Development of a skin test for bovine tuberculosis for differentiating infected from vaccinated animals

Abstract

The tuberculin skin test has been used for the diagnosis of bovine and human tuberculosis (TB) for over a hundred years. However, the specificity of the test is compromised by vaccination with the Mycobacterium bovis-derived vaccine strain bacille Calmette-Guérin (BCG). Since current promising vaccines against bovine TB are based on heterologous prime-boost combinations that include BCG, there is a need for diagnostic tests for differentiating infected from vaccinated animals (DIVA). The application of antigens such as ESAT-6 and CFP-10 for DIVA has so far been realized largely through their application in the blood-based gamma interferon release assay. In the current study, we have reassessed the potential of such antigens as skin test reagents for DIVA in cattle. A cocktail of the Mycobacterium tuberculosis complex recombinant protein antigens ESAT-6, CFP-10, MPB70, and MPB83 elicited delayed-type hypersensitivity (DTH) skin test responses in 78% of naturally infected tuberculin-positive cattle. Importantly, this cocktail induced no skin responses in BCG-vaccinated cattle despite them being sensitized for strong tuberculin responses. Further optimization of skin test antigen combinations identified that the inclusion of Rv3615c (Mb3645c) enhanced skin test sensitivity in naturally infected cattle without compromising specificity. In addition, we demonstrate for the first time the utility of synthetic peptides as promising skin test antigens for bovine TB for DIVA. Our data provide a promising basis for the future development of skin tests for DIVA with practical relevance for TB diagnosis in both veterinary and clinical settings.

FIG. 1.

Protein cocktail-induced skin test responses in cattle. Skin test responses were measured at 72, 96, and 120 h in cattle naturally exposed to M. bovis (n = 37). (A) Comparative SICCT PPD responses (PPD-B − PPD-A). (B) Responses induced by a protein cocktail of 10 μg each of ESAT-6, CFP-10, MPB70, and MPB83. The increase in skin induration for each animal is represented by open circles, and the horizontal line provides the mean (± standard error of the mean [SEM]), with results expressed as the difference in skin thicknesses (mm) between the pre- and post-skin test readings. The statistical difference between responses was determined by using ANOVA (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG. 2.

In vitro recognition of protein antigens. The in vitro capacity of antigens to induce IFN-γ in blood from cattle naturally exposed to M. bovis (n = 37) was determined for ESAT-6, CFP-10, MPB70, and MPB83 either individually (5 μg/ml) or as a combined protein cocktail (5 μg/ml per constituent). Responses induced by PPD-B (10 μg/ml), staphylococcal enterotoxin B (SEB) (1 μg/ml), or the no-antigen control were also determined. The antigen-induced IFN-γ response for each animal is represented by open circles, and the horizontal line provides the mean (±SEM), with results expressed as the background-corrected OD₄₅₀.

FIG. 3.

Skin test protein cocktail dose titration. A skin test dose titration was determined for the protein cocktail comprised of ESAT-6, CFP-10, MPB70, and MPB83 in cattle that were naturally exposed to M. bovis (n = 19). The protein cocktail was administered at concentrations of 10, 5, and 1 μg for each antigen component. Protein cocktail and SICCT (PPD-B − PPD-A) responses measured at 72 h for each animal are represented by open circles, and the horizontal line provides the mean (±SEM), with results expressed as the difference in skin thicknesses (mm) between the pre- and post-skin test readings. The statistical difference between responses induced by the protein cocktails was determined by using ANOVA (*, P < 0.05; ***, P < 0.001).

FIG. 4.

Skin test specificity of a defined protein cocktail in naïve and BCG-vaccinated cattle. Skin test responses were measured for the protein cocktail comprised of ESAT-6, CFP-10, MPB70, and MPB83 (10 μg for each component) and for the SICCT test (PPD-B − PPD-A) in naïve TB-free (n = 19) and BCG-vaccinated (n = 20) cattle. Responses measured at 72 h for each animal are represented by open circles, and the horizontal line provides the mean (±SEM), with results expressed as the difference in skin thicknesses (mm) between the pre- and post-skin test readings.

FIG. 5.

Optimization of skin test response-inducing defined antigen combinations. Skin test responses induced by the SICCT test (PPD-B − PPD-A) and a selection of protein- and peptide-based antigen combinations were determined for cattle naturally exposed to M. bovis (n = 13). Protein combinations of ESAT-6 (E6), CFP-10 (C10), MPB70, MPB83 (83), and/or Rv3615c were all tested at a concentration of 10 μg per constituent protein. The ESAT-6 and CFP-10 (E6/C10) peptide cocktail comprised 21 peptides at a concentration of 10 μg per peptide. Responses measured at 72 h for each animal are represented by open circles, and the horizontal line provides the mean (±SEM), with results expressed as the difference in skin thicknesses (mm) between the pre- and post-skin test readings. The statistical difference between responses induced by the indicated cocktails was determined by using ANOVA (*, P < 0.05).

FIG. 6.

Skin test response-inducing capacity of individual protein antigens. Skin test responses induced by the ESAT-6, CFP-10, MPB83, and Rv3615c proteins were determined either individually (10 μg) or in combination (10 μg per protein) for cattle naturally exposed to M. bovis (n = 12). Additionally, responses to a cocktail of 25 peptides (10 μg per peptide) derived from ESAT-6, CFP-10, MPB83, and Rv3615c were measured in these cattle, along with SICCT skin test responses. Reactions measured at 72 h for each animal are represented by open circles, and the horizontal line provides the mean (± SEM), with results expressed as the difference in skin thicknesses (mm) between the pre- and post-skin test readings.