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Bacillus Calmette-Guérin (BCG) is a routinely used vaccine for protecting children against Mycobacterium tuberculosis that comprises attenuated Mycobacterium bovis . BCG can also be used to protect livestock against M. bovis ; however, its effectiveness has not been quantified for this use. We performed a natural transmission experiment to directly estimate the rate of transmission to and from vaccinated and unvaccinated calves over a 1-year exposure period. The results show a higher indirect efficacy of BCG to reduce transmission from vaccinated animals that subsequently become infected [74%; 95% credible interval (CrI): 46 to 98%] compared with direct protection against infection (58%; 95% CrI: 34 to 73%) and an estimated total efficacy of 89% (95% CrI: 74 to 96%). A mechanistic transmission model of bovine tuberculosis (bTB) spread within the Ethiopian dairy sector was developed and showed how the prospects for elimination may be enabled by routine BCG vaccination of cattle.

Mycobacterium bovis bacille Calmette-Guérin (BCG), an experimental vaccine designed to protect cattle from bovine tuberculosis, was administered for the first time to a newborn baby in Paris in 1921. Over the past century, BCG has saved tens of millions of lives and has been given to more humans than any other vaccine. It remains the sole tuberculosis vaccine licensed for use in humans. BCG provides long-lasting strong protection against miliary and meningeal tuberculosis in children, but it is less effective for the prevention of pulmonary tuberculosis, especially in adults. Evidence mainly from the past two decades suggests that BCG has non-specific benefits against non-tuberculous infections in newborn babies and in older adults, and offers immunotherapeutic benefit in certain malignancies such as non-muscle invasive bladder cancer. However, as a live attenuated vaccine, BCG can cause localised or disseminated infections in immunocompromised hosts, which can also occur following intravesical installation of BCG for the treatment of bladder cancer. The legacy of BCG includes fundamental discoveries about tuberculosis-specific and non-specific immunity and the demonstration that tuberculosis is a vaccine-preventable disease, providing a foundation for new vaccines to hasten tuberculosis elimination., Competing Interests: Declaration of interests SHEK is coinventor of the tuberculosis vaccine VPM1002 and coholder of a patent licensed to Serum Institute of India, Pune, India. MGN is coholder of patents on trained immunity licensed to Trained Therapeutix Discovery. CL reports personal fees from Chiesi, Gilead, Janssen, Novartis, Oxford Immunotec, and Insmed, outside of the submitted work. All other authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

H65, a fusion protein of three pairs of ESX-secreted antigens of Mycobacterium tuberculosis and Mycobacterium bovis, formulated with the liposomal adjuvant CAF01 has been shown to confer protection against M. tuberculosis infection in mice. In this study, we evaluated the impact of combining BCG with H65 + CAF01 immunization in a M. bovis mouse model of infection. We found that a BCG-H65 + CAF01/ H65 + CAF01 prime-boost scheme induced higher protection than BCG and H65 + CAF01 alone. Altogether, H65 antigen formulated in liposomal adjuvant improved the BCG-induced immune protection, thus making this vaccine strategy a promising tool to control bovine tuberculosis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Mycobacterium bovis (M. bovis) is a member of mycobacterium tuberculosis complex (MTBC), and a causative agent of chronic respiratory disease in a wide range of hosts. Bacillus Calmette-Guerin (BCG) vaccine is mostly used for the prevention of childhood tuberculosis. Further substantial implications are required for the development and evaluation of new tuberculosis (TB) vaccines as well as improving the role of BCG in TB control strategies. In this study, we prepared PLGA nanoparticles encapsulated with argF antigen (argF-NPs). We hypothesized, that argF nanoparticles mediate immune responses of BCG vaccine in mice models of M. bovis infection. We observed that mice vaccinated with argF-NPs exhibited a significant increase in secretory IFN-γ, CD4+ T cells response and mucosal secretory IgA against M. bovis infection. In addition, a marked increase was observed in the level of secretory IL-1β, TNF-α and IL-10 both in vitro and in vivo upon argF-NPs vaccination. Furthermore, argF-NPs vaccination resulted in a significant reduction in the inflammatory lesions in the lung's tissues, minimized the losses in total body weight and reduced M. bovis burden in infected mice. Our results indicate that BCG prime-boost strategy might be a promising measure for the prevention against M. bovis infection by induction of CD4 + T cells responses and mucosal antibodies., (Copyright © 2021. Published by Elsevier Masson SAS.)

