RESUMO
Equine herpesvirus 1 (EHV-1) is the causative agent of abortion, perinatal foal mortality, neurological and acute respiratory diseases in horses. Conventional laboratory diagnosis involving viral isolation from aborted foetuses is laborious and lengthy and requires processing of samples within 24 h of collection, which is problematic for samples that come from long distances. The aim of this study was to develop a polymerase chain reaction (PCR) assay useful in Argentina to detect DNA sequences of EHV-1 in different tissues from aborted equine foetuses with variable quality of preservation and without the use of conventional DNA fenolic extraction. Several DNA extraction protocols and primers were evaluated. The amplification method was standardized and its specificity was analysed using 38 foetal samples of variable quality of preservation. Of the 38 different foetal tissues, nine livers, six spleens and two lungs in good preservation and eight livers, one spleen and four lungs in a poor state of preservation were positive for PCR. EHV-1 was recovered only from the nine livers, five spleens and two lungs in good preservation. No virus was isolated from the samples that were poorly preserved. Viral isolation was confirmed by cytopathic effect and indirect immunofluorescence. The specificity of the PCR results was confirmed by the restriction endonuclease digestion of PCR products and hybridization.
Assuntos
Aborto Animal/virologia , Feto/virologia , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/isolamento & purificação , Doenças dos Cavalos/virologia , Aborto Animal/embriologia , Animais , DNA Viral/análise , Infecções por Herpesviridae/embriologia , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/genética , Doenças dos Cavalos/embriologia , Cavalos , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/veterinária , Sensibilidade e EspecificidadeRESUMO
An indirect enzyme linked immunosorbent assay was developed. Infected and non infected allantoic fluids precipitated with polyetilenglycol 6000 were used as antigen and control antigen, respectively. Serum samples were diluted 1/20 and a commercial horse radish peroxidase-labelled rabbit anti-equine IgG was used as second antibody. The reaction was developed using azino-diethylbenzotyazol-sulfonate (ABTS). Cut-off was determined by ratio sample (Rs). The hemagglutination inhibition test was used as a reference test for the 391 samples analyzed. Of these, 301 sera were positive by hemagglutination inhibition test and indirect ELISA, 75 were negative by both techniques, and 15 were positive by indirect ELISA and negative by hemagglutination inhibition test. Using hemagglutination inhibition test as standard, the indirect ELISA showed a relative specificity and sensitivity of 83.3 and 100%, respectively. This indirect ELISA is useful as screening test.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Vírus da Influenza A/isolamento & purificação , Influenza Humana/diagnóstico , Infecções por Orthomyxoviridae/diagnóstico , Animais , Testes de Inibição da Hemaglutinação , Humanos , Coelhos , Sensibilidade e EspecificidadeRESUMO
An indirect enzyme linked immunosorbent assay was developed. Infected and non infected allantoic fluids precipitated with polyetilenglycol 6000 were used as antigen and control antigen, respectively. Serum samples were diluted 1/20 and a commercial horse radish peroxidase-labelled rabbit anti-equine IgG was used as second antibody. The reaction was developed using azino-diethylbenzotyazol-sulfonate (ABTS). Cut-off was determined by ratio sample (Rs). The hemagglutination inhibition test was used as a reference test for the 391 samples analyzed. Of these, 301 sera were positive by hemagglutination inhibition test and indirect ELISA, 75 were negative by both techniques, and 15 were positive by indirect ELISA and negative by hemagglutination inhibition test. Using hemagglutination inhibition test as standard, the indirect ELISA showed a relative specificity and sensitivity of 83.3 and 100, respectively. This indirect ELISA is useful as screening test.
