Field evaluation of a novel differential diagnostic reagent for detection of Mycobacterium bovis in cattle.

In the search for improved tools with which to control bovine tuberculosis, the development of enhanced immunodiagnostic reagents is a high priority. Such reagents are required to improve the performance of tuberculin-based reagents and allow the discrimination of vaccinated cattle from those infected with Mycobacterium bovis. In this study, we identified the immunodominant, frequently recognized peptides from Rv3873, Rv3879c, Rv0288, and Rv3019c, which, together with peptides comprising the current lead diagnostic antigens, ESAT-6 and CFP-10, were formulated into a peptide cocktail. In a test of naturally infected cattle, this cocktail was significantly better than tuberculin was for identifying skin test-negative animals with confirmed bovine tuberculosis. In addition, the specificity of this cocktail was not compromised by Mycobacterium bovis BCG vaccination. In summary, our results prioritize this peptide-based, fully synthetic reagent for assessment in larger trials.

Identification of novel Mycobacterium tuberculosis antigens with potential as diagnostic reagents or subunit vaccine candidates by comparative genomics.

An independent review for the British government has concluded that the development of a cattle vaccine against Mycobacterium bovis holds the best long-term prospects for tuberculosis control in British herds. The development of complementary diagnostic tests to differentiate between vaccinated and infected animals is necessary to allow the continuation of test-and-slaughter-based control policies alongside vaccination. Vaccination with M. bovis bacillus Calmette-Guérin (BCG), the only available vaccine, results in tuberculin purified protein derivative sensitivity and has shown varying vaccine efficacies in cattle. Thus, identification of more-specific reagents to distinguish between vaccination and infection, as well as the identification of subunit vaccine candidates for improved tuberculosis vaccines, is a research priority. In the present study, we applied comparative genomics to identify M. bovis-Mycobacterium tuberculosis antigens whose genes had been deleted in BCG Pasteur. In total, 13 open reading frames (ORFs) from the RD1, RD2, and RD14 regions of the M. tuberculosis genome were selected. Pools of overlapping peptides spanning these ORFs were tested in M. bovis-infected (n = 22), BCG-vaccinated (n = 6), and unvaccinated (n = 10) control cattle. All were recognized in infected cattle, with responder frequencies varying between 16 and 86%. In particular, eight highly immunogenic antigens were identified whose potentials as diagnostic reagents or as subunit vaccines warrant further study (Rv1983, Rv1986, Rv3872, Rv3873, Rv3878, Rv3879c, Rv1979c, and Rv1769).

Immunogenicity of Mycobacterium tuberculosis RD1 region gene products in infected cattle.

Current immuno-diagnostic tests for the detection of Mycobacterium bovis infection in cattle rely on the use of tuberculin PPD as antigens. However, the use of a cattle vaccine is effectively prohibited because BCG, the only potentially available cattle TB vaccine, compromises the current tuberculin test. The main objective of this study was to identify specific antigens, which could increase the test sensitivity to levels achieved with tuberculin. Our approach utilized the availability of the genome sequences of Mycobacterium tuberculosis and BCG by applying principles of comparative genomics to the identification of species-specific antigens. Eight open-reading frames (Rv3871 to Rv3878) encoding for putative antigens in the RD1 region of the M. tuberculosis genome, which is deleted in all strains of BCG, were selected and screened in the form of pools of synthetic peptides for immunological reactivity (antigen induced proliferation and IFN-gamma secretion) with peripheral blood mononuclear cells from cattle experimentally infected with M. bovis. Our results confirm the immunodominant role of two RD1 region products, CFP-10 (Rv3874) and ESAT-6 (Rv3875). In addition, we were able to identify 3 more antigens (Rv3871, Rv3872 and Rv3873), which induced immunological reactivity in PBMC from more than 50%M. bovis of infected cattle.

Use of synthetic peptides derived from the antigens ESAT-6 and CFP-10 for differential diagnosis of bovine tuberculosis in cattle.

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.

Toward the development of diagnostic assays to discriminate between Mycobacterium bovis infection and bacille Calmette-Guérin vaccination in cattle.

A scientific review of the recent sharp increase in bovine tuberculosis in Great Britain has concluded that the development of a cattle vaccine holds the best prospect for long-term disease control. It is important to develop a diagnostic test that differentiates between vaccinated and Mycobacterium bovis-infected animals, to ensure that test-and-slaughter control strategies can continue alongside vaccination. The mycobacterial antigens ESAT-6, MPB64, and MPB83 are expressed at high levels in M. bovis but are expressed at low levels or not at all in bacille Calmette-Guérin (BCG) Pasteur. Promiscuous bovine T cell epitopes of these antigens were identified and formulated into a peptide cocktail. This cocktail and a cocktail composed of recombinant forms of the 3 antigens was able to distinguish cattle infected with virulent M. bovis from those vaccinated with BCG and from those sensitized to avian tuberculin in lymphocyte transformation and interferon-gamma assays.

Effective DNA vaccination of cattle with the mycobacterial antigens MPB83 and MPB70 does not compromise the specificity of the comparative intradermal tuberculin skin test.

The current tuberculin test and slaughter strategy for the control of bovine tuberculosis in cattle has failed to prevent a sharp rise in cases over recent years, especially in the south-west of England. A recent scientific review has concluded that the development of a cattle vaccine holds the best prospect for tuberculosis control in British herds. In order to continue with test and slaughter-based control strategies, the development of TB vaccines that do not compromise the specificity of the tuberculin skin test are required. This report describes results of cattle vaccination experiments with TB DNA vaccines expressing the mycobacterial antigens MPB70, MPB83, and Ag85A and constitutes the first published vaccination study with DNA vaccines undertaken in a target host species. All calves vaccinated with the MPB83 expressing plasmid demonstrated potent cellular immune responses, characterised by CD4(+) T cells producing interferon-gamma as well as humoral immunity characterised by IgG1 biased specific antibodies. Vaccination with MPB70 was less effective with immune responses only observed in half of the vaccinated animals, while vaccination with Ag85A did not result in vaccine-induced immune responses. Intramuscular vaccination was found to stimulate stronger cellular responses than intradermal immunisation. Significantly, the specificity of tuberculin skin testing was not compromised by DNA vaccination since none of the vaccinated calves showed positive skin test reactivity.