Vaccination is a useful approach for the control of disease in wildlife populations. However, its effectiveness is dependent in part on delivery to a sufficient proportion of the target population. Measuring the proportions of wild animal populations that have been vaccinated is challenging and so there is a need to develop robust approaches that can contribute to our understanding of the likely efficacy of wildlife vaccination campaigns. We used a modified capture mark recapture technique to estimate vaccine coverage in a wild population of European badgers (Meles meles) vaccinated by live-trapping and injecting with Bacillus Calmette-Guérin as part of a bovine tuberculosis control initiative in Wales, United Kingdom. Our approach used genetic matching of vaccinated animals to a sample of the wider population to estimate the percentage of badgers that had been vaccinated. Individual-specific genetic profiles were obtained using microsatellite genotyping of hair samples, which were collected directly from trapped and vaccinated badgers and non-invasively from the wider population using hair traps deployed at badger burrows (setts). With two nights of trapping at each sett in an annual campaign, an estimated 50 % (95 % confidence interval 40-60 %) of the badger population received at least one dose of vaccine in a single year. Using a simple population model this suggested that the proportion of the population that would have received at least one dose of vaccine over the course of the four year vaccination campaign was between 67 % and 83 %. This is the first attempt, outside of field trials, to quantify the level of vaccine coverage achieved by trapping and injecting badgers, which is currently the only option for delivering BCG vaccine to this species. The results therefore have specific application to bTB control policy and the novel approach may have wider value in wildlife management and research., (Crown Copyright © 2020. Published by Elsevier B.V. All rights reserved.)

Bovine tuberculosis is an important animal health problem and the predominant cause of zoonotic tuberculosis worldwide. It results in serious economic burden due to losses in productivity and the cost of control programmes. Control could be greatly improved by the introduction of an efficacious cattle vaccine but the most likely candidate, BCG, has several limitations including variable efficacy. Augmentation of BCG with a subunit vaccine booster has been shown to increase protection but the selection of antigens has hitherto been left largely to serendipity. In the present study, we take a rational approach to identify the protective antigens of BCG, selecting a BCG transposon mutant library in naïve and BCG-vaccinated cattle. Ten mutants had increased relative survival in vaccinated compared to naïve cattle, consistent with loss of protective antigen targets making the mutants less visible to the BCG immune response. The immunogenicity of three putative protective antigens, BCG_0116, BCG_0205 (YrbE1B) and BCG_1448 (PPE20) was investigated using peptide pools and PBMCs from BCG vaccinated cattle. BCG vaccination induced PBMC to release elevated levels of IP10, IL-17a and IL-10 in response to all three antigens. Taken together, the data supports the further study of these antigens for use in subunit vaccines., (Copyright © 2020. Published by Elsevier Ltd.)

