Uso de una proteína recombinante del virus de la fiebre aftosa, la polimerasa 3D, en una prueba de inmunodifusión en gel de agar

En este estudio, se examinó la efectividad del uso del polipéptido 3D recombinante, obtenido en su forma nativa en una prueba de IDGA (IDGA-3D), para uso en la detección de anticuerpos específicos de infección con VFA, independientemente de la condición de vacunación. Los resultados indican que en relación a la tradicional prueba de IDGA-VIAA, la IDGA 3D ofrece, particularmente cuando se evalúan sueros de bajo título, un método más consistente, con especificidad comparable, y por lo menos la misma sensibilidad. Ninguno de los antígenos ofreció una ventaja particular con respecto a la definición de las bandas de precipitación. El reemplazo del VIAA por la proteína 3D recombinante tiene considerables atracciones, dado que proporciona un suministro ilimitado de material inocuo, económico, de fácil purificación y consistente, eliminando la presencia potencial de antígenos no específicos de células BHK o componentes de la cápside del VFA.

Distribución histórica de la estomatitis vesicular en Brasil

La estomatitis vesicular es una enfermedad que afecta bovinos, ovinos, porcinos, caprinos y equinos clínicamente indistinguible de la fiebre aftosa. En Brasil fue diagnosticada por primera vez en 1964 en los estados de Alagoas y Pernambuco. En este trabajo se hace un relato histórico a partir de los resultados de diagnósticos de los virus tipos Indiana-2 e Indiana-3 realizados durante los años 1964-1996 en el Centro Panamericano de Fiebre Aftosa.

Obtención de glicoproteínas del virus de la estomatitis vesicular para identificación de anticuerpos

Se describe um método práctico basado en el uso de un detergente no iónico, que se puede remover por diálisis para la extración de las glicoproteínas del virus de la estomatitis vesicular con el objeto de ser usadas para la identificación de anticuerpos por la técnica de ELISA.

Characterization of foot-and-mouth disease virus by monoclonal antibodies.

Monoclonal antibodies (MAbs) were produced against foot-and-mouth disease (FMD) virus types O1 Campos Br1/58, A24 Cruzeiro Br1/55, and C3 Indaial Br1/71, which are the strains used for production of FMD vaccines in the majority of South American countries. Within the library of MAbs produced, a group was selected on the basis of their neutralizing titer in cell culture, protective titer in suckling mice, sensitivity to trypsin, and specificity for virus structural proteins. The MAbs were utilized in an ELISA test format to compare European and South American representative field isolates with vaccine production strains in their r1 relationship as obtained by 50% complement fixation (CF50) with polyclonal antibodies (PAb) and their virus neutralization (VN) relationship obtained with sera from one-time-vaccinated and from revaccinated cattle, respectively. The MAbs selected varied in their reactivity against the different strains and, therefore, enabled us to compare field FMDV strains to those against which the MAbs were produced, with definite advantages over the r1 and VN ratios. Thus, panels of MAb produced with the vaccine strains and appropriately selected are significantly useful for the FMD-control programs because they serve to provide guidance on the immunological coverage provided by the vaccines against FMDV strains circulating in the field. The MAbs are also useful for the differentiation of FMD virus strains.

Foot-and-mouth disease virus typing by complement fixation and enzyme-linked immunosorbent assay using monovalent and polyvalent antisera.

An indirect "sandwich" enzyme-linked immunosorbent assay (ELISA) using polyvalent and monovalent antisera was compared with the 50% complement fixation (CF50) test for the detection of foot-and-mouth disease (FMD) O, A, and C virus types. ELISA was more sensitive than CF50 tests when polyvalent antisera were used for detecting the 3 types of virus in epithelial samples, whereas ELISA using monovalent antisera was the least sensitive technique. The ELISA performed with polyvalent antisera was 9 times more sensitive for detecting FMD virus than that with monovalent antisera. However, viral isolation in cell culture was the most sensitive detection system. The combined use of ELISA with polyvalent antisera and cell culture inoculations was the most effective procedure for identifying FMD virus in epithelial samples from the field.

