Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces human CC- and CXC-chemokines in vitro and in vivo

Both CC- and CXC-chemokines are known to be potent leucocyte activators and chemoattractants and play important roles in inflammatory responses. However, chemokine response to bacillus Calmette-Guérin (BCG) infection remains incompletely defined. In this study, we investigated human CC- [macrophage-derived chemokine (MDC), monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1alpha and eosinophil chemoattractant activity (eotaxin)] and CXC-interferon-inducible protein (IP)-10 chemokine production in response to BCG stimulation. BCG efficiently induced all chemokines tested in the urine of four bladder cancer patients undergoing intravesical BCG immunotherapy. The peak urinary chemokine responses occurred generally between the fourth and sixth weekly treatment, except eotaxin, which was less predictable. To evaluate the effect of BCG on induction of chemokines in vitro, urothelial cell lines and peripheral blood mononuclear cells (PBMCs) were used. Although BCG induced no or marginal chemokines from urothelial SV-HUC-1, RT4 and T24 cells, BCG-derived cytokines [interleukin (IL)-1beta, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha] induced all chemokines tested except eotaxin from these cell lines. BCG also efficiently induced all chemokines tested except eotaxin from PBMCs of both BCG-naive and BCG-vaccinated subjects. MCP-1 and MIP-1alpha emerged at 4-5 h post-BCG exposure (early chemokines); IP-10 elevated at day 1 and peaked at day 2 (intermediate chemokine); and MDC elevated at day 1 and peaked at day 7 (late chemokine). This kinetic pattern was paralleled with that of BCG-induced cytokines [early: TNF-alpha; intermediate: IL-6 and IL-10; and late: IFN-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF)]. Taken together, these results indicate that BCG directly or indirectly induces human CC- and CXC-chemokine production, which may represent one of the mechanisms by which BCG exerts its anti-tumour activity.     

Gammadelta T cells in immunity induced by Mycobacterium bovis bacillus Calmette-Guérin vaccination

Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccination is efficacious for newborns or adults with no previous exposure to environmental mycobacteria. To determine the relative contribution and the nature of gammadelta T-cell receptor-positive T cells in newborns, compared to CD4(+) T cells, in immunity induced by M. bovis BCG vaccination, 4-week-old specific-pathogen-free pigs were vaccinated with M. bovis BCG and monitored by following the gammadelta T-cell immune responses. A flow cytometry-based proliferation assay and intracellular staining for gamma interferon (IFN-gamma) were used to examine gammadelta T-cell responses. Pigs were found to mount Th1-like responses to M. bovis BCG vaccination as determined by immunoproliferation and IFN-gamma production. The gammadelta T-cell lymphoproliferation and IFN-gamma production to stimulation with mycobacterial antigens were significantly enhanced by M. bovis BCG vaccination. The relative number of proliferating gammadelta T cells after stimulating peripheral blood mononuclear cells with Mycobacterium tuberculosis H37Rv culture filtrate protein was higher than that of CD4(+) T cells at an early time point after M. bovis BCG vaccination, but CD4(+) T cells were found to be more abundant at a later time point. Although the gammadelta T-cell responses were dependent on the presence of CD4(+) T cells for the cytokine interleukin-2, the enhanced gammadelta T cells were due to the intrinsic changes of gammadelta T cells caused by M. bovis BCG vaccination rather than being due solely to help from CD4(+) T cells. Our study shows that gammadelta T cells from pigs at early ages are functionally enhanced by M. bovis BCG vaccination and suggests an important role for this T-cell subset in acquired immunity conferred by M. bovis BCG vaccination.     