Bovine tuberculosis (BTB) remains as a costly disease of cattle-herds in the Republic of Ireland (ROI). This persistence is partially attributable to the presence of M. bovis infection in a wildlife reservoir, the European badger (Meles meles). Thus, both area-wide and limited-area targeted-badger-culling have been part of the ROI-BTB control/eradication program to help reduce the future incidence of a cattle-herd BTB breakdown (i.e. a "new herd-level occurrence of BTB"). However, neither badger-culling practice can be sustained as a major component in the ongoing BTB eradication program in the ROI. Vaccination of badgers with Bacille Calmette-Guérin (BCG) has been proposed as an alternative to badger culling. Thus, in 2011, a five-year non-inferiority study was implemented in seven counties in the ROI. This study was designed to compare and contrast the cattle-herd-BTB-incidence in areas where intramuscular badger vaccination would be implemented versus the cattle-herd-BTB-incidence in the remaining area of the same county where targeted-badger-culling was maintained as the standard treatment response to probable badger-sourced BTB breakdowns. Our outcome of interest was a new cattle-herd-BTB-episode (breakdown) with a total of >2 standard skin-test (SICTT) reactors detected during the episode. Treatments (badger vaccination or targeted badger culling) were cluster allocated based on where the majority of the herd owner's land was located. To assess the impact of the two treatments, we compared the incidence-risk, of our defined outcome, for cattle herds in the area under vaccination to the outcome incidence-risk for cattle herds in the remainder of the same county after 4 and 5 years of having implemented badger vaccination. A random-effects logit model with adjustment for clustering by treatment, and statistical control of herd-type, herd-size and five-year prior-BTB-episode history was used for our analyses. Although not included in the logistic model, a relative badger density metric based on the annual number of badgers captured-per-sett-night of capturing effort was developed for each treatment area; this metric indicated that relative badger density was approximately 40 % higher in vaccination areas than in the targeted badger-culling areas during our study. Overall, our study results indicated that vaccination was not inferior to targeted badger-culling in four counties and badger vaccination was deemed to produce ambivalent results in one (County Cork North) of the seven study sites in the ROI. A post-study investigation, in County Galway, where vaccination was deemed inferior to target culling, revealed that widespread purchases of cattle from a nearby cattle mart, by herd owners in the vaccination-area, was associated with the increased herd and vaccination-area risk of BTB. No single "biasing hypothesis" was evident for the apparent vaccine inferiority in the second study site (County Monaghan) where vaccination was deemed inferior to targeted culling; hence no further investigations were conducted., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

In the absence of biomarkers of protective immunity, newly developed vaccines against bovine tuberculosis need to be evaluated in virulent Mycobacterium bovis challenge experiments, which require the use of expensive and highly in demand Biological Safety Level 3 (BSL3) animal facilities. The recently developed bovine BCG challenge model offers a cheaper and faster way to test new vaccine candidates and additionally reduces the severity of the challenge compared to virulent M. bovis challenge in line with the remits of the NC3Rs. In this work we sought to establish the sensitivity of the BCG challenge model by testing a prime boost vaccine regimen that previously increased protection over BCG alone against M. bovis challenge. All animals, except the control group, were vaccinated subcutaneously with BCG Danish, and half of those were then boosted with a recombinant adenoviral vector expressing Antigen 85A, Ad85A. All animals were challenged with BCG Tokyo into the prescapular lymph node and the bacterial load within the lymph nodes was established. All vaccinated animals, independent of the vaccination regimen, cleared BCG significantly faster from the lymph node than control animals, suggesting a protective effect. There was however, no difference between the BCG and the BCG-Ad85A regimens. Additionally, we analysed humoral and cellular immune responses taken prior to challenge for possible predictors of protection. Cultured ELISpot identified significantly higher IFN-ɣ responses in protected vaccinated animals, relative to controls, but not in unprotected vaccinated animals. Furthermore, a trend for protected animals to produce more IFN-ɣ by quantitative PCR and intracellular staining was observed. Thus, this model can also be an attractive alternative to M. bovis challenge models for the discovery of protective biomarkers., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)

