No abstract

In Great Britain an independent scientific review for the government has concluded that the development of a cattle vaccine against Mycobacterium bovis infection holds the best long-term prospect for tuberculosis control in British herds. A precondition for vaccination is the development of a complementary diagnostic test to differentiate between vaccinated animals and those infected with M. bovis so that testing and slaughter-based control strategies can continue alongside vaccination. To date bacillus Calmette-Guérin (BCG), an attenuated strain of M. bovis, is the only available vaccine for the prevention of tuberculosis. However, tests based on tuberculin purified protein derivative cannot distinguish between M. bovis infection and BCG vaccination. Therefore, specific antigens expressed by M. bovis but absent from BCG constitute prime candidates for differential diagnostic reagents. Recently, two such antigens, ESAT-6 and CFP-10, have been reported to be promising candidates as diagnostic reagents for the detection of M. bovis infection in cattle. Here we report the identification of promiscuous peptides of CFP-10 that were recognized by M. bovis-infected cattle. Five of these peptides were formulated into a peptide cocktail together with five peptides derived from ESAT-6. Using this peptide cocktail in T-cell assays, M. bovis-infected animals were detected, while BCG-vaccinated or Mycobacterium avium-sensitized animals did not respond. The sensitivity of the peptide cocktail as an antigen in a whole-blood gamma interferon assay was determined using naturally infected field reactor cattle, and the specificity was determined using blood from BCG-vaccinated and noninfected, nonvaccinated animals. The sensitivity of the assay in cattle with confirmed tuberculosis was found to be 77.9%, with a specificity of 100% in BCG-vaccinated or nonvaccinated animals. This compares favorably with the specificity of tuberculin when tested in noninfected or vaccinated animals. In summary, our results demonstrate that this peptide cocktail can discriminate between M. bovis infection and BCG vaccination with a high degree of sensitivity and specificity.Bovine tuberculosis (BTB) is caused by Mycobacterium bovis. It is a zoonotic disease and was the cause of approximately 6% of total human deaths due to BTB in the 1930s and 1940s and of more than 50% of all cervical lymphadenitis cases in children (13, 23). The introduction of pasteurization of milk in the 1930s dramatically reduced the transmission from cattle to humans, and in 1995 only 1% of 3,200 isolates from patients with TB in Great Britain were identified as M. bovis (23). However, human TB caused by M. bovis is still a major health issue in many developing countries (12,14,15).BTB has severe implications for animal welfare in both developed and developing countries, since it causes reduced productivity and premature death in cattle and since affected farms also suffer severe economic losses. A compulsory eradication program based on the slaughter of infected animals detec...

Bovine tuberculosis persists as a costly zoonotic disease in numerous countries despite extensive eradication and control efforts. Sequential serum samples obtained from Mycobacterium bovis-infected cattle were evaluated for seroreactivity to mycobacterial antigens. Animals received M. bovis by aerosol, intratonsil, intranasal, or intratracheal inoculation. Assays included the multiantigen print immunoassay for determination of antigen recognition patterns, immunoblot analysis for sensitive kinetic studies, and the VetTB STAT-PAK test, a novel, rapid test based on lateral-flow technology. Responses to MPB83 were detected for all M. bovis-infected animals regardless of the route or strain of M. bovis used for inoculation. Other less commonly recognized antigens included ESAT-6, CFP-10, and MPB70. Responses to MPB83 were detectable as early as 4 weeks after inoculation, were boosted upon injection of purified protein derivatives for skin testing, and persisted throughout the course of each of the four challenge studies. MPB83-specific immunoglobulin M (IgM) was detected prior to MPB83-specific IgG detection; however, early IgM responses rapidly waned, suggesting a benefit of tests that detect both IgM-and IgG-specific antibodies. The VetTB STAT-PAK test detected responses in sera from 60% (15/25) of the animals by 7 weeks after challenge and detected responses in 96% (24/25) of the animals by 18 weeks. These findings demonstrate the potential for new-generation antibody-based tests for the early detection of M. bovis infection in cattle.Tuberculosis (TB) in humans may result from exposure to any one of the tubercle bacilli included within the Mycobacterium tuberculosis complex (i.e., M. tuberculosis, M. bovis, M. africanum, M. pinnipedii, and M. microti). Mycobacterium bovis, unlike M. tuberculosis, has a wide host range, is the species most often isolated from tuberculous cattle, and has several wildlife maintenance hosts, including the Eurasian badger (Meles meles), brush-tailed possum (Trichosurus vulpecula), and white-tailed deer (Odocoileus virginianus). Wildlife reservoirs have made M. bovis eradication from national herds in several developed countries, including the United Kingdom, New Zealand, and the United States, particularly difficult (3, 4, 16). Eradication campaigns in these countries have generally relied on test and removal, slaughterhouse surveillance, movement restriction, and/or wildlife reservoir control strategies. The tests most widely used for the detection of TB in humans and cattle include the measurement of delayed-type hypersensitivity (i.e., skin testing) to purified protein derivatives (PPDs) and/or in vitro assays for gamma interferon produced in response to mycobacterial antigen stimulation (i.e., Bovigam [Prionics AG, Schlieren, Switzerland] and Quantiferon Gold [Cellestis Inc., Carnegie, Victoria, Australia]). These tests rely on early cell-mediated responses, a hallmark of TB immunopathogenesis. In contrast, the poor sensitivity of antibodybased tests has prevented the widespre...