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To further understand the epidemic of bovine tuberculosis in Great Britain, Northern Ireland and the Republic of Ireland, we identified 16 mutations that are phylogenetically informative for Mycobacterium bovis strains from these regions. We determined the status of these mutations among a collection of 501 strains representing the molecular diversity found in these three regions of the British Isles. The resulting linear phylogenies from each region were concordant, showing that the same lineage of M. bovis was present. The dominance of this lineage is unique within Europe, and suggests that in the past the populations were homogenous. Comparison of approximately 500 strains isolated in 2005 from each region by spoligotype and 5 locus VNTR profiling, revealed distinct differences in the genotype frequencies and sub-lineage makeup between each region. We concluded that whilst each region shared the same major phylogenetic lineage of M. bovis, more recent evolution had resulted in the development of region-specific populations. Regional differences in the M. bovis populations suggest that it may be possible to identify the movement of strains from one region to another.

There is a requirement for vaccines or vaccination strategies that confer better protection against TB than the current live attenuated Mycobacterium bovis Bacillus Calmette-Guerin (BCG) vaccine for use in cattle. Boosting with recombinant viral vectors expressing mycobacterial proteins, such as Ag85A, has shown a degree of promise as a strategy for improving on the protection afforded by BCG. Experiments in small animal models have indicated that broadening the immune response to include mycobacterial antigens other than Ag85A, such as Rv0288, induced by boosting with Ad5 constructs has a direct effect on the protection afforded against TB. Here, we compared the immunogenicity and protection against challenge with M. bovis afforded by boosting BCG-vaccinated cattle with a human type 5 (Ad5)-based vaccine expressing the mycobacterial antigens Ag85A (Ad5-85A); or Ag85A, Rv0251, Rv0287 and Rv0288 (Ad5-TBF); or with protein TBF emulsified in adjuvant (Adj-TBF). Boosting with TBF broaden the immune response. The kinetics of Ad5-TBF and Adj-TBF were shown to be different, with effector T cell responses from the latter developing more slowly but being more durable than those induced by Ad5-TBF. No increase in protection compared to BCG alone was afforded by Ad5-TBF or Adj-TBF by gross pathology or bacteriology. Using histopathology, as a novel parameter of protection, we show that boosting BCG vaccinated cattle with Ad5-85A induced significantly better protection than BCG alone. © 2013.

Summary Bovine tuberculosis (bTB) is highly prevalent in intensive dairy farms of the urban “milk‐sheds” in Ethiopia, and vaccination could be a cost‐effective disease control strategy. In the present study, the efficacy of Bacillus Calmette–Guerin (BCG) to protect against bTB was assessed in Holstein–Friesian calves in a natural transmission setting. Twenty‐three 2‐week‐old calves were subcutaneously vaccinated with BCG Danish SSI strain 1331, and matched 26 calves were injected with placebo. Six weeks later, calves were introduced into a herd of M. bovis‐infected animals (reactors) and kept in contact with them for 1 year. In vitro and in vivo immunological tests were performed to assess immune responses post‐vaccination and during exposure. Successful vaccine uptake was confirmed by tuberculin skin test and IFN‐γ responses in vaccinated calves. The kinetics of IFN‐γ responses to early secretory antigen target 6 and culture filtrate protein 10 (ESAT6 and CFP10, respectively) and tuberculin skin test responses post‐exposure suggested that the animals were infected early after being placed in contact with the infected herd as immunological signs of infection were measurable between 2 and 4 months post‐initial exposure. Protection was determined by comparing gross and microscopic pathology and bacteriological burden between vaccinated and control calves. BCG vaccination reduced the proportions of tissues with visible pathology in vaccinates compared to control calves by 49% (p

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.

Tuberculosis remains a major public health problem in Nigeria. While human to human transmission of Mycobacterium tuberculosis is clearly of major importance in driving the tuberculosis epidemic in Nigeria, the impact of Mycobacterium bovis transmission from infected cattle is largely unknown. Molecular epidemiology of M. bovis in Nigeria will increase our understanding of this endemic disease and provide tools to assess cattle-to-human transmission. Between 2002 and 2004, molecular techniques including spoligotyping, variable number of tandem repeats (VNTR) typing and deletion typing were used to track and analyze a sample of strains of the M. tuberculosis complex circulating in the cattle population in Ibadan, Southwestern Nigeria. In all, 180 isolates were typed with a view to elucidating epidemiological information on circulating strains, occurrence of transborder transmission and molecular diversity of the M. bovis strains. Results obtained showed that 99% (178/180) of the isolates were M. bovis, while the remaining were M. tuberculosis and M. africanum. In all, strains of M. bovis had 34 different spoligotypes: strains with spoligotype pattern SB0944 (as designated by www.mbovis.org) were the most common (46% of strains). This molecular type is also common in countries neighbouring Nigeria. Strains with this spoligotype pattern could be further divided into 40 different VNTR types. This analysis shows the value of simple molecular epidemiological techniques applied to strains of M. bovis and suggests that further epidemiological studies will shed more light on the transmission dynamics of bovine tuberculosis locally and across neighbouring African countries.

Vaccination of neonatal calves with Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces a significant degree of protection against infection with virulent M. bovis , the causative agent of bovine tuberculosis (bTB). We compared two strains of BCG, Pasteur and Danish, in order to confirm that the current European human vaccine strain (BCG Danish) induced protective immunity in calves, and we assessed immune responses to determine correlates of protection that could assist future vaccine evaluation in cattle. Both vaccine strains induced antigen (purified protein derivate [PPD])-specific gamma interferon (IFN-γ) in whole-blood cultures. These responses were not significantly different for BCG Pasteur and BCG Danish and peaked at week 2 to 4 postvaccination. Vaccination with either BCG Danish or BCG Pasteur induced significant protection against bTB, with reductions in both lesion score and bacteriological burden evident in both groups of vaccinated calves compared with nonvaccinated control calves. Measurement of IFN-γ-expressing T lymphocytes postvaccination and postchallenge revealed both correlates and surrogates of protective efficacy. The frequency of central memory T lymphocytes present at 12 weeks postvaccination (at the time of M. bovis challenge) correlated significantly with protection. Conversely, the number of IFN-γ-expressing effector T cells present after M. bovis challenge was correlated with disease. These results demonstrate that vaccination of neonatal calves with either BCG Pasteur or BCG Danish induces protective immune responses against TB. In addition, we show that measurement of antigen-specific T lymphocyte populations may provide a reliable means for identifying protective vaccine candidates.

