Genomic approach to identification of Mycobacterium bovis diagnostic antigens in cattle

Abstract

Differential delayed-type hypersensitivity skin testing with tuberculin purified protein derivatives from Mycobacterium bovis and M. avium is the standard for diagnosing bovine tuberculosis. However, improved tests based on defined, specific antigens are urgently needed. In the present study, a combination of bioinformatics, molecular biology, and bovine models of infection were used to screen mycobacterial proteins for their potential as diagnostic reagents which could be used in a whole-blood assay for diagnosis of tuberculosis. Initial screening of 28 proteins selected in silico and expressed as recombinants in Escherichia coli indicated that CFP-10, ESAT-6, TB27.4, TB16.2, TB15.8, and TB10.4 induced strong gamma interferon responses in experimentally infected cattle. A more thorough investigation over time in two groups of animals infected with a high (10(6) CFU) and a low (10(4) CFU) dose of M. bovis revealed that, for both groups, the strength of the in vitro response to individual antigens varied greatly over time. However, combining the results for ESAT-6, CFP-10, and TB27.4, possibly supplemented with TB10.4, gave sensitivities at different infection stages close to those obtained with M. bovis purified protein derivative. Importantly, while responsiveness to ESAT-6 and CFP-10 correlated strongly for individual samples, the same was not the case for ESAT-6 and TB27.4 responsiveness. The results suggest that combinations of specific antigens such as these have great potential in development of optimized diagnostic systems for bovine tuberculosis.

FIG. 1.

Initial screening of 28 recombinant mycobacterial proteins. Average ELISA IFN-γ responses and standard deviation for blood samples isolated from six cattle infected with 10⁴ CFU of M. bovis at 126 and 154 days postinfection and three cattle infected with 10⁴ CFU of M. bovis at 182 days postinfection. Antigen numbers on the x axis correspond to the antigen numbers in Table 2. Antigen 29 is the phosphate-buffered saline control. Open circles mark antigens selected for further study.

FIG. 2.

Time kinetic of antigen recognition. A group of six animals were infected with 10⁶ CFU of M. bovis, and antigen recognition in blood samples from these animals was followed continuously. IFN-γ levels were measured after stimulation with the appropriate antigen. Results at −15, 14, 21, 28, 56, 98, 140, and 180 days postinfection are shown. The PPDB results are indicated with a stippled line in each figure. Two of the optical density (OD) values for PPDB at 21 days postinfection were above the maximum detection level. The cutoff value (0.188) is indicated by a horizontal dotted line and was calculated as the average of the phosphate-buffered saline group plus 5 standard deviations.

FIG. 3.

Complementation of individual antigens. After the recognition response has developed (14 days postinfection for the group receiving 10⁶ CFU of M. bovis and 21 days postinfection for the group receiving 10⁴ CFU), 144 samples were collected. For each of these, the recognition signal for ESAT-6 is plotted against the recognition signal for CFP-10 (a) or TB27.4 (b). Solid circles are data points from the group infected with 10⁶ CFU of M. bovis (78 samples), and open circles are from the group infected with 10⁴ CFU of M. bovis (66 samples).