The consequences of vaccination with the Johne's disease vaccine, Gudair, on diagnosis of bovine tuberculosis.

The single intradermal comparative cervical tuberculin skin-test (SICCT) remains the primary surveillance tool to diagnose bovine tuberculosis (BTB) in the UK. Therefore, understanding the potential confounding influences on this test is important. This study investigated the effects of vaccination against Johne's disease (JD) on the immunodiagnosis of BTB using a Mycobacterium bovis BCG vaccination model as a surrogate of M bovis infection. Calves were vaccinated with either BCG (an attenuated live vaccine) or the JD vaccine, Gudair (a heat-inactivated suspension of Mycobacterium avium subspecies paratuberculosis), or a combination of both, and SICCT responses were measured approximately six and 12 weeks postvaccination. Animals vaccinated with Gudair only were negative to the SICCT test, thus supporting the specificity of the SICCT test following Gudair vaccination. However, while animals vaccinated with BCG-only demonstrated a bovine tuberculin-biased response as expected, covaccination with Gudair resulted in a bias towards avian tuberculin in the SICCT test. Therefore, our model demonstrates the potential of the Gudair vaccine to reduce the sensitivity of the SICCT. In addition, while we also demonstrate that Gudair vaccination can compromise the specificity of serological tests to detect JD, the specificity of defined M bovis antigens in serological or interferon gamma-based blood assays was not compromised by the vaccine.

Specificity of the tuberculin skin test is modified by use of a protein cocktail containing ESAT-6 and CFP-10 in cattle naturally infected with Mycobacterium bovis.

The mycobacterial immunodominant ESAT-6 and CFP-10 antigens are strongly recognizable in tuberculosis-infected cattle, and they do not elicit a response in cattle without infection. In addition, they are absent in most environmental mycobacterial species, and therefore, their use can be an alternative to purified protein derivative (PPD) tuberculin in the development of a more specific skin diagnostic test in cattle. The aim of the current study was to assess the potential of an ESAT-6 and CFP-10 (E6-C10) protein cocktail in a skin test format in naturally tuberculosis-infected and paratuberculosis-infected cattle. We also included MPB83 as a third component in one of the protein cocktail preparations. The protein cocktail was tested at different dose concentrations (5, 10, and 15 µg per protein). The best skin response to the E6-C10 protein cocktail was obtained with 10 µg. Subsequently, this concentration was tested in 2 herds with high and low bovine tuberculosis prevalence, the latter with paratuberculosis coinfection. Our data show that the E6-C10 cocktail allows identification of an important proportion of animals that PPDB is not able to recognize, especially in low-prevalence herds. The protein cocktail did not induce reactions in tuberculosis-free cattle or in paratuberculosis-infected cattle. Addition of MPB83 to the protein cocktail did not make any difference in the skin reaction.

Lack of correlation between BCG-induced tuberculin skin test sensitisation and protective immunity in cattle.

Vaccination of cattle with Mycobacterium bovis Bacille Calmette-Guérin (BCG) can provide significant protection against bovine tuberculosis (TB). However, BCG vaccination sensitises animals to respond to the tuberculin skin-test. This provides a potential operational impediment to the use of BCG as a cattle vaccine since the tuberculin skin-test is the primary surveillance tool used by many countries with 'test and slaughter' control strategies. Currently, it is also unclear what BCG-induced skin-test conversion means in respects to BCG's protective immunity. In the current study we first investigated the duration of tuberculin skin-test sensitisation in calves neonatally vaccinated with BCG. BCG vaccination induced strong skin-test responses in calves during their first 6 months. However, a rapid decay in skin-test sensitivity was observed after this time. Between 6 and 9 months this represented a reduction from 80% to 8% of calves providing a positive response in the single intradermal comparative cervical tuberculin test at standard interpretation. We next investigated the relationship between BCG induced skin-test sensitivity and retention of protective immunity. Calves were neonatally vaccinated with BCG and subsequently divided into 2 groups based on retention or loss of tuberculin skin-test responses after 6 months. In contrast to their skin-test responsiveness, these vaccinates maintained their tuberculin specific IFN-γ blood responses. Moreover, irrespective of their pre-challenge skin-test responses, following M. bovis challenge both groups of BCG vaccinated calves demonstrated comparable levels of protection, as evidenced by reduced TB-associated pathology. Therefore, we have demonstrated that following neonatal BCG vaccination of cattle, tuberculin skin-test responder frequencies waned rapidly after 6 months but importantly, loss of skin-test sensitivity did not correlate with loss of protective immunity. These findings could have implications for the practical application of BCG based cattle vaccines.

Immune responses in cattle inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii.

Cattle were inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii to compare the antigen-specific immune responses to various patterns of mycobacterial disease. Disease expression ranged from colonization with associated pathology (M. bovis infection) and colonization without pathology (M. tuberculosis infection) to no colonization or pathology (M. kansasii infection). Delayed-type hypersensitivity and gamma interferon responses were elicited by each mycobacterial inoculation; however, the responses by the M. bovis- and M. tuberculosis-inoculated animals exceeded those of the M. kansasii-inoculated animals. Specific antibody responses were detected in all M. tuberculosis- and M. bovis-inoculated cattle 3 weeks after inoculation. From 6 to 16 weeks after M. tuberculosis inoculation, the antibody responses waned, whereas the responses persisted with M. bovis infection. With M. kansasii inoculation, initial early antibody responses waned by 10 weeks after inoculation and then increased 2 weeks after the injection of purified protein derivative for the skin test at 18 weeks after challenge. These findings indicate that antibody responses are associated with the antigen burden rather than the pathology, cellular immune responses to tuberculin correlate with infection but not necessarily with the pathology or bacterial burden, and exposure to mycobacterial antigens may elicit an antibody response in a presensitized animal.