Bacillus Calmette Guérin (BCG) is the only currently available vaccine against tuberculosis (TB), but it confers incomplete and variable protection against pulmonary TB in humans and bovine TB (bTB) in cattle. Insights into the immune response induced by BCG offer an underexploited opportunity to gain knowledge that may inform the design of a more efficacious vaccine, which is urgently needed to control these major global epidemics. Humoral immunity in TB and bTB has been neglected, but recent studies supporting a role for antibodies in protection against TB has driven a growing interest in determining their relevance to vaccine development. In this manuscript we review what is known about the humoral immune response to BCG vaccination and re-vaccination across species, including evidence for the induction of specific B cells and antibodies; and how these may relate to protection from TB or bTB. We discuss potential explanations for often conflicting findings and consider how factors such as BCG strain, manufacturing methodology and route of administration influence the humoral response. As novel vaccination strategies include BCG prime-boost regimens, the literature regarding off-target immunomodulatory effects of BCG vaccination on non-specific humoral immunity is also reviewed. Overall, reported outcomes to date are inconsistent, but indicate that humoral responses are heterogeneous and may play different roles in different species, populations, or individual hosts. Further study is warranted to determine whether a new TB vaccine could benefit from the targeting of humoral as well as cell-mediated immunity.

The global burden of bovine tuberculosis (bTB) remains poorly characterized, with spill-over impacts on multiple species. The "One Health" concept is especially relevant given the bidirectional risk of cattle infecting humans with Mycobacterium bovis and humans infecting cattle with Mycobacterium tuberculosis. "Test and cull" is the traditional bTB control method, but the strategy may not be economically feasible or culturally acceptable where cattle are highly prized or their killing is a religious taboo; it is also less effective when there are wildlife reservoirs of infection. Vaccination with M. bovis bacille Calmette-Guerin (BCG) provides protection against bTB, but its use in animals has been limited. The Jerusalem One Health workshop considered key bTB knowledge gaps and innovative solutions. Knowledge gaps identified included (a) the poorly quantified prevalence of M. bovis infection and disease in cattle, domestic camelids and human populations in developing countries, (b) the absence of alternatives to a "test and cull" strategy in settings where the killing of infected animals is culturally or economically unacceptable, or where affected species are protected and (c) an understanding of the induction of mucosal immunity against bTB. We summarize discussions on the use of BCG vaccination in domestic animals and wildlife and list potential projects to address the knowledge gaps identified., (© 2018 Blackwell Verlag GmbH.)

Vaccination of cattle with Mycobacterium bovis BCG has been shown to protect against infection with virulent strains of M. bovis, and against resultant bovine tuberculosis (TB). Here we report on a large-scale trial in New Zealand where free-ranging cattle were vaccinated with 3 x 10 5 BCG via injection, a lower dose than any previously trialed in cattle against exposure to a natural force of M. bovis infection. In a multi-year enrolment study involving >800 animals, three cohorts of 1-2 year old cattle were randomised to receive vaccine or to serve as non-vaccinated controls. Cattle were slaughtered and subject to standard abattoir post mortem examination for M. bovis culture-positive TB lesions after up to 3.7 years of in-field exposure; additionally, lymph node samples from approximately half of the cattle were examined further to identify infection in the absence of lesions. Overall TB prevalence, as identified by gross lesions detected at slaughter, was low among farmed cattle at the study site (<4% annually). There were two lesioned cases among 520 vaccinated trial cattle (0.38%) compared to eight among 297 non-vaccinated trial cattle (2.69%). Trial vaccine efficacy was 85.7% against abattoir-detectable TB (statistically significant protection), and 86.7% when adjusted for duration of exposure. BCG vaccination did not significantly affect the response rates of cattle to ante mortem skin- or blood-tests in diagnostic tests conducted >7 months post-vaccination. Use of a reduced, yet effective, dose of BCG would increase the cost effectiveness of using this vaccine in a bovine TB control programme., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