In order to identify cytokines that may be useful as candidates for inclusion in diagnostic tests for Mycobacterium bovis infection in cattle, we compared the levels of gamma interferon (IFN-γ), interleukin 1β (IL-1β), IL-4, IL-10, IL-12, macrophage inflammatory protein 1β (MIP-1β), and tumor necrosis factor alpha (TNF-α) in whole-blood cultures from tuberculosis (TB) reactor animals or TB-free controls following stimulation with M. bovis -specific antigens (purified protein derivative from M . bovis [PPD-B] or ESAT-6/CFP-10). In addition to IFN-γ responses, the production of IL-1β and TNF-α was also statistically significantly elevated in TB reactor cattle over that in uninfected controls following stimulation with PPD-B or ESAT-6/CFP-10 peptides. Thus, we evaluated whether the use of these two additional readouts could disclose further animals not detected by measuring IFN-γ alone. To this end, receiver operating characteristic (ROC) analyses were performed to define diagnostic cutoffs for positivity for TNF-α and IL-1β. These results revealed that for ESAT-6/CFP-10-induced responses, the use of all three readouts (IFN-γ, TNF-α, and IL-1β) in parallel increased the sensitivity of detection of M. bovis -infected animals by 11% but also resulted in a specificity decrease of 14%. However, applying only IFN-γ and IL-1β in parallel resulted in a 5% increase in sensitivity without the corresponding loss of specificity. The results for PPD-B-induced responses were similar, although the loss of specificity was more pronounced, even when only IFN-γ and IL-1β were used as readout systems. In conclusion, we have demonstrated that the use of an additional readout system, such as IL-1β, can potentially complement IFN-γ by increasing overall test sensitivity for the detection of M. bovis infection in cattle.

In developing countries, the conventional test and slaughter strategy for the control of bovine tuberculosis is prohibitively expensive, and alternative control methods such as vaccination are urgently required. In this study, the efficacy of Mycobacterium bovis bacillus Calmette-Guérin (BCG) for protection against bovine tuberculosis (bTB) was evaluated in Holstein calves under field conditions in Ethiopia. Thirteen neonatally vaccinated and 14 control calves were exposed for 10 to 23 months to skin test reactor cows. Gamma interferon (IFN-γ) testing, comparative intradermal tuberculin testing, postmortem examination, and bacteriological culture were used for the evaluation of BCG efficacy. The overall mean pathology score was significantly ( P < 0.05) higher in control calves than in vaccinated calves. Culture positivity for Mycobacterium bovis was higher in the control calves than in the vaccinated calves, and significantly more BCG-vaccinated animals would have passed a standard meat inspection ( P = 0.021). Overall, the protective efficacy of BCG was between 56% and 68%, depending on the parameters selected. Moreover, by measuring gamma interferon responses to the antigens ESAT-6 and CFP-10, which are present in M. bovis but absent from BCG, throughout the experiment, we were able to distinguish between vaccinated animals that were protected against bTB and those animals that were not protected. In conclusion, the present trial demonstrated an encouraging protective effect of BCG against bTB in a natural transmission setting in Ethiopia.

Tuberculosis (TB) remains one of the most important infectious diseases of humans and animals. Mycobacterium bovis BCG, the only currently available TB vaccine, demonstrates variable levels of efficacy; therefore, a replacement or supplement to BCG is required. Protein subunit vaccines have shown promise but require the use of adjuvants to enhance their immunogenicity. Using the protective mycobacterial antigen Rv3019c, we have evaluated the induction of relevant immune responses by adjuvant formulations directly in the target species for bovine TB vaccines and compared these to responses induced by BCG. We demonstrate that two classes of adjuvant induce distinct immune phenotypes in cattle, a fact not previously reported for mice. A water/oil emulsion induced both an effector cell and a central memory response. A cationic-liposome adjuvant induced a central memory response alone, similar to that induced by BCG. This suggests that water/oil emulsions may be the most promising formulations. These results demonstrate the importance of testing adjuvant formulations directly in the target species and the necessity of measuring different types of immune response when evaluating immune responses.

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.

The aim of this study was to obtain a contemporary data set of pathology in tuberculin reactor and in-contact cattle in England and Wales. Four hundred animals (200 reactors and 200 in-contacts) from 242 farms located in 14 counties in Western England and Wales were examined. The mean number of lymph nodes (LNs) with tuberculosis (TB)-like lesions per TB-confirmed animal was 1.7 in reactors and 1.5 in in-contact animals. Tuberculous lesions in both reactor and in-contact animals were most commonly observed in the LNs of the thorax, followed by the head and abdomen, particularly the mediastinal, retropharyngeal and tracheobronchial LNs. Twenty-five reactors had macroscopic lesions in the palatine tonsils. Among TB-confirmed cattle, 27% of reactors and 9% of in-contact animals had gross TB-like lesions in the lungs, particularly in the caudal lobes. Gross lesions that were not TB-confirmed were parasitic granulomas (45%), bacterial or mycotic club-forming pyogranulomas (27%) and bacterial abscesses (23%). Diagnostic sensitivity was maximised when bacteriology and histopathology were used concurrently. Stage IV granulomas, alone or in combination with other stages, constituted 63% of lesions, while 16% of lesions were stage I/II granulomas. Caseous necrosis and calcification were common features of the granulomas encountered in natural Mycobacterium bovis infections, even with pathology limited to a small number of sites. Granulomas often covered large areas of histological sections and typically contained only small numbers of acid fast bacilli.

Bovine tuberculosis (TB), caused by Mycobacterium bovis, has serious consequences for Britain's cattle industry. European badgers (Meles meles) can transmit infection to cattle, and for many years the British government culled badgers in a series of attempts to reduce cattle infections. We investigated the impact of badger culling on the spatial distribution of M. bovis infection in badger and cattle populations in replicated areas in England. M. bovis infection was significantly clustered within badger populations, but clustering was reduced when culls were repeated across wide areas. A significant spatial association between M. bovis infections in badgers and cattle herds likewise declined across successive culls. These patterns are consistent with evidence that badgers are less territorial and range more widely in culled areas, allowing transmission to occur over greater distances. Prior to culling, M. bovis infections were clustered within cattle populations. Where badger culling was localised, and in unculled areas just outside widespread culling areas, cattle infections became less spatially clustered as badger culling was repeated. This is consistent with expanded badger ranging observed in these areas. In contrast, clustering of infection in cattle persisted over time on lands where badgers were repeatedly culled over wide areas. While this lack of a temporal trend must be interpreted with caution, it might reflect persistent infection within, and continued transmission between, cattle herds in areas where transmission from badgers to cattle had been reduced by badger culling. Continued spatial association of infections in cattle and badgers in such areas might partly reflect transmission from cattle. Synthesis and applications: Our findings confirm that badger culling can prompt spatial spread of M. bovis infection, a phenomenon likely to undermine the utility of this approach as a disease control measure. Possible evidence of transmission from cattle, both to other cattle and to badgers, suggests that improved cattle controls might yield multiple benefits for TB management.

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.

