Immunodiagnostic assays and molecular epidemiology of bovine tuberculosis

Abstract

Bovine tuberculosis (TB) is a highly prevalent infectious disease of dairy herds worldwide and is a major issue of both human public health and economics. Annual testing of bovine TB and culling of Mycobacterium bovis-infected cattle has been a back-bone of the bovine TB control programs in Korea as well as many other countries in the world. Tuberculin skin test (TST) has been the international standard for diagnosis of bovine TB in dairy cattle. Recently, to improve sensitivity and specificity of diagnosis for bovine TB, new immunodiagnostic assays using recombinant mycobacterial antigens were introduced in many countries. In this study, an in-house IFN-g assay using an 6 kDa early secreted antigenic target (ESAT6) and 10 kDa culture filtrate protein (CFP10) cocktail antigen and an ELISA-based antibody test using mycobacterial protein from species bovis-70 (MPB70) and mycobacterial protein from species bovis-83 (MPB83) antigens were evaluated for immunodiagnosis of bovine TB in dairy cattle in Korea. Both the IFN-g assay and antibody tests showed clear distinction between TST-reactors and non-reactors from bovine TB-free herds with sensitivity of 87.4% (118/135) and specificity of 100% (100/100) for IFN-g assay and sensitivity of 77.0% (137/178) and specificity of 95.7% (111/116) for antibody tests. However, many TST-negative cattle were positive by immunological assays in dairy cattle herds with recent bovine TB outbreaks within 12 months showing positivity of 36.8% by IFN-g assay and 23.7%–29.8% by antibody tests. A majority of such cattle (11/14) were confirmed to be M. bovis-infected by post-mortem examinations followed by culture and molecular detection of M. bovis. Each M. bovis-infected cattle, however, showed various patterns of immune responses in immunodiagnostic tests. These results suggested that besides TST, other ancillary immunological tests such as

IFN-g assay and antibody tests are required for more effective detection of M. bovis infection in cattle in Korea.In addition, a molecular typing method was established for distinguishing M. bovis strains as an effort to understand transmission of M. bovis among different animal species, particularly between cattle and deer. A total of 133 M. bovis clinical isolates from 59 Holstein dairy cattle, 40 Korean beef cattle, and 34 deer with bovine TB-like lesions were analyzed in this study. Thirty published variable-number tandem repeat (VNTR) markers were applied to these isolates, and 16 of 30 markers showed allelic diversity. The most discriminatory locus for M. bovis isolates in Korea was VNTR 3336 (h = 0.59). Queen’s University Belfast (QUB) 26, mycobacterial interspersed repetitive units (MIRU) 31, VNTR 2401, and VNTR 3171 also showed high discriminatory power (h = 0.42). The combined VNTR loci had an allelic diversity of 0.84. On the basis of the VNTR profiles of 30 VNTR loci, 26 genotypes (A–Z) were identified in Korea. Two genotypes, designated as K and L, were prevalent among all M. bovis isolates (33.1% and 18.8%, respectively). Six genotypes–I, J, Q, R, S, and T–were also common in 2 out of the 3 species. These results suggest that M. bovis interspecies transmission may occur frequently in Korea.In summary, the immunological assays developed in this study were useful in identifying animals infected with M. bovis in substantial portion of TST-negative cattle in the herds with bovine TB outbreaks, thus applicable to the control and eradication programs of bovine TB as ancillary tests to TST. In addition, MIRU-VNTR typing was useful for differentiation of M. bovis molecular types in Korea. Molecular typing data showed a clear evidence of M. bovis inter-herd and interspecies transmission, thus highlighting the importance of bovine TB control programs in

deer as well as in dairy and beef cattle.