Development of an indirect ELISA to detect Corynebacterium pseudotuberculosis specific antibodies in sheep employing T1 strain culture supernatant as antigen

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.

A panel of recombinant proteins for the serodiagnosis of caseous lymphadenitis in goats and sheep

“…The assays developed in this study were compared to those developed by Rebouças et al . () and Seyffert et al . () using serum samples taken in an epidemiological survey of sheep and goats from Sergipe, a Brazilian State.…”

“…The sheep assay using PLD only was also more effective than the immunoenzymatic assay developed by Rebouças et al . (), which used crude C. pseudotuberculosis secreted/excreted molecules as antigens, achieving 99% specificity and 89% sensitivity. Notably, Solanet et al .…”

“…() and Rebouças et al . () for goats and sheep, respectively, which are characterized by 93.5% sensitivity and 98.5% specificity (Seyffert et al ., ), and 89% sensitivity and 99% specificity (Rebouças et al ., ). This result confirmed the highest relative sensitivity of the assays developed in the present study.…”

“…The ELISAs were developed based on a checkerboard procedure, as described by Rebouças et al . (), upon which we established the optimal antigen concentrations, serum samples, and conjugated antibodies dilutions. The parameters that we based on the choice of these conditions revealed optimal positive control OD/negative control OD ratios, with lower values for the blank controls.…”

Draft Genome Sequence of Corynebacterium pseudotuberculosis Strain PA07 Biovar ovis , Isolated from a Sheep Udder in Amazonia

“…Corynebacterium pseudotuberculosis is a Gram-positive, facultative intracellular, pleomorphic, nonsporulating, noncapsulated, nonmotile bacterium that, together with Mycobacterium , Nocardia , and Rhodococcus , forms the CMNR group which shares specifics characteristics, such as (i) high G+C content and (ii) specific cell wall organization, characterized by the presence of peptidoglycan, arabinogalactan, and mycolic acids (1, 2). …”