Assuntos
Humanos , Animais , Coelhos , Ensaio de Imunoadsorção Enzimática , Infecções por Orthomyxoviridae/diagnóstico , Influenza Humana , Vírus da Influenza A/isolamento & purificação , Testes de Inibição da Hemaglutinação , Sensibilidade e EspecificidadeRESUMO
An indirect enzyme linked immunosorbent assay was developed. Infected and non infected allantoic fluids precipitated with polyetilenglycol 6000 were used as antigen and control antigen, respectively. Serum samples were diluted 1/20 and a commercial horse radish peroxidase-labelled rabbit anti-equine IgG was used as second antibody. The reaction was developed using azino-diethylbenzotyazol-sulfonate (ABTS). Cut-off was determined by ratio sample (Rs). The hemagglutination inhibition test was used as a reference test for the 391 samples analyzed. Of these, 301 sera were positive by hemagglutination inhibition test and indirect ELISA, 75 were negative by both techniques, and 15 were positive by indirect ELISA and negative by hemagglutination inhibition test. Using hemagglutination inhibition test as standard, the indirect ELISA showed a relative specificity and sensitivity of 83.3 and 100, respectively. This indirect ELISA is useful as screening test.(AU)
Assuntos
Humanos , Animais , Coelhos , Ensaio de Imunoadsorção Enzimática/métodos , Influenza Humana/diagnóstico , Vírus da Influenza A/isolamento & purificação , Infecções por Orthomyxoviridae/diagnóstico , Testes de Inibição da Hemaglutinação , Sensibilidade e EspecificidadeRESUMO
An indirect enzyme linked immunosorbent assay was developed. Infected and non infected allantoic fluids precipitated with polyetilenglycol 6000 were used as antigen and control antigen, respectively. Serum samples were diluted 1/20 and a commercial horse radish peroxidase-labelled rabbit anti-equine IgG was used as second antibody. The reaction was developed using azino-diethylbenzotyazol-sulfonate (ABTS). Cut-off was determined by ratio sample (Rs). The hemagglutination inhibition test was used as a reference test for the 391 samples analyzed. Of these, 301 sera were positive by hemagglutination inhibition test and indirect ELISA, 75 were negative by both techniques, and 15 were positive by indirect ELISA and negative by hemagglutination inhibition test. Using hemagglutination inhibition test as standard, the indirect ELISA showed a relative specificity and sensitivity of 83.3 and 100
, respectively. This indirect ELISA is useful as screening test.
RESUMO
Um sistema de western blotting (WB) foi desenvolvido para detecçäo de anticorpos contra o vírus da leucose em soros bovinos. Foram utilizadas amostras de soros de 233 animais naturalmente infectados e soros de vacas experimentalmente infectadas. O teste de imunodifusäo em ágar (AGID) foi usado para comparaçäo dos resultados. Graus diferentes de reatividade foram observados em soros positivos ao AGID, quando testados em WB frente a um ou aos dois antígenos mais importantes (gp51 e p24). Outras proteínas (gp30, p15, p12 e p10) näo foram detectadas por nenhum soro positivo ao AGID, sendo que três bandas correspondentes à proteína p24 foram observadas ocasionalmente. Em soros obtidos por inoculaçäo experimental, anticorpos contra a proteína p24 foram detectados entre a segunda e a quarta semanas após a inoculaçäo e, em alguns animais, detectaram-se anticorpos anti-gp51 mais tardiamente. O estudo de soros de campo com AGID e WB mostrou concordância de 90,9 por cento sendo que apenas 1,7 por cento dos soros negativos pelo AGID foram positivos ao WB e 7,2 por cento dos resultados näo conclusivos por AGID foram definidos por WB (4,2 por cento como positivos e 3 por cento como negativos)
Assuntos
Animais , Masculino , Feminino , Western Blotting , Diagnóstico , Leucose Enzoótica Bovina , BovinosRESUMO
A western blotting (WB) procedure has been developed for detecting antibodies to bovine leukosis virus (BLV) in cattle sera. Two hundred and thirty three serum samples from naturally infected cattle with BLV virus and serial bleedings from experimentally BLV infected cows were used. An agar gel immunodiffusion test (AGID) was used for comparing with the results obtained by WB. The AGID positive sera showed a different degree of reactivity by WB test against the two most important viral antigens (gp51 and p24), or against one of them. Other proteins (gp30, p15, p12 and p10) were not detected with any AGID positive sera, being observed occasionally three bands corresponding to the p24 protein. Using sera obtained by BLV experimental inoculation, the antibodies directed to p24 appeared early (between the 2nd and 4th week post inoculation) and thereafter antibodies to gp51were detected in some animals. The analysis of field serum samples by AGID as compared to WB showed an agreement of 90.9%. Only 1.7% of sera were negative by AGID and positive by WB and 7.2% that were not conclusive by AGID and were defined by WB (4.2% as positive and 3.0% as negative).