Aislamiento del virus de la fiebre aftosa en animales de laboratorio. II. Susceptibilidad comparativa de seis especies

Los hallazgos del presente estudio demuestran que el hamster es más susceptible a la infección por el virus de la fiebre aftosa que el ratón lactante, habitualmente empleado para efectuar aislamientos de este agente con fines diagnósticos. El hamster mostró una susceptibilidad superior a la inoculación de Aphthovirus obtenidos de bovinos infectados naturalmente. La comparación se basó en las manifestaciones clínicas, el tiempo medio de supervivencia, el porcentaje de mortalidad, la relación entre título y mortalidad, y el comportamiento de la infección en animales destetados. Le siguieron en orden descendente de susceptibilidad los meriones, conejos y cobayos lactantes, mientras que las ratas resistieron a la infección. Los resultados se analizan en términos de su implicancia diagnóstica para estudios epidemiológicos y el control de la enfermedad.

The present findings demonstrate that the hamster is more susceptible to infection with foot-and-mouth disease virus than the sucking mouse, traditionally used for isolating this agent. Hamsters were more sensitive to the inoculation of Aphthovirus obtained from bovines with natural infections. The comparison was based on clinical manifestations, mean survival time, percent mortality, relationship between titer and mortality, and evolution of infection in weanlings. Following in decreasing order of sisceptibility were suckling gerbils, rabbits and guinea pigs, while rats were refractory. The results are discussed in terms of their diagnostic implications for epidemiologic studies and disease control.

Anticuerpos monoclonales específicos para herpesvirus bovino tipo-1 (cepa Cooper)

Anticuerpos monoclonales fueron preparados contra herpesvirus bovino tipo 1 (BHV-1) purificado y se seleccionaron varios de acuerdo con su habilidad de neutralizar la infectividad viral. El virus fue purificado por dos métodos alternativos de concentración, con polietilenglicol seguido de ultracentrifugación sobre un colchón de sacarosa al 25 por ciento (p/p) o usando un gradiente linear de tartrato de potasio. De un total de 204 líneas celulares que segregaban anticuerpos para el virus, obtenidas en dos fusiones, se seleccionaron y expandieron clonalmente 39 hibridomas. La selección se basó en su reactividad específica por ELISA. De éstos, 11 anticuerpos monoclonales fueron capaces de neutralizar BHV-1 total o parcialmente, con o sin el agregado de complemento. Siete anticuerpos monoclonales fueron producidos (en la forma de fluido ascítico) y purificados por precipitación en sulfato de amonio. De éstos, dos fueron capaces de prevenir la penetración de virión luego de su adherencia a la células. Asimismo, tres anticuerpos monoclonales fueron seleccionados para ser usados en las pruebas de inmunoperoxidasa para la detección del BHV-1 en cultivos celulares infectados.

Monoclonal antibodies (McAbs) were prepared against purified Bovine Herpesvirus Type-(BHV-1) and selected for their ability to neutralize viral infectivity. Viral purification was performed by polyethylene glycol concentration and ultracentrifugation on a 25% (w/w) sucrose cushion, and by potassium tartrate linear gradient. Out of 204 cell lines expressing antobodies to the virus, obtained in two fusions, 39 hybridomas were selected and cloned based on enzyme-linked immunosorbent assay (ELISA) reactivity. Eleven McAbs were able to neutralize BHV-1 partially or totally, with or without complement. Seven McAbs were produced as ascitic fluid, and ammonium sulfate-purified. Of these, two were able to prevent virion penetration into the cell after attachment. Three neutralizing McAbs were selected for use in immunoperoxidase tests for detecting BHV-1 in infected cell cultures.

Development and evaluation of an enzyme-linked immunosorbent assay for detection, typing, and subtyping of vesicular stomatitis virus.

An indirect sandwich enzyme-linked immunosorbent assay (ELISA) has been used for vesicular stomatitis virus (VSV) typing using sets of monovalent and polyvalent rabbit/guinea pig antisera for identification of VSV types New Jersey (VNJ) and Indiana (VIND). The VIND polyvalent antiserum (VIND-P) detects any strain of the 3 subtypes of the VIND type (VIND-1, VIND-2, and VIND-3) with the same strong reactivity. It is also possible to subtype the VIND strains using VIND-P rabbit antiserum as capture antibody and monovalent VIND-1, VIND-2, or VIND-3 guinea pig antisera as detector. The ELISA proposed has about 10 times more sensitivity and provides 10% more positive results than does the complement fixation 50% (CF50) test when epithelial samples are tested.