Isoniazid treatment of Mycobacterium bovis in cattle as a model for human tuberculosis.

Cattle infected with Mycobacterium bovis spoligotype 9 were treated with Isoniazid (INH) from three to 14 weeks post infection, rested for fourweeks to allow INH depletion and then challenged with M. bovis spoligotype 35. Post mortem examination (PME) 35 weeks after the initial infection showed partial protection against infectious challenge following INH-attenuated infection compared with the spoligotype 35 challenge controls. Antigen-specific IFN-gamma responses decreased over time with INH therapy, following a similar pattern to that observed in the treatment of M. tuberculosis infection in humans. Following cessation of therapy, specific IFN-gamma responses increased more strongly in those calves that were visibly lesioned at PME. IFN-gamma responses were also used to identify two antigens, TB10.4 and Acr2, that induced anamnestic responses in INH-treated, re-challenged calves, suggesting a role for both antigens in protective immunity. Specific IL-10 responses were observed in all calves following treatment with INH suggesting a role for IL-10 in the resolution of infection.

Shedding of Mycobacterium bovis in the nasal mucus of cattle infected experimentally with tuberculosis by the intranasal and intratracheal routes.

Four groups of six calves were infected experimentally with either a low dose of approximately 10(4) colony-forming units (cfu) or a high dose of approximately 10(6) cfu of Mycobacterium bovis. Each dose was delivered by the intranasal and intratracheal routes. More severe disease was observed in the groups inoculated with the high dose. Visible lesions were identified in 21 of the 24 animals, all of which also gave positive skin tests and interferon-gamma (IFN-gamma) responses. Nasal shedding was detected in 15 of the 24 animals and the frequency of shedding was influenced by both the route and the dose of infection; no shedding was observed in the group infected intratracheally with the low dose. Two of the 15 confirmed shedders had no visible lesions at postmortem examination; both of these calves gave IFN-gamma responses but only one was skin test positive.

Modulation of the bovine delayed-type hypersensitivity responses to defined mycobacterial antigens by a synthetic bacterial lipopeptide.

The use of defined protein and peptide antigens can overcome specificity limitations of purified protein derivatives in the detection of bovine tuberculosis when the antigens are used in blood-based tests. Since the use of these specific antigens as skin test reagents could have practical advantages, we investigated the potential of Mycobacterium bovis-specific antigens to stimulate delayed-type hypersensitivity (DTH) responses in cattle experimentally infected with M. bovis. A cocktail of the recombinant antigens ESAT-6, MPB83, and MPB64 failed to stimulate in vivo DTH in cattle that had been experimentally infected with M. bovis despite the fact that the antigens were recognized in vitro by the same animals. However, it was possible to stimulate antigen-specific bovine DTH responses by using ESAT-6 in combination with a synthetic bacterial lipopeptide. This lipopeptide stimulated the release of the proinflammatory cytokine tumor necrosis factor alpha from monocyte-derived bovine dendritic cells in vitro, thereby providing a possible mechanism for its DTH-enhancing properties.

Generation of antibodies to the signal peptide of the MPT83 lipoprotein of Mycobacterium tuberculosis.

The mpt83 gene (Rv2873) encodes the exported MPT83 lipoprotein of Mycobacterium tuberculosis. The corresponding identical mpb83 gene of Mycobacterium bovis is expressed to varying extents in different substrains of M. bovis Bacille Calmette Guerin (BCG), BCG Tokyo and BCG Moreau being high producers and BCG Danish 1331, a low producer of the MPB83 protein. Immunization with the 13-mer N-terminal part of the signal peptide of MPT83, MINVQAKPAAAASC, coupled to keyhole limpet haemocyanin (KLH) through the added C-terminal cysteine resulted in rapid antibody formation monitored by enzyme-linked immunosorbent assay (ELISA) with free immunizing peptide on the solid phase. In ELISA, with four 20-mer overlapping peptides covering the N-terminal part of the MPT83 sequence, three polyclonal rabbit antisera reacted only with the N-terminal peptide. Antigenic signal peptide could not be detected in sonicates of BCG Tokyo and BCG Moreau. After SDS-PAGE and blotting, the antibodies reacted with sonicates of recombinant Escherichia coli containing the entire mpt83 gene including the signal sequence, but not with the 22 kDa form of native MPB83 purified from BCG culture filtrate. In partition chromatography the recMPT83 partitioned in the water phase while 26 kDa MPB83 in BCG culture filtrate partitioned in the lipid phase confirming that lipidation at the N-terminal cysteine residue occurs after the splitting of the polypeptide chain by signal peptidase II.

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.

Antigen specificity in experimental bovine tuberculosis.

This report describes the kinetics of T-cell responses to a panel of mycobacterial antigens (PPD-M, PPD-A, ESAT-6, Ag85, 38kD, MPB64, MPB70, MPB83, hsp16.1, hsp65, and hsp70) following experimental infection of cattle with Mycobacterium bovis. Increased antigen-specific lymphocyte proliferation, gamma interferon, and interleukin-2 responses were observed in all calves following infection. Positive lymphocyte proliferation and cytokine responses to PPD-M and ESAT-6 were observed throughout the infection period studied. In contrast, responses to all other antigens were more variable and were not constantly present, suggesting that antigen cocktails rather than individual antigens should be used for immunodiagnosis. The detection of cytokine responses in the absence of lymphocyte proliferation, particularly during the early stages of infection, suggests a role for antigen-specific cytokine readout systems in the early identification of M. bovis infection in cattle.

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.