ABSTRACT
Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis (CLA), a chronic disease that affects goats and sheep, characterized by granuloma formation in subcutaneous and internal lymph nodes. CLA causes significant economic losses to commercial goat herds. In this study, we aimed to test secreted antigens secreted from T1 strain bacteria grown in brain heart infusion (BHI) broth in an indirect ELISA system to determine the presence of specific immunoglobulins against C. pseudotuberculosis. We analyzed the BHI antigen electrophoretic profile and the recognition pattern by infected sheep sera samples. The ELISA results were compared with multiplex PCR assay and IFN-gamma production. The ELISA was able to discriminate between negative and positive animals, with a sensitivity of 89% and a specificity of 99%, using microbiological isolation as gold standard. When this assay was compared with multiplex PCR and specific IFN-gamma quantification, six discrepant results were found among thirty-two samples. We concluded that the ELISA using antigens secreted from C. pseudotuberculosis T1 strain growth in BHI broth culture can be used for the serodiagnosis of CLA in sheep.
Corynebacterium pseudotuberculosis é o agente etiológico da linfadenite caseosa (LC) uma doença crônica que afeta ovinos e caprinos caracterizada pela formação de granulomas em linfonodos. A LC causa perdas econômicas significativas em criações de pequenos ruminantes. Este trabalho teve como objetivo testar antígenos secretados da cepa T1 da bactéria em um sistema de ELISA indireto para detecção de anticorpos específicos contra C. pseudotuberculosis. O perfil eletroforético do antígeno foi analisado, bem como o padrão de reconhecimento por soros de animais infectados. Os resultados do ELISA foram comparados com ensaio de multiplex PCR e com teste de indução de produção específica de IFN-gama. O ELISA foi capaz de discriminar animais positivos de animais negativos, com sensibilidade de 89% e especificidade de 99%, usando o isolamento microbiológico como padrão ouro. Quando o ensaio foi comparado com o multiplex PCR e a produção específica de IFN-gama, somente seis resultados discrepantes foram encontrados em trinta e duas amostras. Pode-se concluir que o ensaio de ELISA desenvolvido pode ser utilizado com alto grau de confiança para o diagnóstico da LC em ovinos.
Subject(s)
Animals , Antibodies, Bacterial/isolation & purification , Goats/immunology , Corynebacterium pseudotuberculosis/isolation & purification , Enzyme-Linked Immunosorbent Assay/veterinary , Sheep/immunology , Diagnostic Techniques and Procedures/veterinary , Electrophoresis, Polyacrylamide Gel/veterinaryABSTRACT
In the current study, the applicability of the quantification of gamma interferon (IFN-γ) levels for the detection of animals infected with Corynebacterium pseudotuberculosis and for determining caseous lymphadenitis (CLA) clinical status was evaluated. Peripheral blood leukocytes were collected from CLA nonendemic areas animals, from CLA seropositive animals without clinical signs of the disease, and from seropositive animals presenting CLA clinical signs. The leukocytes were stimulated with C. pseudotuberculosis-secreted antigens that were concentrated by the three-phase partitioning technique. An ovine IFN-γ enzyme-linked immunosorbent assay was used to quantify IFN-γ production. Goats and sheep with CLA had higher IFN-γ levels than uninfected seronegative animals. Leukocytes from sheep with CLA chronic abscesses produced higher IFN-γ levels when compared with seropositive sheep without CLA clinical signs, but this difference was not significant in goats. The sensitivity of the assay was 55.8% and 56%, whereas the specificity was 100% and 93%, for goats and sheep, respectively. In conclusion, IFN-γ is a potential marker for the determination of CLA infection status in small ruminants; however, further research is needed to improve assay sensitivity.
