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.     

Protection by a Recombinant Mycobacterium bovis Bacillus Calmette-Guérin Vaccine Expressing Shiga Toxin 2 B Subunit against Shiga Toxin-Producing Escherichia coli in Mice

We have developed a novel vaccine against Shiga toxin (Stx)-producing Escherichia coli (STEC) infection using a recombinant Mycobacterium bovis BCG (rBCG) system. Two intraperitoneal vaccinations with rBCG expressing the Stx2 B subunit (Stx2B) resulted in an increase of protective serum IgG and mucosal IgA responses to Stx2B in BALB/c mice. When orally challenged with 10³ CFU of STEC strain B2F1 (O91: H21), the immunized mice survived statistically significantly longer than the nonvaccinated mice. We suggest that intraperitoneal immunization with rBCG expressing Stx2B would be a potential vaccine strategy for STEC.     

A Single Dose of a DNA Vaccine Encoding Apa Coencapsulated with 6,6′-Trehalose Dimycolate in Microspheres Confers Long-Term Protection against Tuberculosis in Mycobacterium bovis BCG-Primed Mice

Mycobacterium bovis BCG prime DNA (Mycobacterium tuberculosis genes)-booster vaccinations have been shown to induce greater protection against tuberculosis (TB) than BCG alone. This heterologous prime-boost strategy is perhaps the most realistic vaccination for the future of TB infection control, especially in countries where TB is endemic. Moreover, a prime-boost regimen using biodegradable microspheres seems to be a promising immunization to stimulate a long-lasting immune response. The alanine proline antigen (Apa) is a highly immunogenic glycoprotein secreted by M. tuberculosis. This study investigated the immune protection of Apa DNA vaccine against intratracheal M. tuberculosis challenge in mice on the basis of a heterologous prime-boost regimen. BALB/c mice were subcutaneously primed with BCG and intramuscularly boosted with a single dose of plasmid carrying apa and 6,6′-trehalose dimycolate (TDM) adjuvant, coencapsulated in microspheres (BCG-APA), and were evaluated 30 and 70 days after challenge. This prime-boost strategy (BCG-APA) resulted in a significant reduction in the bacterial load in the lungs, thus leading to better preservation of the lung parenchyma, 70 days postinfection compared to BCG vaccinated mice. The profound effect of this heterologous prime-boost regimen in the experimental model supports its development as a feasible strategy for prevention of TB.     

CD30 Is Required for Activation of a Unique Subset of Interleukin-17A-Producing γδ T Cells in Innate Immunity against Mycobacterium bovis Bacillus Calmette-Guérin Infection

Interleukin-17A (IL-17A)-producing γδ T cells are known to be activated following Mycobacterium bovis bacillus Calmette-Guérin (BCG) infection. Here, we show that CD30, a member of the tumor necrosis factor (TNF) receptor superfamily, is important for activation of IL-17A-producing γδ T cells after BCG infection. Vγ1⁻ Vγ4⁻ γδ T cells preferentially expressing Vγ6/Vδ1 genes were identified as the major source of IL-17A in the peritoneal cavity during the early stage of BCG infection. The number of IL-17A-producing Vγ1⁻ Vγ4⁻ γδ T cells bearing Vγ6 increased in peritoneal exudate cells (PEC) of wild-type (WT) mice but not in those of CD30 knockout (KO) mice in response to BCG infection. Consistently, CD30 ligand (CD30L) or CD30 expression, predominantly by Vγ1⁻ Vγ4⁻ γδ T cells, was rapidly upregulated after BCG infection. Inhibition of CD30L/CD30 signaling by in vivo administration of a soluble CD30 and immunoglobulin fusion protein (CD30-Ig) severely impaired activation of IL-17A-producing Vγ1⁻ Vγ4⁻ γδ T cells in WT mice, while stimulating CD30L/CD30 signaling by in vivo administration of agonistic anti-CD30 monoclonal antibody (MAb) restored IL-17A production by Vγ1⁻ Vγ4⁻ γδ T cells in CD30L KO mice after BCG infection. These results suggest that CD30 signaling plays an important role in the activation of IL-17A-producing Vγ1⁻ Vγ4⁻ γδ T cells bearing Vγ6 at an early stage of BCG infection.