Interferon-alphabeta mediates partial control of early pulmonary Mycobacterium bovis bacillus Calmette-Guérin infection

The role of type I interferon (IFN-alphabeta) in modulating innate or adaptive immune responses against mycobacterial infection in the lung is unclear. In this study we investigated the susceptibility of IFN-alphabeta-receptor-deficient (IFN-alphabetaR-/-) mice to pulmonary infection with aerosolized Mycobacterium bovis bacillus Calmette-Guérin (BCG). During early infection (2-3 weeks), enhanced growth of BCG was measured in the lungs of IFN-alphabetaR-/- mice compared to wild-type mice. However, during late infection the burden of BCG was similar in the lungs of IFN-alphabetaR-/- and wild-type mice. Although control of BCG growth was delayed, recruitment and activation of T and natural killer cells, production of IFN-gamma, and cytokine expression were all similar in wild-type and IFN-alphabetaR-/- mice. However, decreased expression of nitric oxide in bronchoalveolar lavage fluids from IFN-alphabetaR-/- mice correlated with enhanced growth of BCG. Bone marrow-derived macrophages from IFN-alphabetaR-/- mice also produced less nitric oxide following infection with BCG in vitro. These findings suggest that IFN-alphabeta contributes to innate immunity to pulmonary mycobacterial infection by augmenting production of nitric oxide.     

Improved immunogenicity of recombinant Mycobacterium bovis bacillus Calmette-Guérin strains expressing fusion protein Ag85A-ESAT-6 of Mycobacterium tuberculosis

Early secretory antigen target 6 (ESAT-6) is a dominant target for cell-mediated immunity in the early phase of tuberculosis (TB) in patients with TB, causing T-cell proliferation and gamma interferon (IFN-γ) production, which has been considered to be a protective antigen that can be used for future vaccine development. Ag85A is the most essential component for bacterial survival within macrophages and has been used in numerous vaccine preparations, which can induce strong cellular immune responses. In this study, we constructed a new recombinant bacilli Calmette-Guérin (BCG) strain (rBCG-AE) that could express fusion protein Ag85A-ESAT-6 of Mycobacterium tuberculosis and evaluated its immunogenicity in BALB/c mice. There was no evidence for increased virulence of this rBCG. Our experiments illustrated that the rBCG-AE was able to induce higher titer of antibody and elicit more long-lasting and stronger Th1 type cellular immune responses than the parental BCG strain, or rBCG-A (expressing Ag85A) strain, or rBCG-E (expressing ESAT-6) strain, which are characterized by the strong antibody response, the proliferation rate of splenocytes, the ratio of CD4(+) T and CD8(+) T cells stimulated by tuberculin-purified protein derivative and elevated levels of IFN-γ in antigen-stimulated splenocyte cultures. The results show that rBCG-AE is an improved TB vaccine for further study.     

Interleukin-15 as an immune adjuvant to increase the efficacy of Mycobacterium bovis bacillus Calmette-Guérin vaccination

Interleukin-15 (IL-15) transgenic mice which had been inoculated with Mycobacterium bovis bacillus Calmette-Guérin (BCG) 24 weeks previously showed resistance against airborne infection with Mycobacterium tuberculosis H37Rv accompanied by an increased CD8(+)-Tc1-cell response. IL-15 may be used as an immune adjuvant given with BCG vaccination to enhance its biologic efficacy.     

Human leucocytes response to viable, extended freeze-drying or heat-killed Mycobacterium bovis bacillus Calmette-Guérin

We investigated the effects of viable, extended freeze-drying (EFD) or heat-killed (HK) Mycobacterium bovis bacillus Calmette-Guérin (BCG) in respiratory burst activity, gene expression of CYBB and NCF1 encoding components of the human phagocyte nicotinamide adenine dinucleotide (NADPH) oxidase, TLR2 expression, and in IL-10 and TNF-α cytokine production by human peripheral blood mononuclear cells (PBMCs). Viable BCG significantly inhibited TLR2 and CYBB gene expression, as well as superoxide release by human PBMC. All BCG stimuli augmented IL-10 release, but only HK BCG or viable BCG increased TNF-α release by PBMCs. Our studies show that viable BCG can impair the NADPH oxidase system activation and the TLR2 route in human PBMCs. As well, different BCG preparations can distinctly influence cytokine production by human PBMCs.     