European badgers (Meles meles) are a wildlife reservoir for Mycobacterium bovis (M. bovis) in parts of England, Wales and Ireland, constituting a potential source of tuberculosis (TB) infection for cattle. Vaccination of badgers against TB is one of the tools available for helping reduce the prevalence of bovine TB in badgers, made possible by the licensing in 2010 of Bacillus Calmette-Guérin (BCG) vaccine for intramuscular administration to badgers (BadgerBCG). However, practical limitations associated with administering an injected vaccine to wild animals make an oral, bait-delivered form of the vaccine highly desirable. Evaluation of the safety of oral BCG to badgers and the environment is a mandatory step on the road to licensing an oral vaccine. This study had the following objectives: (a) to determine whether adverse effects followed the oral administration of BCG vaccine to badgers; (b) to measure the quantity and frequency of BCG excreted in the faeces of vaccinated badgers; and (c) to assess whether there was evidence of the vaccine spreading to unvaccinated, 'sentinel' badgers sharing the same environment as vaccinated animals. We report here that the oral administration per badger of ≥6.4 × 10 9  cfu BCG, followed 14 days later by a single oral dose of ≥6.4 × 10 7  cfu BCG caused no adverse physical effects and did not affect the social behaviour and feeding habits of the vaccinated animals. BCG was cultured from the faeces of two of nine vaccinated animals (372 cfu/g and 996 cfu/g, respectively) approximately 48 h after the higher dose of BCG was administered and by one of the nine vaccinated animal (80 cfu/g) approximately 24 h after receiving the lower dose of BCG. We found no evidence for the transmission of BCG to unvaccinated, sentinel, badgers housed with the vaccinated animals despite the occasional excretion of BCG in faeces., (Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.)

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 < .001) with 56%, 43%, 72%, and 38% reductions in the proportion of lesioned tisues in head, thoracic, abdominal lymph nodes, and lungs, respectively (p-values .029-.0001). In addition, the lesions were less severe grossly and microscopically in vaccinated calves than in non-vaccinated calves (p < .05). The reduction in the overall incidence rates of bTB was 23%, 28%, and 33% on the basis of the absence of gross pathology, M. bovis culture positivity, and histopathology, respectively, in vaccinated animals. In conclusion, BCG vaccination reduced the frequency and severity of the pathology of bTB significantly, which is likely to reduce onwards transmission of the disease., (© 2017 The Authors. Transboundary and Emerging Diseases Published by Blackwell Verlag GmbH.)