Um sistema de western blotting (WB) foi desenvolvido para detecção de anticorpos contra o vírus da leucose em soros de bovinos. Foram utilizadas amostras de soros de 233 animais naturalmente infectados e soros de vacas experimentalmente infectadas. O teste de imunodifusão em ágar (AGID) foi usado para comparação dos resultados. Graus diferentes de reatividade foram observados em soros positivos ao AGID, quando testados em WB frente a um ou aos dois antígenos mais importantes (gp51 e p24). Outras proteínas (gp30, p15, p12 e p10) não foram detectadas por nenhum soro positivo ao AGID, sendo que três bandas correspondentes à proteína p24 foram observadas ocasionalmente. Em soros obtidos por inoculação experimental, anticorpos contra a proteína p24 foram detectados entre a segunda e a quarta semanas após a inoculação e, em alguns animais, detectaram-se anticorpos anti-gp51 mais tardiamente. O estudo de soros de campo com AGID e WB mostrou concordância de 90,9% sendo que apenas 1,7% dos soros negativos pelo AGID foram positivos ao WB e 7,2% dos resultados não conclusivos por AGID foram definidos por WB (4,2% como positivos e 3% como negativos).
RESUMO
The genomes of 10 equine herpesvirus 1 (EHV-1) strains isolated in Argentina from 1979 to 1991, and a Japanese HH1 reference strain were compared by restriction endonuclease analysis. Two restriction enzymes, BamHI and BglII, were used and analysis of the electropherotypes did not show significant differences among isolates obtained from horses with different clinical signs. This suggests that the EHV-1 isolates studied, which circulated in Argentina for more than 10 years, belong to a single genotype.
Assuntos
Proteínas de Bactérias , Variação Genética , Genoma , Herpesvirus Equídeo 1/genética , Argentina , Desoxirribonuclease BamHI , Desoxirribonucleases de Sítio Específico do Tipo II , Eletroforese , Herpesvirus Equídeo 1/isolamento & purificaçãoRESUMO
The genomes of 10 equine herpesvirus 1 (EHV-1) strains isolated in Argentina from 1979 to 1991, and a Japanese HH1 reference strain were compared by restriction endonuclease analysis. Two restriction enzymes, Bam HI and Bgl II, were used and analysis of the electropherotypes did not show significant differences among isolates obtained from horses with different clinical signs. This suggests that the EHV-1 isolates studied, which circulated in Argentina for more than 10 years, belong to a single genotype
Assuntos
Variação Genética , Genoma , Herpesvirus Equídeo 1/genética , Argentina , Desoxirribonuclease BamHI , Desoxirribonucleases de Sítio Específico do Tipo II , Eletroforese , Herpesvirus Equídeo 1/isolamento & purificaçãoRESUMO
Genomes of four Argentine isolates of Aujeszky's disease virus (ADV) (Rio Cuarto/79, Mercedes, Chanar Ladeado-7 and Chanar Ladeado-15) from pigs were characterized and compared with four ADV strains obtained from U.S.A. (Indiana-S), Sweden (Sweden 66), France (Alfort) and Japan (Yamagata-S81) by restriction endonuclease (RE) analysis. Although three Argentine isolates were classified into type I of BamHI cleavage pattern, one isolate, Mercedes, belonged to type II, according to the classification by Herrmann et al. [6]. Since this type II virus was first isolated in 1981, no outbreak of ADV infection by this type has so far been reported in Argentina. This may imply that the immediate measures by total slaughter of pigs in the farm led successful eradication of the type II ADV infection in Argentina. This report is the first epidemiological study using RE analysis on ADV strains in this country.