Maturation of human dendritic cells by cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guérin: involvement of toll-like receptors

The constituents of mycobacteria are an effective immune adjuvant, as observed with complete Freund's adjuvant. In this study, we demonstrated that the cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guérin (BCG-CWS), a purified noninfectious material consisting of peptidoglycan, arabinogalactan, and mycolic acids, induces maturation of human dendritic cells (DC). Surface expression of CD40, CD80, CD83, and CD86 was increased by BCG-CWS on human immature DC, and the effect was similar to those of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), heat-killed BCG, and viable BCG. BCG-CWS induced the secretion of TNF-alpha, IL-6, and IL-12 p40. CD83 expression was increased by a soluble factor secreted from BCG-CWS-treated DC and was completely inhibited by monoclonal antibodies against TNF-alpha. BCG-CWS-treated DC stimulated extensive allogeneic mixed lymphocyte reactions. The level of TNF-alpha secreted through BCG-CWS was partially suppressed in murine macrophages with no Toll-like receptor 2 (TLR 2) or TLR4 and was completely lost in TLR2 and TLR4 double-deficient macrophages. These results suggest that the BCG-CWS induces TNF-alpha secretion from DC via TLR2 and TLR4 and that the secreted TNF-alpha induces the maturation of DC per se.     

Oral recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing HIV-1 antigens as a freeze-dried vaccine induces long-term,HIV-specific mucosal and systemic immunity

Induction of HIV-1-specific immune responses was evaluated using a recombinant BCG (rBCG) vector-based vaccine expressing HIV-1 Env V3 peptide (rBCG-pSOV3J1). rBCG-pSOV3J1 was manufactured as a freeze-dried preparation based on good laboratory practice guidelines. Guinea pigs were immunized with the freeze-dried rBCG vaccine by oral administration to test the effectiveness of what is generally considered the most convenient and practical route for vaccination. While delayed-type hypersensitivity (DTH) skin reactions to purified protein derivative were not detected in any of the animals receiving oral rBCG-pSOV3J1, HIV-1 V3J1 antigen-specific DTH responses were detected in all of the immunized guinea pigs 1.5 years after immunization. In addition, significant proliferative responses against HIV-1 V3J1 antigen were measured in peripheral blood mononuclear cells and splenocytes from all animals receiving oral rBCG. Interestingly, intestinal intraepithelial lymphocytes from the animals also exhibited high levels of proliferative activity against HIV-1 V3J1 antigen. These results suggest that oral vaccination of guinea pigs with freeze-dried rBCG-pSOV3J1 induces high levels of functional T cells specific for HIV-1 antigens in both mucosal and systemic compartments and suggest that this approach has potential for use as a vaccine against HIV-1.     

Virulence,immunogenicity, and protective efficacy of two recombinant Mycobacterium bovis bacillus Calmette-Guérin strains expressing the antigen ESAT-6 from Mycobacterium tuberculosis

We constructed two recombinant Mycobacterium bovis BCG (rBCG) strains expressing ESAT-6 of Mycobacterium tuberculosis, named rBCG-1 and rBCG-2. rBCG-1 contained the ESAT-6 gene linked to BCG hsp60 and expressed a fusion protein, while rBCG-2, with a secretory sequence, could secret ESAT-6 into the culture medium. There was no evidence for increased virulence of the two rBCG strains when we made a comparison between them and BCG with regard to organ bacterial loads, lung histology, and survival time. rBCG-1 induced significantly higher specific antibody titers and stronger cellular immune response than BCG, whereas rBCG-2 had immunogenicity similar to that of the parental BCG strain. Both rBCG-1 and rBCG-2 conferred marked protection against M. tuberculosis infection, yet in terms of protective efficacy, they showed no significant improvements upon conventional BCG vaccine.     

The in vivo immunomodulatory effect of recombinant tumour necrosis factor-alpha in guinea pigs vaccinated with Mycobacterium bovis bacille Calmette-Guérin

A void in understanding primary biliary cirrhosis (PBC) is the absence of appropriate animal models. Our laboratory has studied a murine model of autoimmune cholangitis induced following immunization with 2-octynoic acid (2OA), an antigen identified following extensive quantitative structural activity relationship (QSAR) analysis, using human autoantibodies and three-dimensional analysis of the mitochondrial autoantigen, the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2). Mice immunized with 2OA coupled to bovine serum albumin (BSA) develop anti-mitochondrial antibodies (AMAs) of the identical specificity as humans with PBC, and in addition develop inflammatory portal cell infiltrates in liver. However, the natural history of disease is less severe than in humans and does not include fibrosis. Data from human and autoimmune murine models suggest that environmental and/or infectious agents can exacerbate autoimmune reactions, and a model of PBC has been described in which polyinosinic-polycytidylic acid (poly I:C), a viral RNA mimetic and Toll-like receptor 3 (TLR-3) agonist induces low-titre AMAs and in mild portal infiltrates. We took advantage of our established model to determine whether immunization with 2OA-BSA coupled with poly I:C alters the disease process. Indeed, the addition of poly I:C produces a profound exacerbation of autoimmune cholangitis, including a significant increase in CD8(+) infiltrating T cells, as well as a marked increase of proinflammatory cytokines. In addition, mice have evidence of fibrosis. These findings lend support to the concept that besides breakdown of self-tolerance, there is a requirement of a second 'hit' during the breakdown process that leads to disease which more faithfully mimics human PBC.     