The development of novel vaccine strategies supplementingMycobacterium bovisBCG (BCG) constitutes an urgent research challenge. To identify potential subunit vaccine candidates, we have tested a series of eight recently identifiedMycobacterium tuberculosisantigens inM. bovis-infected and BCG-vaccinated cattle. These antigens were characterized on the basis of their ability to induce in vitro gamma interferon responses in infected or BCG-vaccinated calves. We were able to establish a hierarchy of these antigens based on how frequently they were recognized in both groups of animals. In particular, we were able to prioritize frequently recognized proteins like Rv0287, Rv1174, and Rv1196 for future evaluation as subunit vaccines to be used in BCG-protein heterologous prime-boost vaccination scenarios. In addition, the antigen most dominantly recognized inM. bovis-infected cattle in this study, Rv3616c, was significantly less frequently recognized by BCG vaccinees and could be a target to improve BCG, for example, by increasing its secretion, in a recombinant BCG vaccine.

Bovine tuberculosis caused by Mycobacterium bovis remains an economically important problem in Great Britain with potential zoonotic consequences, and the incidence is rising exponentially. In 1997 an independent scientific review recommended that the best option for disease control in Great Britain was the development of a cattle vaccine. Bovine tuberculosis remains a significant problem in countries of the developing world. Indeed, more than 94% of the world's population live in countries in which the control of bovine tuberculosis in buffalos or cattle is limited or absent. Effective vaccination strategies would have a major impact in countries that cannot afford expensive test and slaughter-based control strategies. Here, we present a review of progress toward that goal, and discuss how this progress has shaped our research strategy for the development of a vaccine.

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.

In-bred strains of mice are commonly used to model pathogenic infections due to their cost and utility. In order to understand better the nature of experimental tuberculosis in mice, we infected BALB/c mice with a virulent field isolate of Mycobacterium bovis. Mice were sacrificed at intervals in order to visualise the pathological lesions in major internal organs. Pathological lesions in tissues increased in number and severity over time and replicated many of the salient features observed in badgers and cattle infected with M. bovis. These similarities are discussed. Examination of pathological lesions at terminal stages of infection enabled us to suggest the lethal effects of M. bovis mediated through the host response. We conclude that the mouse is a relevant surrogate species in which to study the virulence of M. bovis, as well as the influence of vaccination on its pathogenicity.

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.

1. Control of zoonotic disease is difficult to achieve when populations of multiple hosts, particularly wildlife, become persistently infected. Bovine tuberculosis (TB) is one such disease: its causative agent, Mycobacterium bovis, infects cattle, humans and multiple wildlife species including European badgers Meles meles. 2. In Britain, from 1974 to 1998 various strategies for the control of cattle TB involved culling badgers in the immediate vicinity of TB-affected herds. However, patterns of association between cattle and badgers had not been investigated at a local scale. 3. Using data from the Randomized Badger Culling Trial, an ongoing large-scale study of TB dynamics and control, we investigated local geographical associations between M. bovis infection in badgers and cattle. 4. Mycobacterium bovis infections were locally clustered within both badger and cattle populations. 5. We show, for the first time, that M. bovis infections in badgers and cattle are spatially associated at a scale of 1-2 km. Badgers and cattle infected with the same strain type of M. bovis are particularly closely correlated. These observational data support the hypothesis that transmission occurs between the two host species; however, they cannot be used to evaluate the relative importance of badger-to-cattle and cattle-to-badger transmission. 6. Synthesis and applications. The close associations between M. bovis infections in cattle and badgers suggest that localized badger culling could reasonably be expected to reduce the risks of cattle TB infection; however, experimental culls have found no such beneficial effects over the time-scale on which they were tested. This demonstrates the difficulty of predicting the outcome of management interventions, and reinforces the need for well-designed empirical assessments of future control strategies.

More accurate tests are required to test cattle which have reacted positively in the tuberculin skin test. For this purpose, a range of mycobacterial antigens, MPB59, MPB64, MPB70, MPB83, ESAT-6 and CFP10, were used either as recombinant proteins or as synthetic peptides in the whole blood interferon-gamma (IFN-gamma) test. Groups of uninfected cattle with typical 'non-specificity' problems were targeted, in particular animals with skin tuberculosis, animals vaccinated against Johne's disease and animals that were positive in the standard purified protein derivative (PPD)-based IFN-gamma test. The two study groups consisted of 74 Mycobacterium bovis-culture positive animals and 72 uninfected animals, all of which tested positive in the caudal fold tuberculin skin test eight to 28 days before the blood test. The use of combinations of ESAT-6 and CFP10 antigens, either as recombinant proteins or peptides, detected similar percentages of M bovis-infected animals as the PPD-based IFN-gamma test, but produced significantly fewer false positive reactions. The PPD-based IFN-gamma test was very effective in differentiating animals vaccinated against Johne's disease that were skin-test positive from those with bovine tuberculosis, and the use of PPD or specific mycobacterial antigens minimised the number of false positive reactions in animals with skin tuberculosis.

A panel of species cross-reactive antibodies was established for the immunohistochemical labelling of phagocytic and lymphoid cells in formalin-fixed normal badger tissues. These reagents were used to investigate the immunopathogenesis of both tuberculous and non-tuberculous granulomas in badgers. In normal badger tissues, antisera specific for the CD79a and CD79b epitopes strongly labelled follicular B lymphocytes and plasma cells in lymph nodes, bronchus-associated lymphoid tissue and Peyer's patches. Rabbit anti-dog IgG, IgM and IgA, and goat anti-human lambda light chain strongly labelled plasma cells, but goat anti-ferret IgA produced weak labelling. Interfollicular and occasional follicular lymphocytes and gut intraepithelial lymphocytes expressed the CD3 epitope. Mouse anti-human HLA-DR (MHC Class II) antigen strongly labelled macrophages, some follicular lymphocytes and some intestinal and respiratory epithelial cells. Mouse anti-human calprotectin (MAC387) labelled a limited number of macrophages. In infected badgers, all fusiform to angular macrophages (epithelioid cells) of all tuberculous granulomas strongly expressed HLA-DR antigen, but only a small, variable proportion of these were labelled by MAC387 antiserum. Lymphocytes in the peripheral rims of granulomas and those scattered sparsely amongst the epithelioid cells were labelled primarily with CD3 antiserum. Peripheral plasma cells were more common in larger than in smaller tubercles and usually expressed IgA or IgG. Small unencapsulated siliceous granulomas, which were present in both tuberculous and non-tuberculous badgers, consisted of aggregates of round to polyhedral epithelioid cells expressing the MHC Class II but not the MAC387 epitope. Granulomas caused by infection with presumed fungal adiaspores of Chrysosporium sp. consisted of aggregates of variably shaped macrophages that expressed MHC Class II antigen, but only a proportion expressed MAC387 antigen. The majority of lymphocytes within the peripheral rims of these granulomas were T cells, accompanied by sparse to moderate numbers of plasma cells that primarily expressed IgG or IgA. In conclusion, species cross-reactive antibodies can be used to identify the cellular components of tuberculous and non-tuberculous granulomas. Immunohistochemical examination failed to distinguish small tuberculous granulomas from adiaspiromycotic granulomas.

