RESUMO
Sheep and goat pox (SGP) with peste des petits ruminants (PPR) are transboundary viral diseases of small ruminants that cause huge economic losses. Recombinant vaccines that can protect from both infections have been reported as a promising solution for the future. SGP was used as a vector to express two structural proteins hemagglutinin or the fusion protein of PPRV. We compared immunity conferred by recombinant capripoxvirus vaccines expressing H or F or both HF. Safety and efficacy were evaluated in goats and sheep. Two vaccine doses were tested in sheep, 104.5TCDI50 in 1ml dose was retained for the further experiment. Results showed that the recombinant HF confers an earlier and stronger immunity against both SGP and PPR. This recombinant vaccine protect also against the disease in exposed and unexposed sheep. The potential Differentiating Infected from Vaccinated Animals of recombinant vaccines is of great advantage in any eradication program.
Assuntos
Capripoxvirus/imunologia , Doenças das Cabras/prevenção & controle , Peste dos Pequenos Ruminantes/virologia , Vírus da Peste dos Pequenos Ruminantes/imunologia , Infecções por Poxviridae/veterinária , Doenças dos Ovinos/prevenção & controle , Proteínas Virais/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/imunologia , Capripoxvirus/genética , Capripoxvirus/isolamento & purificação , Capripoxvirus/fisiologia , Doenças das Cabras/imunologia , Doenças das Cabras/virologia , Cabras , Hemaglutininas/administração & dosagem , Hemaglutininas/genética , Hemaglutininas/imunologia , Vírus da Peste dos Pequenos Ruminantes/genética , Vírus da Peste dos Pequenos Ruminantes/isolamento & purificação , Vírus da Peste dos Pequenos Ruminantes/fisiologia , Infecções por Poxviridae/imunologia , Infecções por Poxviridae/prevenção & controle , Infecções por Poxviridae/virologia , Ovinos , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/virologia , Proteínas Virais de Fusão/administração & dosagem , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologia , Proteínas Virais/administração & dosagem , Proteínas Virais/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
BACKGROUND: In this study, we developed a SYBR Green-based real-time PCR assay for the detection of sheep pox virus using a plasmid construct carrying one of the highly conserved genes encoding the virion envelope protein (P32) as a template. RESULTS: The method was demonstrated to be highly sensitive, allowing a precise SPV DNA quantitation over a range of nine orders of magnitude (from 101 to 109 copies of standard DNA). Then, specimens from SPV suspected sheep were analyzed by conventional gel-based PCR, real-time PCR and sequence analysis. CONCLUSION: Comparison between these different techniques revealed that real-time PCR is more sensitive than conventional gel-based PCR, allowing detection low viral titers of SPV in infected sheep.
Assuntos
Capripoxvirus/isolamento & purificação , Infecções por Poxviridae/veterinária , Reação em Cadeia da Polimerase em Tempo Real , Doenças dos Ovinos/diagnóstico , Animais , Capripoxvirus/genética , Infecções por Poxviridae/diagnóstico , Valores de Referência , Sensibilidade e Especificidade , Ovinos , Proteínas do Envelope Viral/genéticaRESUMO
The development of a multiplex polymerase chain reaction (PCR) method with amplification of capripoxvirus in a single-step procedure from skin biopsies using three primer pairs, two specific for capripoxvirus and one specific for alpha-tubulin is described. A sensitive multiplex PCR was achieved by optimization of parameters such as the primer concentrations, magnesium and dNTPs concentrations. False negative results that sometimes arise due to inhibitors of DNA amplification may be avoided by the inclusion in the assay of alpha-tubulin primers. The results reported on 42 skin biopsies from sheep suspected to have poxvirus infection, indicated that the assay could monitor simultaneously DNA extraction from skin biopsy samples and allow improved detection of capripoxvirus within 24 h of specimen receipt in the laboratory.