Identificación de anticuerpos de la lengua azul por la técnica de inmunodifusión en gel de agar

Se preparó un antígeno soluble del virus de la lengua azul (VLA) para ser utilizado en pruebas de inmunodifusión en gel de agar (IDGA). Dicho antígeno es grupo específico, y es capaz de detectar anticuerpos inducidos por cualquiera de los 24 serotipos del VLA. Fue producido a partir del VLA serotipo 4 y controlado en IDGA frente a antígeno y sueros de referencia (NVSL, Ames, EUA; LARA, Campinas, Brasil; Veterinary Diagnostic Technology, Inc., EUA) y por la técnica inmunoenzimática (ELISA) con anticuerpo monoclonal 3-17-A3 (IADR, Pirbright, Inglaterra). Todas las pruebas mostraron una reacción de total identidad con los reactivos controles.

A soluble antigen of the bluetongue virus (BTV) was prepared for use in immunodifusion in agar gel tests (IDAG). The antigen is group specific and is capable of detecting antibodies induced by any of the 24 BTV serotypes. It was produced from type 4 BTV serotype and controled in IDAG against reference antigens and sera (NVSL, Ames, USA; LARA, Campinas, Brazil; Veterinary Diagnostic Technology, Inc. USA) and by the enzyme-linked immunosorbent assay (ELISA) with 3-17-A3 monoclonal antibody (IADR, Pirbright, England). All tests yielded a reaction of total identify with the control reagents.

Aplicación de la técnica inmunoenzimática (ELISA) para el diagnóstico de los virus de la fiebre aftosa y estomatitis vesicular en comparación con la prueba de fijación del complemento

Se ha comparado la técnica inmunoenzimática (ELISA) "sandwich" indirecta con la prueba de fijación del complemento 50 por ciento (FC50) para detectar los serotipos O, A y C del virus de la fiebre aftosa (VFA) y New Jersey (NJ) e Indiana(IND) del virus de la estomatitis vesicular (VEV). Para las cepas O, A, C e IND se usaron antisueros polivalentes como detectores y para las cepas NJ fueron utilizados antisueros monovalentes. La prueba de Elisa mostró ser un procedimiento más satisfactorio para identificar VFA y VEV en muestras de epitelios de animales afectados por enfermedad vesicular.

Caracterización epidemiológica, antigénica, inmunogénica y bioquímica del virus de la fiebre aftosa A-81 Argentina/87

Estudios seroepidemiológicos llevados a cabo en muestras de campo, realizados a fines de 1986 por el Laboratorio de Diagnóstico y Referencia para las Américas en el Centro Panamericano de Fiebre Aftosa (CPFA), mostraron una variación antigénica de virus de la fiebre aftosa tipo A en relación con el usual A24 y aislamientos de campo A-79 en la Argentina.

Seroepidemiologic studies constantly carried out on field samples by the Diagnostic Reference Laboratory for te Americas at the Pan American Foot-and-Mouth Disease Center (PAFMDC) revealed, late in 1986, an antigenic variation of FMDV type A in relation to the usual A24 and A-79 field isolates in Argentina.

Actualizacion de cepas del virus de la fiebre aftosa de importancia epidemiologica en America del Sur / Updating on food-and-mouth disease virus strains of epidemiological importance in South America

The high variability of foot-and-mouth disease, due to genetic recombination and the selective pressure of antibodies in areas of systematic vaccination, is the reason that one strain of virus causes only one epidemic wave in the field. The list of subtypes should therefore be revised periodically to eliminate those which are not longer found in the field. The diagnostic laboratories have to support the control campaigns of the disease by relating antigenically and immunologically the field strains with those used in the production of vaccines. The coverage of the vaccine strains, furthermore, is of great usefulness and interest for vaccines banks. One test which can be recommended for this type of study is the expected percentage of protection (EPP) determined through the serum protection test with sera from vaccinated and revaccinated cattle. An EPP of less than 75 percent with sera from revaccinated cattle is an indication of a low protection level in the field