In the UK and Ireland, Bacille Calmette-Guérin (BCG) vaccination of badgers has been suggested as one of a number of strategies to control or even eradicate Mycobacterium bovis infection in badgers. In this manuscript, we present the results of a badger field trial conducted in Ireland and discuss how the novel trial design and analytical methods allowed the effects of vaccination on protection against infection and, more importantly, on transmission to be estimated. The trial area was divided into three zones North to South (A, B and C) where vaccination coverages of 0, 50 and 100%, respectively, were applied. Badgers were trapped over a 4year period. Badgers were assigned to either placebo or vaccine treatment, with treatment allocation occurring randomly in zone B. Blood samples were collected at each capture, and serology was performed in these samples using a chemiluminescent multiplex ELISA system (Enfer test). The analysis aimed to compare new infections occurring in non-infected non-vaccinated badgers to those in non-infected vaccinated ones, while accounting for the zone in which the badger was trapped and the infection pressure to which this individual badger was exposed. In total, 440 records on subsequent trappings of individual non-infected badgers were available for analysis. Over the study period, 55 new infections occurred in non-vaccinated (out of 239=23.0%) and 40 in vaccinated (out of 201=19.9%) badgers. A Generalized Linear Model (GLM) with a cloglog link function was used for analysis. Statistical analysis showed that susceptibility to natural exposure with M. bovis was reduced in vaccinated compared to placebo treated badgers: vaccine efficacy for susceptibility, VE S , was 59% (95% CI=6.5%-82%). However, a complete lack of effect from BCG vaccination on the infectivity of vaccinated badgers was observed, i.e. vaccine efficacy for infectiousness (VE I ) was 0%. Further, the basic reproduction ratio as a function of vaccination coverage (p) (i.e. R(p)) was estimated. Given that the prevalence of M. bovis infection in badgers in endemic areas in Ireland is approximately 18%, we estimated the reproduction ratio in the unvaccinated population as R(0)=1.22. Because VE S was now known, the reproduction ratio for a fully vaccinated population was estimated as R(1)=0.50. These results imply that with vaccination coverage in badgers exceeding 30%, eradication of M. bovis in badgers in Ireland is feasible, provided that the current control measures also remain in place., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Cattle vaccination against bovine tuberculosis (bTB) has been proposed as a supplementary method to help control the incidences of this disease. Bacillus Calmette-Guérin (BCG) is currently the only viable candidate vaccine for immunization of cattle against bTB, caused by Mycobacterium bovis (M. bovis). In an attempt to characterize the differences in the immune response following M. bovis infection between BCG-vaccinated and non-vaccinated animals, a combination of gross pathology, histopathology and immunohistochemical (IHC) analyses was used. BCG vaccination was found to significantly reduce the number of gross and microscopic lesions present within the lungs and lymph nodes. Additionally, the microscopically visible bacterial load of stages III and IV granulomas was reduced. IHC using cell surface markers revealed the number of CD68+ (macrophages), CD3+ (T lymphocytes) and WC1+ cells (γδ T cells) to be significantly reduced in lymph node granulomas of BCG-vaccinated animals, when compared to non-vaccinated animals. B lymphocytes (CD79a+) were significantly increased in BCG-vaccinated cattle for granulomas at stages II, III and IV. IHC staining for iNOS showed a higher expression in granulomas from BCG-vaccinated animals compared to non-vaccinated animals for all stages, being statistically significant in stages I and IV. TGFβ expression decreased alongside the granuloma development in non-vaccinated animals, whereas BCG-vaccinated animals showed a slight increase alongside lesion progression. IHC analysis of the cytokines IFN-γ and TNF-α demonstrated significantly increased expression within the lymph node granulomas of BCG-vaccinated cattle. This is suggestive of a protective role for IFN-γ and TNF-α in response to M. bovis infection. Findings shown in this study suggest that the use of BCG vaccine can reduce the number and severity of lesions, induce a different phenotypic response and increase the local expression of key cytokines related to protection., (© 2016 Crown copyright.)

Conventional control and eradication strategies for bovine tuberculosis (BTB) face tremendous difficulties in developing countries; countries with wildlife reservoirs, a complex wildlife-livestock-human interface or a lack of veterinary and veterinary public health surveillance. Vaccination of cattle and other species might in some cases provide the only suitable control strategy for BTB, while in others it may supplement existing test-and-slaughter schemes. However, the use of live BCG has several limitations and the global rise of HIV/AIDS infections has furthermore warranted the exploration of inactivated vaccine preparations. The aim of this study was to compare the immune response profiles in response to parenteral vaccination with live BCG and two inactivated vaccine candidates in cattle. Twenty-four mixed breed calves (Bos taurus) aged 4-6 months, were allocated to one of four groups and vaccinated sub-cutaneously with live M. bovis BCG (Danish 1331), formalin-inactivated M. bovis BCG, heat-killed M. bovis or PBS/Montanide™ (control). Interferon-γ responsiveness and antibody production were measured prior to vaccination and at weekly intervals thereafter for twelve weeks. At nine weeks post-priming, animals were skin tested using tuberculins and MTBC specific protein cocktails and subsequently challenged through intranodular injection of live M. bovis BCG. The animals in the heat-killed M. bovis group demonstrated strong and sustained cell-mediated and humoral immune responses, significantly higher than the control group in response to vaccination, which may indicate a protective immune profile. Animals in this group showed reactivity to the skin test reagents, confirming good vaccine take. Lastly, although not statistically significant, recovery of BCG after challenge was lowest in the heat-killed M. bovis group. In conclusion, the parenteral heat-killed M. bovis vaccine proved to be clearly immunogenic in cattle in the present study, urging further evaluation of the vaccine in challenge studies using virulent M. bovis and assessment of vaccine efficacy in field conditions.

