Use of rMPB70 protein and ESAT-6 peptide as antigens for comparison of the enzyme-linked immunosorbent, immunochromatographic, and latex bead agglutination assays for serodiagnosis of bovine tuberculosis

Abstract

Current assays used to detect Mycobacterium bovis infection lack accuracy, especially for recently infected animals, or are impractical for rapid field diagnostic applications. To overcome these limitations with serological assays, a synthetic peptide derived from early secretory antigenic target 6 (ESAT6-p) and a recombinant major secreted immunogenic protein (rMPB70) of M. bovis were used in an enzyme-linked immunosorbent assay (EIA), an immunochromatographic assay (ICGA), and a latex bead agglutination assay (LBAA). Sera from noninfected, M. bovis-infected, or M. avium subsp. paratuberculosis-infected (by natural and experimental routes) animals were evaluated. Receiver operating characteristic analysis comparing optical density values from the EIA with results of bacterial culture or skin test, the reference test, established suitable cutoff values for assessing sensitivity and specificity. The EIA and LBAA, respectively, had sensitivities of 98.6 and 94.8%, specificities of 98.5 and 92.6%, and kappa values of 0.97 and 0.88 with ESAT6-p. The EIA, ICGA, and LBAA, respectively, had sensitivities of 96.8, 83.0, and 86.7%, specificities of 90.1, 99.4, and 97.8%, and kappa values of 0.87, 0.85, and 0.83 with rMPB70. Examination of serial samples of sera collected from experimentally M. bovis-infected cattle and deer revealed that ESAT6-p-specific responses developed early after infection whereas responses to rMPB70 developed later in the course of disease. The advantage of the LBAA and ICGA as initial tests for multiple species is a rapid reaction obtained in 2 to 3 h by LBAA or 20 min by ICGA without species-specific secondary antibodies under field conditions, thus allowing immediate segregation of suspect animals for further testing before culling.

FIG. 1.

ROC curves for EIA with ESAT6-p (A) or rMPB70 (B). Sensitivity and specificity, respectively, were calculated as the number of serum samples showing positive results by the reference test and the EIA at each cutoff value divided by the number of reference test positive results and as the number of serum samples showing negative results by the reference test and the EIA at each cutoff value divided by the number of reference test negative results, based on the results of M. bovis culture or a skin test as the reference test and a comparison of those two tests (reference test versus EIA).

FIG. 2.

Spearman rank correlation analysis between OD values from the EIA and the intensity of agglutination from the LBAA utilizing ESAT-6 (A) or rMPB70 (B) and between OD values and the intensity of the test line band from the ICGA with rMPB70 (C). The EIA and LBAA turned out to be positively correlated (P < 0.0001), with r = 0.73 (95% CI, 0.70 to 0.77) or 0.68 (95% CI, 0.64 to 0.72) when using ESAT-6 or rMPB70 as the detecting antigen, respectively. A statistically significant correlation between the ICGA and EIA utilizing rMPB70 was also found, with r = 0.87 (95% CI, 0.85 to 0.88, P < 0.0001).

FIG. 3.

ODs from the EIA with ESAT6-p or rMPB70 and sera from experimentally M. bovis-infected calves (A) or deer and control deer (B). Detailed information on how M. bovis was inoculated experimentally into calves or deer and how blood was collected from them is given in Materials and Methods. ODs from EIAs using different capture or detecting antigens and sera from control or M. bovis-infected deer are shown on the y axes (EIA using ESAT-6 on the left y axis and EIA with rMPB70 on the right y axis [B]). D, days; wk, week.