Optimizing antigen cocktails for detection of Mycobacterium bovis in herds with different prevalences of bovine tuberculosis: ESAT6-CFP10 mixture shows optimal sensitivity and specificity

Abstract

Bovine tuberculosis is a major problem in many countries; hence, new and better diagnostic tools are urgently needed. In this work, we have tested ESAT6, CFP10, PE13, PE5, MPB70, TB10.4, and TB27.4 for their potentials as diagnostic markers in field animals from Northern Ireland, Mexico, and Argentina, regions with low, medium, and high prevalences of bovine tuberculosis, respectively. At all three sites, ESAT6 and CFP10 were superior diagnostic antigens, while their combination performed even better at the two sites where the combination was tested, providing the best coverage for the detection of diseased populations. The high sensitivity in the skin test reactor groups, combined with the high specificity in the tuberculosis-free groups, indicated that a diagnosis could correctly be made for 85% of the infected animals, based on their responses to these two antigens. Furthermore, TB10.4, PE13, and PE5 have the potential to supplement ESAT6 and CFP10 in a future five-component diagnostic cocktail.

Figures

FIG. 1.

Quality control of CFP10. Fifty micrograms of recombinant protein was loaded onto reverse-phase C4 columns and eluted by using a linear acetonitrile gradient. The elution profile was monitored by continuously measuring the absorbance at 214 nm (mAU, milli-absorbance units) (A). Over the 0 to 100% elution gradient, 20 fractions were collected and applied onto SDS-polyacrylamide gels, followed by silver staining. Lanes M, SDS-polyacrylamide gel electrophoresis size marker (97, 66, 45, 31, 21, and 14 kDa); lanes 1 to 20, the 20 eluted fractions, respectively. Fraction 1 corresponds to 0% acetonitrile on the chromatogram, and fraction 20 corresponds to 100% acetonitrile (B).

FIG. 2.

Whole-blood IFN-γ release assay with single antigens. Blood collected from skin test reactors (○) or TB-free animals (×) was incubated with different mycobacterial antigens, and the amount of IFN-γ released was measured by ELISA after 20 h of incubation in a humidified incubator at 37°C with 5% CO2. Samples were collected from animals from Northern Ireland (A), Argentina (B), and Mexico (C). The results are given as ODI units. Horizontal bars represent median values. The numbers of animals were 49, 39, and 36 for reactors in Northern Ireland, Argentina and México, respectively, and 20, 18, and 9 for TB-free animals from the three countries, respectively. The data for the PPDB-PPDA group are calculated by subtraction of the value for PPDA from the value for PPDB for a given animal.

FIG. 3.

IFN-γ cytokine release in a one-well assay with ESAT6 and CFP10. ESAT6 and CFP10 were mixed in equal amounts (total, 4 μg per well), and the ability to release IFN-γ from peripheral blood mononuclear cells was compared to those for PPDB and PPDA after incubation with blood from skin test reactors (○) or TB-free animals (×) for 20 h at 37°C with 5% CO2. The amount of IFN-γ released was measured by ELISA. The animals came from Northern Ireland (A) and Argentina (B). The results are given as ODI units. Horizontal bars represent the median value. The numbers of animals from each location are as provided in the legend to Fig. 2. The data for the PPDB-PPDA group were calculated by subtraction.

FIG. 4.

Amount of IFN-γ released in blood after stimulation with PE5, PE13, TB10.4, ESAT6, or CFP10 for 20 h at 37°C with 5% CO2. Blood was collected from nine skin test reactors and was incubated with single mycobacterial antigens at a concentration of 4 μg/ml, after which the release of IFN-γ was measured by ELISA. All antigens were tested singly. Note that the use of PE5, PE13, and TB10.4 can improve the assay readout. (A to I) Results for animals A to I, respectively.