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.

BCG is used experimentally as a vaccine against tuberculosis (TB), induced by Mycobacterium bovis, in cattle (bTB). However, the efficacy of BCG is variable in humans, cattle and guinea pigs. An adenoviral vector expressing Antigen 85A (Ad5Ag85A) has enhanced protection against TB in mice when used in combination with BCG for prime-boost experiments. However, the route of immunisation affects the degree of protection seen. This work examines the immunogenicity of a new vectored vaccine (Ad5-TBF) that expresses Ag85A, Rv0287, Rv0288 and Rv0251c to explore the effects of dose of adenoviral boost and route of inoculation on immunogenicity. We found that 2×109 infectious units (iu) delivered intradermally conferred the most consistent and strongest responses of the different regimes tested. © 2014 .

SummaryLocal immune reactivity in the lungs of BALB/c mice was studied following (i) intranasal (i.n.) vaccination with Mycobacterium bovis BCG, (ii) intravenous (i.v.) challenge with a virulent M. bovis field isolate and (iii) i.n. vaccination with M. bovis BCG followed by i.v. challenge with an M. bovis field isolate. The results demonstrated that i.n. vaccination with BCG induced a high degree of protection against systemic M. bovis challenge, and that this protection correlated with a rapid production of IFN-γ after M. bovis challenge by lung T cells from vaccinated mice.

SUMMARY Nucleic acid (DNA) vaccination against tuberculosis in the European badger (Meles meles) is one approach to addressing the escalating problem of bovine tuberculosis in Great Britain. The aim of vaccination is to reduce the burden of tuberculosis within the badger population and the shedding of Mycobacterium bovis to levels that would break the transmission of infection to cattle. To this end, the vaccine would be required to limit the amount of disseminated tuberculosis in the badger, especially dissemination to the kidney from where M bovis can be shed in the urine. A promising candidate DNA vaccine encoding a 26 kDa major antigen (MPB83) of M bovis was evaluated in a mouse model of disseminated M bovis infection. Using the DNA vaccine, protection against infection of the kidney was found to be greater than that achieved with the current live vaccine, Bacille Calmette-Guerin( BCG). Kidney tissue and skeletal muscle from the badger was used to derive primary cell cultures in which to examine the expression of MPB83 following transfection with the DNA vaccine. Kidney cortex gave rise to a monotypic culture of epithelial cells whilst the muscle gave rise to a mixed culture of fibroblasts and myoblasts. During culture the myoblasts differentiated into multinucleated myotubes, verified by immunofluorescent detection of mammalian desmin. Successful expression of MPB83 by transfected epithelial and myotube cells was confirmed by immunofluorescence using a monoclonal antibody specific to the protein. These observations fulfil the early requirements for the development of a DNA vaccine for badger tuberculosis. NATIONAL bovine tuberculosis programmes based on regular tuberculin testing and removal of infected ani- mals have had a profound influence on reducing the incidence of bovine tuberculosis in cattle and farmed deer herds. However, in a number of countries, the pre- valence of bovine tuberculosis in certain geographical areas has remained stubbornly high. Since the 1970s, accumulating evidence suggests that the European badger (Meles meles) acts as a maintenance host for M bovis infection and as an important source of infec- tion for cattle in the UK (Gallagher et al 1976, Little et al 1982). Lesions in the kidney resulting from haematogenous spread of M bovis, have been described as characteristic of badger tuberculosis (Gallagher and Clifton-Hadley 2000) and large numbers of bacilli can be shed in the urine of affected badgers (Gallagher and Clifton-Hadley 2000, MAFF 1979). However, the precise route of transmission and the factors that influence it, have not been established. The intention of vaccinating badgers against infection with M bovis would be to reduce the burden of infection within the badger popul- ation and reduce the level of shedding of the organism to levels that would break the potential transmission of infection to cattle. To this end, the vaccine would be required to limit the amount of disseminated disease in the badger but not necessarily to prevent primary infection.

The lack of a typing system for Mycobacterium bovis has, until recently, been an impediment to undertaking sophisticated epidemiological studies to assist in the control and eradication of tuberculosis in domestic animals. Molecular biology techniques for mycobacteria have been in development since the mid-1980s, leading to the availability of a number of genetic typing systems for M. bovis. The authors summarise the available techniques, identify those which are most useful at present and those which might prove useful in the future. The present recommendation is to use spoligotyping analysis for rapid, large scale screening of M. bovis isolates, and to use restriction fragment length polymorphism analysis using the polymorphic guanine and cytosine-rich repeat sequences probe where greater differentiation of isolates is required. In the future, systematic analysis of the genome sequence of M. bovis will allow the development of improved techniques that combine good discrimination with ease of use.

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.

The applications of genotyping of Mycobacterium bovis are reviewed. Published research to date has been conducted predominantly within the context of validating typing methods, and few studies have been specifically epidemiological. This is contrasted with the situation in human tuberculosis, where the application of restriction fragment length polymorphism typing using insertion sequence IS6110 has successfully led to insights into the epidemiology and molecular evolution of the pathogen. Based upon the medical experience, the adoption of an integrated approach which combines epidemiology and molecular biology is recommended for future studies. Accordingly, clear identification and explanation of type clustering should be possible, which should facilitate decisions related to disease control.

This study shows that gamma interferon (IFN-γ) and interleukin-4 (IL-4) cytokine responses are produced by peripheral blood cells in cattle infected withMycobacterium bovis. The different kinetics of the IFN-γ and IL-4 responses to bovine tuberculin and to ESAT-6 following experimental intratracheal infection withM. bovisare described. An early increase in IFN-γ was observed that was maintained throughout the period studied. In contrast, the IL-4 response was delayed and confined to a peak of activity lasting 6 to 8 weeks. Interestingly, an experimental challenge of cattle with a lower dose ofM. boviswhich did not result in the development of lesions, positive DTH skin test, or substantial IFN-γ responses nevertheless generated strong specific IL-4 responses. Investigation of naturally infectedM. bovisfield reactors showed increased IFN-γ and IL-4 responses compared to uninfected cattle and that both of these cytokines were equally able to differentiate infected from uninfected animals. The magnitude of theM. bovis-induced IL-4 responses were found to be similar to the antigen-specific IL-4 responses of cattle infected with the parasitic nematodeOnchocerca ochengi, further supporting the presence of this type 2 cytokine in 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.