PD-1-PD-L1 pathway impairs T(h)1 immune response in the late stage of infection with Mycobacterium bovis bacillus Calmette-Guérin

A major concern still prevails as to the reason why various mycobacteria are able to persist within infected host in which protective immunity is generated. To address this question, we monitored the generation of protective T cells during infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG). CD4(+) T cells obtained 3 weeks after infection conferred protection against Mycobacterium tuberculosis challenge and produced IFN-γ and tumor necrosis factor (TNF)-α upon antigen stimulation. However, these abilities were decreased after 6 weeks of infection even though BCG was not thoroughly eliminated from the host. We analyzed the expression of ligands for the CD28/CTLA-4 family receptors on antigen-presenting cells and found that the expression of PD-L1, a ligand for programmed cell death-1 (PD-1), was up-regulated later than 3 weeks of infection. We also found that bacterial numbers in the spleen of PD-1-deficient mice were significantly reduced compared with wild-type mice at 6 and 12 weeks after BCG infection. Furthermore, CD4(+) T cells of PD-1-deficient mice showed a higher ability to confer protection and produce IFN-γ and TNF-α even at 12 weeks after infection. These results indicate that the PD-1-PD-L1 pathway impairs T(h)1 immunity in the late stage of BCG infection, thereby facilitating the bacterial persistence in the host.     

Simultaneous blocking of human Toll-like receptors 2 and 4 suppresses myeloid dendritic cell activation induced by Mycobacterium bovis bacillus Calmette-Guérin peptidoglycan

The Mycobacterium bovis bacillus Calmette-Guérin (BCG) cell wall skeleton (CWS) consists of mycolic acids, arabinogalactan, and peptidoglycan (PGN) and activates Toll-like receptor 2 (TLR2) and TLR4. Here we investigated the ability of the essential portion of highly purified BCG CWS to support the TLR agonist function by using the following criteria: myeloid dendritic cell (DC) maturation, i.e., tumor necrosis factor alpha (TNF-alpha) production and CD83/CD86 up-regulation. The purified PGN region was sufficient to activate TLR2 and TLR4 in mouse DCs and macrophages; in TLR2 and TLR4 double-knockout cells the BCG PGN-mediated TNF-alpha production ability was completely impaired. Likewise, stimulation with BCG CWS of HEK293 cells expressing either human TLR2 or TLR4, MD-2, and CD14 resulted in NF-kappa B activation as determined by a reporter assay. Notably, specific blockers of extracellular human TLR2 (an original cocktail of monoclonal antibodies TLR2.45 and TH2.1) and TLR4 (E5531) inhibited BCG CWS-mediated NF-kappa B activation by 80%. Using this human TLR blocking system, we tested whether human myeloid DC maturation was TLR2 and TLR4 dependent. BCG PGN-mediated DC maturation was blocked by 70% by suppression of both TLR2 and TLR4 and by 30 to 40% by suppression of either of these TLRs. Similar but less profound suppression of BCG CWS-mediated DC maturation was observed. Hence, the presence of BCG PGN is a minimal requirement for activation of both TLR2 and TLR4 in human DCs, unlike the presence of PGNs of gram-positive bacteria, which activate only TLR2. Unexpectedly, however, BCG PGN, unlike BCG CWS, barely activated NF-kappa B in HEK293 cells coexpressing TLR2 plus TLR1, TLR2 plus TLR4, TLR2 plus TLR6, or TLR2 plus TLR10, suggesting that PGN receptors other than TLR2 and TLR4 present on human DCs but not on HEK293 cells are involved in TLR signaling for DC activation.     

