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Journal of Clinical Microbiology, September 2005, p. 4498-4506, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4498-4506.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Hye Cheong Koo,1 Yong Ho Park,1 Jongsam Ahn,1 W. Ray Waters,2 Mitch V. Palmer,2 Mary Jo Hamilton,3 George Barrington,4 Abdelaziz A. Mosaad,5 Kun Taek Park,1 Woo Kyung Jung,1 In Yeong Hwang,1 Sang-Nae Cho,6 Sang Jae Shin,7 and William C. Davis3*

Department of Microbiology, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul National University,1 Department of Microbiology, College of Medicine, Yonsei University, Seodaemoon-gu, Seoul, Republic of Korea,6 Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Ames, Iowa,2 Department of Veterinary Microbiology and Pathology,3 Department of Veterinary Clinical Sciences, Washington State University, Pullman, Washington,4 Department of Bacteriology, Immunology and Mycology, College of Veterinary Medicine, Minufiya University, Minufiya, Egypt,5 Department of Population Medicine and Diagnostic Science, College of Veterinary Medicine, Cornell University, Ithaca, New York7

Received 18 May 2005/ Accepted 24 June 2005

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.

* Corresponding author. Mailing address: Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Bustad Hall, Room 326, Washington State University, Pullman, WA 99164-7040. Phone: (509) 335-6051. Fax: (509) 335-8328. E-mail: davisw{at}vetmed.wsu.edu.

Journal of Clinical Microbiology, September 2005, p. 4498-4506, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4498-4506.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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