This report describes a comparison of immune responses in the peripheral blood and at the site of active disease in cattle 20 weeks after experimental infection with Mycobacterium bovis. Lymphocyte proliferation, and the production of interferon-gamma (IFN-gamma) and interleukin (IL)-2 were measured in response to tuberculin and a number of mycobacterial antigens, including ESAT-6, MPB64, MPB70, MPB83, hsp 16.1, hsp 65, hsp 70 and the 38 000 MW lipoprotein antigen. The level of transforming growth factor-beta (TGF-beta) was measured following stimulation of cells with tuberculin. Our results suggest little difference in the responses of peripheral blood and lymph node cells to most of the antigens used. However, tuberculin purified protein derivative (PPD) and ESAT-6 elicited stronger responses in the peripheral blood compared with lymph node cells. Investigation of the responding T-cell subpopulations in the peripheral blood showed that both CD4+ and, to a lesser extent, gammadelta T-cell receptor-positive (TCR+) T cells contributed to these responses. This is the first report to compare peripheral and local immune responses in bovine tuberculosis. Unlike cases of human tuberculosis where immune activity at the site of disease and anergy in the peripheral blood have been reported, our results suggest that for bovine tuberculosis immune responses occurring in the peripheral blood reflect those at the site of disease.

Based on our N -terminal amino acid sequence of MPT53 and a deduced DNA sequence, we searched for the corresponding gene in the Mycobacterium tuberculosis genomic sequence at the Sanger centre, localizing mpt53 close to mpt70 and mpt83. The gene was cloned and expressed, followed by purification of MPT53 to homogeneity from recombinant M. smegmatis culture fluid. In MPT53 there is 60 % identity with the active site of thioredoxin of M. tuberculosis (MPT46) with two cysteins in a CXXC motif, but MPT53 could not serve as an alternative substrate for thioredoxin reductase. Testing for IgM and IgG1 anti-MPT53 in cattle sera showed that MPT53 is immunogenic following natural and experimental infection with M. bovis. Cloning of mpt53 represents cloning of the last of the 10 proteins originally defined as "secreted proteins" of M. tuberculosis and M. bovis based on determination of their "Localization index" (LI) (J Gen Microbiol 1991;137 : 875-84). The need for a precise definition of the term "secreted protein" is discussed. So far we have observed full concordance between occurrence of an LI value indicating secretion of a protein and occurrence of a signal sequence in the corresponding gene. Signal sequence independent protein secretion in mycobacteria may occur for a limited number of proteins and remains to be established.

BOVINE tuberculosis (TB) is a disease of economic and zoonotic importance caused by the bacterial pathogen Mycobacterium bovis. In the UK, infected cattle are identified using the single intradermal comparative tuberculin test (SICTT). Recent studies have demonstrated that the parallel use of a commercial test that detects antigen-stimulated bovine interferon-γ (IFN-γ) in whole blood cultures, the Bovigam assay (Prionics) (Wood and others 1991), can improve the sensitivity for detecting infected animals compared with the use of either of the tests used in isolation (Ryan and others 2000, Collins 2002). The Bovigam IFN-γ ELISA is approved for use in bovine TB control programmes in the European Union, Australia, New Zealand and the USA. Factors that can influence the strength of IFN-γ responses include the delay between taking blood and setting up the cultures, and the interval between sampling and a recent skin test (Rothel and others 1992, Ryan and others 2000, Whipple and others 2001). In a study using cattle experimentally infected with M bovis, Whelan and others (2004) reported a reduction in the strength of IFN-γ responses if blood samples were stored overnight before testing, and also if samples were collected and tested on the day that skin-test reactions were read. These factors did not alter the diagnostic outcome of the IFN-γ test when the comparative use of bovine and avian tuberculins as test antigens was applied. However, a reduction in test sensitivity was observed when a cocktail of peptides derived from ESAT-6 and CFP-10 was applied (Whelan and others 2004); these antigens have been shown to be useful in overcoming the limitations of specificity of purified protein derivative (PPD) (Pollock and Andersen 1997, Vordermeier and others 1999, 2001). A potential limitation of the previous study was that immune responses in experimentally infected cattle are highly synchronised and may not reflect fully the range of responses in naturally infected animals. This short communication describes a study designed to address this issue, by investigating the effects of a recent skin test and the overnight storage of blood on IFN-γ responses in blood from cattle naturally infected with M bovis. Naturally infected tuberculin reactor cattle (bovine PPD [PPD-B] – avian PPD [PPD-A] >4 mm) were recruited to the study during routine UK SICTT surveillance operations. They were housed in secure accommodation and were repeat skintested up to three times, at 60-day intervals. Blood samples were collected not more than seven days before each skin test, which was performed on day 0 and again on days 3 and 10 after the skin test. Whole blood cultures were set up on the day of blood collection (fresh blood cultures) and also following the overnight storage of blood at ambient room temperature (24-hour blood cultures). Whole blood (250 μl) was cultured in duplicate in the presence of 10 μg/ml of PPDA and PPD-B (VLA – Weybridge), or a cocktail of 5 μg/ml of each of 21 overlapping 16-mer peptides mapping the entire sequences of ESAT-6 and CFP-10 (Louisiana State University Health Services Center), or in the absence of antigen. Plasma supernatants were harvested following 20 hours’ incubation at 37°C in 5 per cent carbon dioxide, and tested for IFN-γ using the Bovigam ELISA kit. The infection status of all the animals studied was confirmed by the culture of M bovis (data not shown). Fig 1 shows the IFN-γ responses of the PPDand peptide cocktail-stimulated cultures. In comparison to the responses of blood samples taken before the skin test (day 0 samples), no significant differences were observed for the day 3 or day 10 responses stimulated by PPD or peptide antigens, either for the fresh blood or 24-hour blood cultures (two-tailed paired t test), although a trend was observed towards stronger IFN-γ responses on day 3 than day 0 in all cases, a finding that was also reported by Whipple and others (2001). The measurement of IFN-γ responses in fresh blood cultures at days 24 to 31, 38 to 45 and 59 after the skin test also demonstrated no significant differences from those seen at day 0 (data not shown). This observation is consistent with the findings of Gormley and others (2004), in a similar study of naturally infected cattle in the Republic of Ireland. The absence of a suppression of IFN-γ production at day 3 is in contrast to previous observations in experimentally infected cattle (Whelan and others 2004). This may reflect differences in the stage or progression of disease in these animals, since the experimentally infected cattle had more extensive pulmonary pathology compared with the naturally infected reactors in this study (data not shown). Whipple and others (2001) reported a boost in the IFN-γ response following the application of a caudal fold test, and that IFN-γ responses remained elevated for up to 59 days. Similarly, Ryan and others (2000) also reported elevated responses to the Bovigam test eight to 28 days after a caudal fold test. These observations contrast with the present findings and those of Gormley and others (2004); one possible reason for this is that the caudal fold test may be more effective at boosting IFN-γ responses than the SICTT. Overnight storage of the blood did result in a lower response (Fig 1), but this had little influence on the diagnostic outcome of the assay when applying the comparative use of PPD as test antigens (Table 1). Studies in New Zealand and the USA have concluded that approximately equal proportions of animals could be defined as positive when the IFN-γ assay was performed either on the day of sampling or 24 hours after sampling, despite a similar drop in signal strength (Rothel and others 1992, Whipple and others 2001). Those results were obtained in reactor animals with confirmed TB (Rothel and others 1992) or with experimentally sensitised steers (Whipple and others 2001), and formed the basis of the New Zealand and USA policy of starting the assay on the day after sampling (within 24 hours). It is interesting to note that the PPD responder frequency for the 24-hour blood was actually higher than for fresh blood, due to the resolution of a PPD-A bias for some of the cattle (Table 1). An increase in specificity following overnight storage has also been reported by Ryan and others (2000). In contrast to these findings, which suggest that overnight storage of blood has no significant impact on the sensitivity of the IFN-γ test, Gormley and others (2004) reported that the delay in culturing the blood reduced the test sensitivity significantly. In that study, three groups of IFN-γ test-positive animals, namely, SICTT-reactors with confirmed TB (group 1), a group of SICTT-positive animals from surveillance operations (group 2) six of 29 of which had confirmed TB and a group of 60 SICTT-negative animals on which no postmortem examinations were performed (group 3), were assessed. Applying the Bovigam test to the group 1 animals within eight or 24 hours did not decrease the sensitivity of the test significantly. However, in the other Veterinary Record (2007) 160, 660-662