Human CD56bright and CD56dim natural killer cell subsets respond differentially to direct stimulation with Mycobacterium bovis bacillus Calmette-Guérin

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is capable of directly stimulating several effector functions of human natural killer (NK) cells in the absence of interleukin-12 and professional antigen presenting cells. To assess the contribution of two main human NK-cell subsets (CD56(dim) and CD56(bright)) to the overall in vitro NK-cell response to BCG, peripheral blood mononuclear cells depleted of nylon wool-adherent cells or purified NK cells were stimulated with live BCG. By combining intranuclear bromodeoxyuridine (BrdU) staining and analysis of CD56 marker intensity, statistically higher percentages of BrdU(+) cells were found among the CD56(bright) subset than the CD56(dim) subset after 6 days of stimulation with BCG. Similarly, evaluation of intracellular interferon-gamma (IFN-gamma) revealed that CD56(bright) cells were those mainly involved in IFN-gamma production in response to BCG. In contrast, the CD56(dim) subset contained higher levels of perforin and granzyme A, two key molecules for exocytosis-mediated cytotoxicity, than the CD56(bright) subset. Although 16-20-h stimulation with BCG did not substantially alter the expression of cytotoxic molecules by the two subsets, a decrease in perforin content was observed in the CD56(dim), but not in the CD56(bright) subset, following 4-h incubation with the NK-sensitive target K562 cell line. This decrease in perforin content correlated with the induction by BCG-stimulated NK cells, of early markers of apoptosis on target cells to a greater extent than unstimulated cells suggesting a major role for the CD56(dim) subset in cytotoxic activity in response to BCG. Taken together, these results demonstrate that CD56(bright) and CD56(dim) human NK-cell subsets exert different functional activities in response to a live bacterial pathogen.     

Recombinant Mycobacterium bovis bacillus Calmette-Guérin (BCG) expressing mouse IL-18 augments Th1 immunity and macrophage cytotoxicity

Interleukin-18 (IL-18) has been demonstrated to synergize with BCG for induction of a T-helper-type 1 (Th1) immune response. Since successful treatment of superficial bladder cancer with BCG requires proper induction of Th1 immunity, we have developed a recombinant (r) BCG strain that functionally secretes mouse (m) IL-18. This rBCG-mIL-18 strain significantly increased production of the major Th1 cytokine IFN-gamma in splenocyte cultures, at levels comparable to that elicited by control BCG plus exogenous rIL-18. IFN-gamma production by splenocytes was eliminated by addition of neutralizing anti-IL-18 antibody. Endogenous IL-12 played a favourable role whereas IL-10 played an adverse role in rBCG-mIL-18-induced IFN-gamma production. Enhanced host antimycobacterial immunity was observed in mice infected with rBCG-mIL-18 which showed less splenic enlargement and reduced bacterial load compared to control mice infected with BCG. Further, splenocytes from rBCG-mIL-18-infected mice, in response to BCG antigen, displayed increased production of IFN-gamma and GMCSF, decreased production of IL-10, elevated cellular proliferation and higher differentiation of IFN-gamma-secreting cells. rBCG-mIL-18 also enhanced BCG-induced macrophage cytotoxicity against bladder cancer MBT-2 cells in a dose-dependent manner. Neutralizing all endogenous macrophage-derived cytokines tested (IL-12, IL-18 and TNF-alpha) as well as IFN-gamma severely diminished the rBCG-mIL-18-induced macrophage cytolytic activity, indicating a critical role for these cytokines in this process. Cytokine analysis for supernatants of macrophage-BCG mixture cultures manifested higher levels of IFN-gamma and TNF-alpha in rBCG-mIL-18 cultures than in control BCG cultures. Taken together, this rBCG-mIL-18 strain augments BCG's immunostimulatory property and may serve as a better agent for bladder cancer immunotherapy and antimycobacterial immunization.     