Caracterizacion antigenica e inmunogenica de varias cepas del serotipo Indiana de estomatitis vesicular aisladas en Brasil / Antigenic and immunogenic characterization of various strains of the Indiana serotype of vesicular stomatitis isolated in Brazil

Antigenic studies performed with vesicular stomatitis (VS) virus strains of the serotype Indiana (IND), Rancharia-Brazil/66 and Riberao-Brazil/78, isolated in the state of Sao Paulo, Brazil in 1966 and 1979, respectively, indicated that they have to be classified within the subtype IND-2 and that they are related to virus strains Salto-Argentina/63 and Cocal-Trinidad/61. The Espinoza-Brazil/77 strain isolated in 1977 in cattle of the state of Minas Gerais is closely related to the Alagoas-Brazil/64 strain, subtype IND-3. Studies of sera from recovered animals and immunodiffusion gel agar test confirmed the results obtained by 50 percent complement fixation tests. Field pathogenicity indicated that strains from the subtype IND-2 only affected horses, while the IND-3 subtype viruses affected cattle to a lower degree in comparison with horses. The northeast states and Minas Gerais, in Brazil, are affected by VS virus subtype IND-3, while in Sao Paulo and Rio Grande do Sul subtype IND-2 strains are identified

Produccion y control de calidad de la vacuna antiaftosa en America del Sur / Production and quality control of foot-and-mouth disease vaccines in South America

In the production of foot-and-mouth disease (FMD) vaccines in South America, the 1950's 60's and part of the 70's were dedicated to creating an infrastructure of production laboratories capable of supplying sufficient quantities of FMD vaccine to meet the needs of the programs. That infrastructure has developed significantly in both the private and official sectors. Most of the South American countries now have qualified FMD vaccines laboratories and advanced production technology. Concomitantly, the infrastructure of the official control laboratoraies was established. The standards of the production laboratories and the vaccines they produce were determined. Quality control that should be applied to the vaccines and the minimum requirements of the protection tests for approval of each series were also defined. At the present the most important step to control and eradicate FMD is to improve the epidemiological surveillance as well as the rational and strategic application of the vaccine, according to the ecosystems of the disease in the Continent

Caracterizacion antigenica e inmunogenica de varias cepas del serotipo Indiana de estomatitis vesicular aisladas en Brasil / Antigenic and immunogenic characterization of various strains of the Indiana serotype of vesicular stomatitis isolated in Brazil

Antigenic studies performed with vesicular stomatitis (VS) virus strains of the serotype Indiana (IND), Rancharia-Brazil/66 and Riberao-Brazil/78, isolated in the state of Sao Paulo, Brazil in 1966 and 1979, respectively, indicated that they have to be classified within the subtype IND-2 and that they are related to virus strains Salto-Argentina/63 and Cocal-Trinidad/61. The Espinoza-Brazil/77 strain isolated in 1977 in cattle of the state of Minas Gerais is closely related to the Alagoas-Brazil/64 strain, subtype IND-3. Studies of sera from recovered animals and immunodiffusion gel agar test confirmed the results obtained by 50 percent complement fixation tests. Field pathogenicity indicated that strains from the subtype IND-2 only affected horses, while the IND-3 subtype viruses affected cattle to a lower degree in comparison with horses. The northeast states and Minas Gerais, in Brazil, are affected by VS virus subtype IND-3, while in Sao Paulo and Rio Grande do Sul subtype IND-2 strains are identified

Serological and immunological relationships among type A foot-and-mouth disease strains in South America.

Field strains of foot-and-mouth disease (FMD) virus which differ immunogenically from vaccine strains appear from time to time and may present serious problems for FMD campaigns. In order that appropriate measures can be taken it is important to determine the degree of differences between the vaccine strain and the variant strain soon after its appearance. With the A strains studied it was observed that a serological relationship by complement fixation test of greater than or equal to 0.40 would indicate sufficient protection of the vaccine strain for the field strain. With a lower relationship, however, the vaccine strains usually do not fully protect against the field strain. The mouse protection test can be used to determine how serious the lack of protection may be. With this test routine it is possible to issue alerts for intensified epidemiological surveillance within 2-3 weeks after the field strain is received at the Center.