das lesöes foi relacionada com a concentraçäo do ácido utilizado. Todos os ratos com colite difusa induzida pelo ácido acético a 50 por cento morreram em sete dias. Näo houve mortalidade no grupo controle. O exame endoscópico foi utilizado para demonstrar a presença e a gravidade das lesöes. Näo houve diferença do ponto de vista morfológico entre as lesöes induzidas pelo ácido acético a 25 por cento, 35 por cento e 50 por cento. Rodas elas foram graves com megacólon tóxico e/ou perfuraçäo. No grupo controle, as lesöes limitaram-se à mucosa e submucosa, com ausência de complicaçäo

Comparaçäo entre ELISA com anticorpos policlonais e monoclonais para avaliaçäo do conteúdo de glicoproteina em vacina contra a raiva tipo Fuenzalida & OPalacios / Comparison betwen ELISA with polyclonal and monoclonal antibodies for evaluating glycoprotein content in \"Fuenzalida & Palacios\" rabies vaccine

Enzyme immunoassay is a valuable, rapid, and reproducible technique for the quantitative determination of rabies antigens in Fuenzalida & Palacios Rabies Vaccines. This study was performed in paltes sensitized with purified whole virus antibodies and the antigen seizured has been demonstrated with three different conjugates: anti whole total virus, anti-glycoprotein monoclonal and polyclonal antibodies conjugated woth peroxidase. The objective of this study was quantify the glycoprotein content of Fuenzalida & Palcios Rabies Vaccine and to compare thoses results wiwth the National Institute of Health Test (NIH)