RESUMO
A full-length cDNA clone of genome segment 7 of African Horse Sickness Virus, serotype 9 (AHSV9) was obtained using the PCR technique. The clone was sequenced and found to be 98.27% homologous to the previously published sequence of the equivalent cDNA clone from AHSV4 at the nucleotide level and to exhibit 99.7% identity at the amino acid level. The cDNA clone was transferred to pGEX-2T (Pharmacia), a bacterial expression vector, such that the reading frame of AHSV9 VP7 was continuous with that of the bacterial glutathione-S-transferase (GST) protein, under the control of the bacterial tac promoter. On induction with IPTG a fusion protein consisting of GST and VP7 was synthesised, which was readily purified on a GST-sepharose column (Pharmacia). The fusion protein reacted equally well in an indirect ELISA using monoclonal antibodies specific for AHSV9 VP7 or polyclonal guinea pig antisera raised against AHSV9 infectious sub-viral particles. This protein was also shown to be a suitable substitute for virus antigen, prepared from infected BHK cell extracts, in a competitive ELISA. Antibodies titres recorded for AHSV9 positive and negative horse sera were similar in the competitive ELISA using either bacterial AHSV VP7 or BHK extracted virus as the source of antigen, in combination with monoclonal or polyclonal antibodies, respectively, as the detectors.
Assuntos
Vírus da Doença Equina Africana/imunologia , Antígenos Virais , Proteínas do Capsídeo , Capsídeo/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Doença Equina Africana/diagnóstico , Doença Equina Africana/imunologia , Vírus da Doença Equina Africana/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Anticorpos Antivirais , Antígenos Virais/biossíntese , Antígenos Virais/genética , Sequência de Bases , Capsídeo/biossíntese , Capsídeo/genética , Clonagem Molecular , DNA Viral/genética , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/genética , Estudos de Avaliação como Assunto , Genes Virais , Vetores Genéticos , Cavalos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Virologia/métodosRESUMO
A monoclonal antibody based competitive enzyme-linked immunosorbent assay (MC-ELISA) for the detection of antibodies against epizootic haemorrhagic disease of deer viruses (EHDV) is described. Test sera were competed with a monoclonal antibody against the VP7 protein of EHDV serotype 1. The assay was capable of detecting antibodies to all serotypes of EHDV but unlike the agar gel immunodiffusion (AGID) test, gave no cross-reactions with antibodies against bluetongue, Palyam or Tilligery viruses. The MC-ELISA was more sensitive than a polyclonal based ELISA reported previously (Thevasagayam et al., 1995b) and would be ideal for epidemiological surveys since it is suitable for the examination of antisera from all animal species without the need for individual anti-species enzyme conjugates.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/análise , Antígenos Virais , Proteínas do Capsídeo , Capsídeo/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Vírus da Doença Hemorrágica Epizoótica/isolamento & purificação , Animais , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Bovinos , Linhagem Celular , Cricetinae , Cervos/virologia , Vírus da Doença Hemorrágica Epizoótica/imunologia , Camundongos , Camundongos Endogâmicos BALB C , OvinosRESUMO
An abattoir survey of sheep and goats was carried out in The Gambia for one year. A total of 1248 goats and 438 sheep, predominantly young females, were slaughtered and sampled. Sixty per cent of the females of both species were pregnant. There were no significant differences between the dressing percentages of different breeds and age groups. Sex and stage of pregnancy had a significant influence on carcase yields in both species. In goats the highest carcase yields were obtained during the early dry season. Most of the animals were clinically healthy and there were few pathological findings postmortem. In both species, there was a seasonal fluctuation of packed cell volume (PCV), with a minimum during the rains, and although the prevalence of trypanosomiasis was low it reduced the PCV. Faecal egg counts of Trichostrongylidae were highest during the rainy season and goats had higher faecal egg and coccidial oocyst counts than sheep. In sheep, a breed difference was observed for PCV and an age difference for egg excretion. The peak or higher rates of egg excretion occurred during the rains in both species. The immune status against peste des petits ruminants was significantly lower in goats (39 per cent) than in sheep (49.5 per cent). Antibodies against bluetongue virus were found in 62.6 per cent of goats and 55.8 per cent of sheep.