Culture fluids after growth of Mycobacterium bovis BCG on Sauton medium contain actively secreted proteins and proteins released by bacterial lysis. BCG culture fluids and sonicates of Mycobacterium tuberculosis and Mycobacterium paratuberculosis were tested after separation by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The localization of marker proteins was determined by enzyme-linked immunosorbent assay and Western blotting with selected monoclonal antibodies of known specificities. Soluble secreted proteins (MPB64 and proteins of the antigen 85 complex) and three heat shock proteins (DnaK, GroEL, and GroES) were recovered in a single peak after gel filtration, indicating their occurrence as a free monomer in the culture fluid and cytosol, respectively. Other constituents eluted in two distinct peaks during gel filtration. The first peak corresponded to the void volume, indicating complex formation between several proteins or attachment to lipids in the surface layer or the cytoplasmic membrane; the second peak corresponded to the expected monomer size indicated by SDS-PAGE under conditions that separate proteins from each other during sample preparation. The two-peak group contained constituents with known lipid contents, the 19- and 38-kDa lipoproteins and lipoarabinomannan. The 26-kDa form of MPB83 behaved similarly. After extraction with Triton X-114, these constituents entered into the detergent phase, confirming the lipoprotein nature of 26-kDa MPB83. The MPB83 molecule was shown to be available on the surface of BCG Tokyo bacilli for reaction with monoclonal antibody MBS43 by flow cytometry.

The control of bovine tuberculosis remains a significant problem in the United Kingdom, especially in the south west of England where the rising prevalence of the disease is attributed to a reservoir of Mycobacterium bovis infection in badgers. The possibility of controlling the disease by the vaccination of cattle has been unpopular with veterinarians largely because of the potential compromise of existing diagnostic tests. However, the vaccination of badgers to reduce the risk of transmission to cattle is an attractive option and has now been adopted as a potential control strategy. Recent research has led to important advances in the molecular genetics of mycobacteria and in the understanding of protective immune responses. These advances mean that it may be feasible to design and develop effective mycobacterial vaccines. This review summarises the current understanding of protective immunity against M bovis infection and discusses the possible strategies for the development of vaccines.

Vaccination of neonatal calves withMycobacterium bovisbacillus Calmette-Guérin (BCG) induces a significant degree of protection against bovine tuberculosis, caused by infection with virulentM. bovis. In two independent experiments, we assessed the duration of the protective immunity induced in calves by neonatal vaccination with BCG Danish. Protection from disease was assessed at 12 and 24 months postvaccination in cattle challenged via the endotracheal route withM. bovis. We also assessed antigen-specific immune responses to assess their utility as correlates of protection. At 12 months postvaccination, significant reductions in lung and lymph node pathologies were observed compared to nonvaccinatedM. bovis-challenged control cattle. At 24 months post-BCG vaccination, there was a reduction in lung and lymph node pathology scores and in bacterial burden. However, when comparing vaccinated and control groups, this did not reach statistical significance. Vaccination induced long-lived antigen (purified protein derivative [PPD])-specific gamma interferon (IFN-γ) release in whole-blood cultures, which remained above baseline levels for more than 20 months (approximately 90 weeks). The number of antigen-specific IFN-γ-secreting central memory T cells present at the time ofM. bovischallenge was significantly higher in vaccinated than in control animals at 12 months postvaccination, but not at 24 months. Vaccination of neonatal calves with BCG Danish induced protective immune responses against bovine TB which were maintained for at least 12 months postvaccination. These studies provide data on the immunity induced by BCG vaccination in calves; the results could inform vaccination strategies for the control of bovine TB in United Kingdom cattle herds.

The intradermal tuberculin tests and the interferon-gamma (IFN-γ) assay are the principal tests used worldwide for the ante-mortem diagnosis of bovine tuberculosis. The conventional reagent currently in use in these tests is purified protein derivative (PPD) tuberculin obtained from Mycobacterium bovis culture. The components of PPD are poorly characterized and difficult to standardize. To overcome this issue, antigens specific to the Mycobacterium tuberculosis complex are being studied. Here we have assessed the biological potency of ESAT-6, CFP-10 and Rv-3615c presented as peptide or recombinant protein cocktails in comparison with the standard bovine PPD used routinely in Spanish eradication campaigns. The study was performed in cattle (n= 23) from a herd with natural M. bovis infection. Animals were simultaneously injected with PPD and the peptide and protein cocktails. The percentages of cattle reacting positively to single intradermal test were 60.9% (bovine PPD), 47.8% (peptide cocktail) and 60.9% (protein cocktail), with no significant difference between the actual skin fold thickness increases (p> 0.05). The IFN-γ assay detected 60.9% of animals when stimulation was performed with bovine PPD, but decreased to 52.2% when stimulation was performed with the peptide cocktail and to 47.8% when stimulation was performed with the protein cocktail. However, no significant differences were found between IFN-γ responder frequencies (p> 0.05). These results show a potential use of these defined reagents for in vivo tuberculosis diagnosis., This study was funded by the EU Project FP7-KBBE-2007-1 “Strategies for the eradication of bovine tuberculosis (TB-STEP)”. C. Casal is recipient of a research contract assigned by Comunidad de Madrid (FINNOVA II Programme).

Summary: The gene encoding an immunodominant secreted antigen, MPB70, of Mycobacterium bovis was cloned into the plasmid vector pBluescript II KS+ along with its native ribosome-binding site. In this construct translation of the protein in Escherichia coli was from the native AUG initiation codon and was directed by the mycobacterial ribosome-binding site. Two different molecular mass forms (26 kDa and 22 kDa) of MPB70 were observed in whole-cell pellets of recombinant E. coli. The difference in size indicates cleavage of the signal peptide of MPB70 by an endopeptidase of E. coli. MPB70 was secreted into the periplasm of recombinant E. coli, where the 22 kDa form of the protein was predominant. The culture filtrate contained only the 22 kDa form of the protein, which was soluble. The passage of MPB70 from the periplasm into the growth medium was found to be due, at least in part, to non-specific leakage of periplasmic proteins across the outer membrane associated with the expression of recombinant MPB70.