Functional characterization of human natural killer cells responding to Mycobacterium bovis bacille Calmette-Guérin

The kinetics of activation and induction of several effector functions of human natural killer (NK) cells in response to Mycobacterium bovis bacille Calmette-Guérin (BCG) were investigated. Owing to the central role of monocytes/macrophages (MM) in the initiation and maintenance of the immune response to pathogens, two different experimental culture conditions were analysed. In the first, monocyte-depleted nylon wool non-adherent (NW) cells from healthy donors were stimulated with autologous MM preinfected with BCG (intracellular BCG). In the second, the NW cells were directly incubated with BCG, which was therefore extracellular. In the presence of MM, CD4+ T lymphocytes were the cell subset mainly expressing the activation marker, CD25, and proliferating with a peak after 7 days of culture. In contrast, in response to extracellular BCG, the peak of the proliferative response was observed after 6 days of stimulation, and CD56+ CD3- cells (NK cells) were the cell subset preferentially involved. Such proliferation of NK cells did not require a prior sensitization to mycobacterial antigens, and appeared to be dependent upon contact between cell populations and bacteria. Following stimulation with extracellular BCG, the majority of interferon-gamma (IFN-gamma)-producing cells were NK cells, with a peak IFN-gamma production at 24-30 hr. Interleukin (IL)-2 and IL-4 were not detectable in NK cells or in CD3+ T lymphocytes at any time tested. IL-12 was not detectable in the culture supernatant of NW cells stimulated with extracellular BCG. Compared to the non-stimulated NW cells, the NW cells incubated for 16-20 hr with BCG induced the highest levels of expression of apoptotic/death marker on the NK-sensitive K562 cell line. BCG also induced expression of the activation marker, CD25, and proliferation, IFN-gamma production and cytotoxic activity, on negatively selected CD56+ CD3- cells. Altogether, the results of this study demonstrate that extracellular mycobacteria activate several NK-cell functions and suggest a possible alternative mechanism of NK-cell activation as the first line of defence against mycobacterial infections.     

Field Evaluation of the Efficacy of Mycobacterium bovis Bacillus Calmette-Guérin against Bovine Tuberculosis in Neonatal Calves in Ethiopia

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.Bovine tuberculosis (bTB), caused mainly by Mycobacterium bovis, is characterized by the formation of granulomatous lesions primarily in the lymph nodes (LNs) and lungs. More than 50 million cattle are infected with M. bovis, resulting in economic losses of approximately $3 billion annually worldwide (19), and in developing countries, M. bovis infection is thought to account for 5 to 15% of human TB (2). Conventionally, the control of bTB is based on a test and slaughter strategy, which has reduced the incidence and prevalence of the disease in developed countries, except in those with wildlife reservoirs such as the United Kingdom and New Zealand. However, this method is too costly to be applicable to most of the developing world. Vaccination of cattle represents an alternative intervention strategy to reduce the impact of bTB on livestock productivity and human health in developing countries. The only currently available vaccine against tuberculosis is the human vaccine M. bovis bacillus Calmette-Guérin (BCG). BCG has been used in cattle in a large number of experiments and trials with variable efficacies (reviewed by Buddle et al. [8]). A series of field trials in East Germany and Malawi found no evidence of protection (3, 4, 11). In contrast, field trials in Malawi and Madagascar reported protective efficacies of 25% and 45%, respectively (9, 23), and other workers have reported 50% protection in field trials in Malawi and the United Kingdom, as well as New Zealand (10, 25). Higher levels of protection have been reported in the United Kingdom by Vordermeier et al. (20). Experimental infection studies over the last 15 years using intratracheal or aerosol challenge routes have optimized the use of BCG in cattle and reaffirmed the ability of BCG to protect cattle against bTB (5, 6, 24). However, few recent studies have evaluated the ability of BCG to protect cattle in a more natural cattle-to-cattle transmission setting using naturally infected donor cattle as the source of infection.In this study, we assessed the efficacy of neonatal BCG vaccination in a natural transmission setting by introducing BCG-vaccinated and control calves into a herd composed of tuberculin skin test reactor animals in a farm in central Ethiopia. The farm was managed under routine Ethiopian intensive husbandry conditions, and protective efficacy was determined after 10 to 23 months in contact with infected animals by postmortem examination and M. bovis culture. We also evaluated whether prototype reagents for the differential diagnosis of infected and vaccinated animals (DIVA reagents) were able to distinguish vaccinated and protected animals from those that were vaccinated but not protected.