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 of anti-Cryptosporidium polyclonal antibodies and standardization of direct immunofluorescence for detecting oocysts in water / Produção de anticorpos policlonais anti-Cryptosporidium e padronização da imunofluorescência direta para a detecção de oocistos na água

INTRODUCTION: The production of anti-Cryptosporidium polyclonal antibodies and its use in direct immunofluorescence assays to determine the presence of Cryptosporidium in water are described in the present work. METHODS: Two rabbits were immunized with soluble and particulate antigens from purified Cryptosporidium oocysts. The sera produced were prepared for immunoglobulin G extraction, which were then purified and conjugated with fluorescein isothiocyanate (FITC). Slides containing known amounts of oocysts were prepared to determine the sensitivity of the technique. To test the specificity, slides containing Giardia duodenalis cysts were prepared. RESULTS: The conjugate was successfully used in water samples experimentally contaminated with Cryptosporidium oocysts, and it was possible to detect up to five oocysts/spot, corresponding to contamination of 250 oocysts/mL. CONCLUSIONS: The three immunizations performed in the rabbits were enough to produce antibodies against Cryptosporidium, the standard direct immunofluorescence assay permitted the detection of five oocysts in 20 percent of the samples, and no cross-reaction with Giardia duodenalis cysts occurred.

INTRODUÇÃO: A produção de anticorpos policlonais anti-Cryptosporidium e sua utilização na imunofluorescência para determinar a presença de Cryptosporidium em água são descritas no presente trabalho. MÉTODOS: Dois coelhos foram imunizados com antígeno solúvel e particulado provenientes de oocistos purificados de Cryptosporidium. O soro produzido foi preparado para a extração de imunoglobulinas G, que foram purificadas e conjugadas com isotiocianato de fluoresceína (FITC). Lâminas contendo quantidades conhecidas de oocistos foram preparadas para determinar a sensibilidade da técnica. Para testar a especificidade foram preparadas lâminas contendo cistos de Giardia duodenalis. RESULTADOS: O conjugado foi usado com sucesso em amostras de água contaminadas experimentalmente com oocistos de Cryptosporidium, sendo capaz de detectar até cinco oocistos/spots que corresponde a uma contaminação de 250 oocistos/mL. CONCLUSÕES: As três imunizações realizadas nos coelhos foram suficientes para produção de anticorpos contra Cryptosporidium; a reação de imunofluorescência direta padronizada permitiu a detecção de cinco oocistos em 20 por cento das amostras; não houve reação cruzada com cistos de Giardia duodenalis.

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.

Isolamento e caracterizaçäo de cepa moderada virulência do vírus de peste suína clássica no Estado do Paraná, Brasil / Isolation and characterization of a moderate virulence strain of classical swine fever virus, in State of Paraná, Brazil

Trata o presente trabalho do isolamento viral realizado em outubro de 1997, no Laboratório de Virologia do Centro de Diagnóstico "Marcos Enrietti", de uma cepa de moderada virulência do vírus da Peste Suína Clássica (PSC), a partir de fragmentos de órgäos provenientes de granjas de suínos do município de Säo Pedro do Iguaçu, Paraná, Brasil. Foram empregadas técnicas de imunofluorescência e imunoperoxidase indiretas em cortes histológicos, ensaio imunoenzimático (ELISA) para detecçäo de antígeno viral e isolamento viral em cultivo celular da linhagem PK15 (Porcine Kidney, Sus scrofa). Os resultados positivos obtidos pelas diferentes técnicas empregadas mostraram grande precisäo para o diagnóstico da Peste Suína Clássica. Com base nesses resultados, o Serviço de Sanidade Animal recomendou o abate de cerca de 2.000 suínos em sete propriedades envolvidas no foco e a tomada de medidas zoosanitárias adequadas para prevenir a disseminaçäo da doença