Eurasian badgers ( Meles meles ) are a reservoir host of Mycobacterium bovis and are implicated in the transmission of tuberculosis to cattle in Ireland and Great Britain. The development of a vaccine for use in badgers is considered a key element of any long-term sustainable campaign to eradicate the disease from livestock in both countries. The aim of this study was to investigate the protective response of badgers vaccinated orally with Bacille Calmette–Guerin (BCG) encapsulated in a lipid formulation, followed by experimental challenge with M. bovis . A group of badgers was vaccinated by inoculating the BCG–lipid mixture containing approximately 10 8 colony forming units (cfu) of BCG into the oesophagus. The control group was sham inoculated with the lipid formulation only. Thirteen weeks after vaccination all the badgers were challenged with approximately 10 4 cfu of M. bovis delivered by endobronchial inoculation. Blood samples were taken throughout the study and the cell mediated immune (CMI) responses in peripheral blood were monitored by the IFN-γ ELISA and ELISPOT assay. At 17 weeks after infection all the badgers were examined post-mortem to assess the pathological and bacteriological responses to challenge. All badgers in both groups were found to be infected. However, a significant protective effect of BCG vaccination was measured as a decrease in the number and severity of gross lesions, lower bacterial load in the lungs, and fewer sites of infection. The analysis of immune responses showed that vaccination with BCG did not generate any detectable CMI immunological responses, however the levels of the responses increased in both groups following M. bovis infection. The results of the study showed that vaccination with oral BCG in the lipid formulation generated a protective effect in the badgers.

Summary The urgency for new and improved cattle vaccines and diagnostic reagents has been acknowledged by the UK Government, and development of cattle vaccine is a research priority. Significant progress has been made to develop specific antigens that allow the differentiation of bacille Calmette-Guerin (BCG) vaccinated and M. bovis-infected cattle [diagnosis of infected from vaccinated individuals (DIVA) test]. This progress has been greatly facilitated by the completion of the genome sequences of Mycobacterium tuberculosis, M. bovis and BCG Pasteur. In this study, we describe how we applied this knowledge, through comparative genome and transcriptome analysis, to define DIVA antigens that complemented the prototype DIVA antigens ESAT-6 and CFP-10 by increasing their test sensitivity. In addition, we draw general conclusions from our experience, and discuss potential future approaches in this area.

Mycobacterium tuberculosis continues to kill about 3 million people every year, more than any other single infectious agent. This is attributed primarily to an inadequate immune response towards infecting bacteria, which suffer growth inhibition rather than death and subsequently multiply catastrophically. Although the bacillus Calmette-Guerin (BCG) vaccine is widely used, it has major limitations as a preventative measure. In addition, effective treatment requires that patients take large doses of antibacterial drug combinations for at least 6 months after diagnosis, which is difficult to achieve in many parts of the world and is further restricted by the emergence of multidrug-resistant strains of M. tuberculosis. In these circumstances, immunotherapy to boost the efficiency of the immune system in infected patients could be a valuable adjunct to antibacterial chemotherapy. Here we show in mice that DNA vaccines, initially designed to prevent infection, can also have a pronounced therapeutic action. In heavily infected mice, DNA vaccinations can switch the immune response from one that is relatively inefficient and gives bacterial stasis to one that kills bacteria. Application of such immunotherapy in conjunction with conventional chemotherapeutic antibacterial drugs might result in faster or more certain cure of the disease in humans.

Tuberculous infections caused by mycobacteria, especially tuberculosis of humans and cattle, are important both clinically and economically. Human populations can be vaccinated withMycobacterium bovisbacille Calmette-Guérin (BCG), and control measures for cattle involving vaccination are now being actively considered. However, diagnostic tests based on tuberculin cannot distinguish between genuine infection and vaccination with BCG. Therefore, identification of differential diagnostic antigens capable of making this distinction is required, and until now sequence-based approaches have been predominant. Here we explored the link between antigenicity and mRNA expression level, as well as the possibility that we may be able to detect differential antigens by analyzing quantified global transcriptional profiles. We generated a list of 14 candidate antigens that are highly expressed inMycobacterium tuberculosisandM. bovisunder a variety of growth conditions. These candidates were screened inM. bovis-infected and naïve cattle for the ability to stimulate a gamma interferon (IFN-γ) response. We identified one antigen, Rv3615c, which stimulated IFN-γ responses in a significant proportion ofM. bovis-infected cattle (11 of 30 cattle [37%] [P< 0.01]) but not in naïve or BCG-vaccinated animals. Importantly, the same antigen stimulated IFN-γ responses in a significant proportion of infected cattle that did not respond to the well-characterized mycobacterial antigens ESAT-6 and CFP-10. Therefore, use of the Rv3615c epitope in combination with previously described differential tests based on ESAT-6 and CFP-10 has the potential to significantly increase diagnostic sensitivity without reducing specificity in BCG-vaccinated populations.

A lateral-flow immunoassay (BrockTB Stat-Pak) for detecting tuberculosis in Eurasian badgers was 49% sensitive and 93% specific against culture for M. bovis ( n = 1,464) at necropsy. However, the sensitivity was significantly higher (66 to 78%) in animals with more severe tuberculosis, indicating that the BrockTB Stat-Pak may be useful for the detection of badgers with the greatest risk of transmitting disease.

Real-time PCR was used to detect and quantify Mycobacterium bovis cells in naturally infected soil and badger feces. Immunomagnetic capture, immunofluorescence, and selective culture confirmed species identification and cell viability. These techniques will prove useful for monitoring M. bovis in the environment and for elucidating transmission routes between wildlife and cattle.

SummaryWe assessed the effect of exposure to Mycobacterium avium on the development of immune responses and the pathogenesis of disease observed following Mycobacterium bovis challenge. A degree of protection against M. bovis was observed in calves which were pre-exposed to M. avium as assessed by the extent of lesions and bacterial load compared to the M. bovis alone group. The immune response following M. bovis challenge in cattle previously inoculated with M. avium was biased towards antigens (PPD) present in M. avium, whereas the response following M. bovis alone was biased towards antigens present in M. bovis, indicating an imprinting of memory to avian antigens on T lymphocytes. A consequence of the memory to M. avium antigens was failure to diagnose M. bovis infection by the skin test or the IFNγ assay in some of the animals which had lesions of tuberculosis at necropsy. The use of M. bovis specific antigens ESAT-6 and CFP-10 increased IFNγ test specificity in animals previously exposed to M. avium but the responses to these antigens were lower than those observed in animals exposed to M. bovis alone. The implication is that responses to M. avium, although providing some immunity, may mask diagnosis of M. bovis infection, even when specific antigens are employed, potentially contributing to disease transmission in the field.

