ELISA using a recombinant chimera of ESAT-6/MPB70/MPB83 for <i>Mycobacterium bovis</i> diagnosis in naturally infected cattle

Abstract

Bovine tuberculosis (bTB) control programs generally rely on intradermal tuberculin tests for the antemortem diagnosis of Mycobacterium bovis infection in cattle, but these tests detect only a portion of the infected animals. The aim of the present study was to evaluate the diagnostic coverage of a combination of the bTB antemortem techniques known as the comparative intradermal tuberculin test (CITT) and an ELISA based on a recombinant chimera of ESAT-6/MPB70/MPB83 as the antigen in cattle. The results were compared to postmortem findings based on M. bovis culturing and PCR. Paired comparisons of all data (n=92) demonstrated that ELISA and LST results compared to the culturing results did not present significant differences (P=0.27 on McNemar’s test and P=0.12 on Fisher’s exact test, respectively). Using culturing as the gold standard, the sensitivity and specificity of ELISA were 79.5% (95% CI: 64.5–89.2%) and 75.5% (95% CI: 62.4–85.1%), respectively, whereas LST demonstrated 100% sensitivity (95% CI: 91.03–100%) and 92.5% specificity (95% CI: 82.1–97.0%). The ELISA results did not reveal significant differences in relation to the LST results (P>0.99 on Fisher’s exact test). Using the latter as the gold standard, the sensitivity and specificity of ELISA were 79.1% (95% CI: 64.8–88.6%) and 79.6% (95% CI: 66.4–88.5%), respectively. The use of ELISA with the recombinant chimera of ESAT-6/MPB70/MPB83 as the antigen complements the diagnostic coverage provided by CITT and increases the removal of infected animals from herds.

Comparing recombinant MPB70/SahH and native 20-kDa protein for detecting bovine tuberculosis using ELISA

“…ELISAs using various recombinant proteins (MPB70, MPB83, and ESAT6) as well as native antigens (P22, 20 kDa, CMP, and PPD) have been developed as a supplementary strategy for bTB serological diagnosis [ 6 , 7 , 14 , 16 , 30 ]. Initially, native proteins such as MPB70, MPB64, MPB59, P32, P70, P65, and CMP70 were evaluated as bTB ELISA antigens [ 6 , 11 , 12 , 15 , 38 ].…”

“…In this study, the purified 20-kDa antigen also showed higher sensitivity and specificity than those of PPD. Immunospecific-recombinant proteins of M. bovis , such as MPB70, MPB83, ESAT6, CFP10, Mb0143, PE5, PE13, TB10.4, TB15.3, Rv3615c, Rv3020c, and ESAT6/MPB70/MPB83 have been previously used as bTB ELISA antigens [ 13 , 29 , 30 , 32 , 34 , 35 ]. Here, we mixed MPB70 with SahH as a novel recombinant antigen combination.…”

“…Previously, the sensitivity and specificity of bTB ELISA using recombinant proteins were reported as 63.0–83.2% and 75.5–98.0%, respectively [ 13 , 29 , 30 , 34 ]. MPB70 and MPB83 are the most important recombinant antigens in ELISA [ 1 , 3 , 19 , 22 ].…”

“…Currently, test-and-cull and abattoir surveillance as well as milk pasteurization are used to prevent the transmission of tuberculosis from animals to humans in South Korea [ 5 , 7 , 17 ]. bTB has been detected through cell-mediated immunity-based diagnosis (CEMID) such as intradermal skin tests (IST) and γ-interferon assays [ 2 , 14 , 30 ]. However, CEMID has a bTB diagnostic window with a high false-negative rate and is laborious, requiring two farm visits for injection and interpretation [ 1 , 8 , 37 ].…”

Application of Transcriptomics to Enhance Early Diagnostics of Mycobacterial Infections, with an Emphasis on Mycobacterium avium ssp. paratuberculosis

“…MPB70 and MPB83 were constitutively expressed to high levels in MB, but only very lowly in some BCG strains [57]. Therefore, these proteins are among the most frequently investigated antigens for the differential diagnosis of bovine tuberculosis and they have been examined as diagnostic biomarkers [72,73,74]. Another study also screened for highly expressed mycobacterial genes that are lowly expressed in the BCG strain, and identified Rv3615c as a useful differential diagnostic antigen [56].…”