Assuntos
Capripoxvirus/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Infecções por Poxviridae/veterinária , Doenças dos Ovinos/diagnóstico , Pele/virologia , Animais , Biópsia , Capripoxvirus/genética , Primers do DNA , DNA Viral/análise , Infecções por Poxviridae/diagnóstico , Infecções por Poxviridae/virologia , Sensibilidade e Especificidade , Ovinos , Doenças dos Ovinos/virologiaAssuntos
Camelus , Capripoxvirus/isolamento & purificação , Doenças dos Bovinos/virologia , Ensaio de Imunoadsorção Enzimática/veterinária , Doenças dos Cavalos/virologia , Infecções por Poxviridae/veterinária , Animais , Bovinos , Doenças dos Bovinos/diagnóstico , Diagnóstico Diferencial , Ensaio de Imunoadsorção Enzimática/normas , Doenças dos Cavalos/diagnóstico , Cavalos , Técnicas Imunoenzimáticas/veterinária , Infecções por Poxviridae/diagnóstico , Valor Preditivo dos Testes , Sensibilidade e EspecificidadeRESUMO
A simple test based on the polymerase chain reaction (PCR) was used to detect capripoxvirus DNA in tissue culture supernatants and biopsy samples. The identity of the PCR products was confirmed by restriction enzyme analysis. The test has greater sensitivity and good specificity compared to an antigen trapping enzyme-linked immunosorbent assay which uses a detector antibody raised against a recombinant capripoxvirus-specific antigen. The reagents for the PCR-based test are all available commercially and the test provides a valuable addition to the current methods of virus detection.
Assuntos
Capripoxvirus/isolamento & purificação , Doenças dos Bovinos/virologia , DNA Viral/análise , Doenças das Cabras/virologia , Reação em Cadeia da Polimerase/métodos , Infecções por Poxviridae/veterinária , Doenças dos Ovinos/virologia , Animais , Biópsia/veterinária , Capripoxvirus/genética , Bovinos , Cabras , Infecções por Poxviridae/patologia , Infecções por Poxviridae/virologia , OvinosRESUMO
An immunocapture enzyme-linked immunosorbent assay (IC-ELISA) for the detection of goat poxvirus (GPV) antigen in skin biopsy samples obtained from healthy and experimentally infected goats as well as from goats from field was evaluated. The assay was 80-100% specific and 70-86% sensitive, and was compared with a commonly used diagnostic test, namely, the counter immunoelectrophoresis (CIE) test in the efficacy for goat pox. Although IC-ELISA was marginally more sensitive than the CIE test, the diagnosis of goat pox could be successfully done provided both the tests were combined in screening scab suspensions. The IC-ELISA could also diagnose sheep poxvirus (SPV) infection.
Assuntos
Capripoxvirus/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/veterinária , Doenças das Cabras/diagnóstico , Cabras , Infecções por Poxviridae/veterinária , Doenças dos Ovinos/diagnóstico , Animais , Antígenos Virais/análise , Capripoxvirus/imunologia , Contraimunoeletroforese , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Doenças das Cabras/virologia , Masculino , Infecções por Poxviridae/diagnóstico , Infecções por Poxviridae/virologia , Sensibilidade e Especificidade , Ovinos , Doenças dos Ovinos/virologia , Pele/virologiaRESUMO
A trapping ELISA for the detection of capripoxvirus antigen in tissue culture supernatant and biopsy material was developed, using a guinea-pig polyclonal detector antiserum raised against a recombinant capripoxvirus specific antigen, expressed in Escherichia coli using the plasmid vector pGEX-2T. The ELISA detected antigen in tissue culture samples that on virus titration contained equal to or in excess of 10(2.8) TCID50/ml. Virus isolation and ELISA were compared for the detection of capripoxvirus in skin biopsy samples from sheep, goats and cattle. The ELISA compared well with virus isolation, and has applications as a diagnostic test. This assay reduces the reliance of diagnostic laboratories on tissue culture facilities, and can be used to confirm the presence of capripoxvirus in tissue culture.