The early, preclinical stages of bovine TB can be detected in live animals by the use of tests of cellular immunity (the skin, gamma-interferon and lymphocyte transformation tests). Tests of humoral (antibody) immunity, Mycobacterium bovis PCR probes on early tissue cultures or live cattle specimens, and tests based on "electronic nose" technology have been developed more recently. The key measure of diagnostic test accuracy is the relationship between sensitivity and specificity, which determines the false-positive and false-negative proportions. None of the tests currently available for the diagnosis of bovine TB allow a perfectly accurate determination of the M. bovis infection status of cattle. Although various factors can reduce the sensitivity and specificity of the skin tests, these remain the primary ante mortem diagnostic tools for TB in cattle, providing a cost-effective and reliable means of screening entire cattle populations. Despite the inescapable limitations of existing diagnostic tests, bovine TB has been effectively eradicated from many developed countries and regions with the implementation of sound programmes of regular tuberculin skin testing and removal of reactors, coupled with slaughterhouse surveillance for undetected infections, repeat testing and culling of infected herds, cattle movement restrictions to prevent introduction of infected animals and occasional slaughter of entire herds with intractable breakdowns. This is likely to remain the mainstay of bovine TB control programmes for the foreseeable future. Additionally, newer ancillary in vitro diagnostic assays are now available to TB control programme managers to supplement the skin tests in defined circumstances according to the specific disease situation in each country or region. The strategic deployment of ancillary in vitro tests alongside the primary skin tests has enhanced the detection of M. bovis-infected cattle and reduced the number of animals slaughtered as false positives.

It is estimated that more than 50 million cattle are infected withMycobacterium bovisworldwide, resulting in severe economic losses. Current diagnosis of tuberculosis (TB) in cattle relies on tuberculin skin testing, and when combined with the slaughter of test-positive animals, it has significantly reduced the incidence of bovine TB. The failure to eradicate bovine TB in Great Britain has been attributed in part to a reservoir of the infection in badgers (Meles meles). Accurate and reliable diagnosis of infection is the cornerstone of TB control. Bacteriological diagnosis has these characteristics, but only with samples collected postmortem. Unlike significant wild animal reservoirs ofM. bovisthat are considered pests in other countries, such as the brushtail possum (Trichosurus vulpecula) in New Zealand, the badger and its sett are protected under United Kingdom legislation (The Protection of Badgers Act 1992). Therefore, an accurate in vitro test for badgers is needed urgently to determine the extent of the reservoir of infection cheaply and without destroying badgers. For cattle, a rapid on-farm test to complement the existing tests (the skin test and gamma interferon assay) would be highly desirable. To this end, we have investigated the potential of an electronic nose (EN) to diagnose infection of cattle or badgers withM. bovis, using a serum sample. Samples were obtained from both experimentally infected badgers and cattle, as well as naturally infected badgers. Without exception, the EN was able to discriminate infected animals from controls as early as 3 weeks after infection withM. bovis, the earliest time point examined postchallenge. The EN approach described here is a straightforward alternative to conventional methods of TB diagnosis, and it offers considerable potential as a sensitive, rapid, and cost-effective means of diagnosingM. bovisinfection in cattle and badgers.

SummaryVaccination of neonates with Mycobacterium bovis bacillus Calmette–Guérin (BCG) may be a strategy that overcomes reduced vaccine efficacy associated with exposure to environmental mycobacteria in humans and cattle. Preliminary comparisons indicated that 2-week-old calves produced an immune response to vaccination at least as intense as that observed in adults. Subsequently, five gnotobiotic hysterotomy derived calves aged 1 day were inoculated with BCG and 3 months later were challenged intranasally with virulent M. bovis. The number of tissues with lesions and the pathological extent of these lesions was reduced significantly in vaccinates. Furthermore, lesions were evident in the lung or associated chest lymph nodes of four of five controls but none of five vaccinates. BCG vaccination reduced significantly the level of bacterial colonization. However, lesions in the head associated lymph nodes were observed in three of five BCG-vaccinated cattle. Levels of interferon gamma (IFN-γ) detected by enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunospot (ELISPOT) in individual vaccinated animals at challenge did not correlate with subsequent resistance and in general immune responses post-challenge were lower in vaccinated calves. Low IL-10 responses were evident but IL-4 was not detected. Responses to ESAT-6 and/or CFP-10 were evident in four of four control calves that had lesions. Two of the BCG vaccinates with lesions did not produce a response to ESAT-6 and CFP-10, indicating that these antigens did not distinguish vaccinated immune animals from vaccinated animals with lesions. Overall, vaccination of neonatal calves with BCG induced significant protection against disease and has potential as a strategy for the reduction of the incidence of bovine tuberculosis.

The sharp rise of bovine tuberculosis (TB) in Great Britain and the continuing problem of wild life reservoirs in countries such as New Zealand and Great Britain have resulted in increased research efforts into the disease. Two of the goals of this research are to develop (1) cattle vaccines against TB and (2) associated diagnostic reagents that can differentiate between vaccinated and infected animals (differential diagnosis). This review summarises recent progress and describes efforts to increase the protective efficacy of the only potential TB vaccine currently available, Mycobacterium bovis BCG, and to develop specific reagents for differential diagnosis. Vaccination strategies based on DNA or protein subunit vaccination, vaccination with live viral vectors as well as heterologous prime-boost scenarios are discussed. In addition, we outline results from studies aimed at developing diagnostic reagents to allow the distinction of vaccinated from infected animals, for example antigens that are not expressed by vaccines like Mycobacterium bovis Bacille-Calmette-Guerin, but recognised strongly in Mycobacterium bovis infected cattle.

The European badger (Meles meles) has been identified as a reservoir for Mycobacterium bovis and is implicated in the maintenance and transmission of tuberculosis in cattle. There is a need for a sensitive test of M. bovis infection in badgers and the current serodiagnostic test used for this purpose has low sensitivity. As observed for other species, assay of interferon-gamma (IFNgamma) produced in response to M. bovis antigens is a more sensitive test of tuberculosis. With this objective in sight, we report the first step in the development of an ELISA for badger IFNgamma. The badger IFNgamma gene was cloned and sequenced and used to generate a specific polyclonal antibody to the cytokine. The gene sequence demonstrated regions that were conserved within the IFNgamma genes of other mammals. The badger sequence was most similar to the canine, showing similar structural organisation of the gene and 88% amino acid identity. Rabbits were immunised with DNA encoding badger IFNgamma and the resulting polyclonal antiserum demonstrated specificity for canine IFNgamma by immunoblot of a commercial recombinant canine IFNgamma. The antiserum was used to detect intracellular badger IFNgamma by flow cytometry analysis of badger lymphocytes stimulated with mitogen.