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Journal of Clinical Microbiology, August 2003, p. 3719-3728, Vol. 41, No. 8
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.8.3719-3728.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Genomic Approach to Identification of Mycobacterium bovis Diagnostic Antigens in Cattle

Claus Aagaard,1* Marc Govaerts,2 Limei Meng Okkels,1 Peter Andersen,1 and John M. Pollock3

Department of Infectious Disease Immunology, Statens Serum Institute, Copenhagen, Denmark,1 Department of Veterinary Science, Queen's University Belfast,2 Veterinary Sciences Division, Department of Agriculture and Rural Development, Stormont, Belfast, United Kingdom3

Received 16 September 2002/ Returned for modification 12 January 2003/ Accepted 8 May 2003

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 (106 CFU) and a low (104 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.

* Corresponding author. Mailing address: Department of Infectious Disease Immunology, Statens Serum Institute, Artillerivej 5, DK-2300 Copenhagen, Denmark. Phone: 45 3268 8297. Fax: 45 3268 3035. E-mail: caa{at}ssi.dk.

Journal of Clinical Microbiology, August 2003, p. 3719-3728, Vol. 41, No. 8
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.8.3719-3728.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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