Human tuberculosis remains a significant cause of mortality and morbidity throughout the world. The global economic impact of bovine TB is considerable. An effective vaccine would be the most cost-effective way to control both epidemics, particularly in emerging economies. TB vaccine research would benefit from the identification of an immune correlate of protection with which vaccines could be gated at both preclinical and clinical levels. In-vitro mycobacterial growth inhibition assays (MGIA) are functional assays that include most aspects of the complex host immune response to mycobacteria, and they may serve as functional immune correlates for vaccine development. We applied to cattle an MGIA that was developed for use with human and murine samples. Several technical difficulties were encountered while transferring it to the cattle model. However, our data demonstrate that the assay was not discriminatory in cattle and further work is needed before using it for bovine TB vaccine development., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Vaccination of cattle against bovine tuberculosis could be a valuable control strategy, particularly in countries faced with intractable ongoing infection from a disease reservoir in wildlife. A field vaccination trial was undertaken in New Zealand. The trial included 1286 effectively free-ranging cattle stocked at low densities in a remote 7600ha area, with 55% of them vaccinated using Mycobacterium bovis BCG (Danish strain 1311). Vaccine was administered orally in all but 34 cases (where it was injected). After inclusion, cattle were exposed to natural sources of M. bovis infection in cattle and wildlife, most notably the brushtail possum (Trichosurus vulpecula). Cattle were slaughtered at 3-5 years of age and were inspected for tuberculous lesions, with mycobacteriological culture of key tissues from almost all animals. The prevalence of M. bovis infection was 4.8% among oral BCG vaccinates, significantly lower than the 11.9% in non-vaccinates. Vaccination appeared to both reduce the incidence of detectable infection, and to slow disease progression. Based on apparent annual incidence, the protective efficacy of oral BCG vaccine was 67.4% for preventing infection, and was higher in cattle slaughtered soon after vaccination. Skin-test reactivity to tuberculin was high in vaccinates re-tested 70days after vaccination but not in non-vaccinates, although reactor animals had minimal response in gamma-interferon blood tests. In re- tests conducted more than 12 months after vaccination, skin-test reactivity among vaccinates was much lower. These results indicate that oral BCG vaccination could be an effective tool for greatly reducing detectable infection in cattle., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The control of tuberculosis (TB) in cattle in the UK and Ireland is compromised by transmission of Mycobacterium bovis to cattle from the European badger (Meles meles), which acts as a wildlife reservoir. Vaccination of badgers could potentially contribute to TB control but the only licensed vaccine is injectable BadgerBCG which requires the live-capture of badgers. Current research is aimed at developing an oral TB vaccine (where vaccine is contained within bait) that is intended to be more cost-effective to deploy over large areas. In order to identify a lead product, candidate baits identified from captive badger studies were evaluated in three successive bait screening studies with wild badgers. A fourth field study, using the lead candidate bait and biomarkers, investigated the effectiveness of different carriers for their potential to deliver liquid payloads (vaccine surrogate). In each field study, bait disappearance was monitored daily for ten days and remote video surveillance was used to determine preference (i.e. the order in which baits were taken). In the carrier study, biomarkers were used to determine what proportion of subsequently trapped badgers had ingested the bait and the vaccine-carrier biomarker payload. Across all four studies, 79% (3397/4330) of baits were taken by badgers although the number varied significantly by badger social group and bait type. In all studies, bait disappearance increased over time, with 75-100% of baits being taken by day ten. In the carrier study, 75% (9/12) of trapped badgers tested positive for at least one of the biomarkers and the type of carrier did not influence bait attractiveness. Together with data from complementary laboratory and captive animal studies, this study identified a highly attractive and palatable bait (peanut-based paste bait; PT) and vaccine-carrier (hydrogenated peanut oil; HPO) combination with the potential to deliver a liquid vaccine to wild badgers., (Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.)

Bovine tuberculosis is a zoonotic disease of global public health concern. Development of diagnostic tools to improve test accuracy and efficiency in domestic livestock and enable surveillance of wildlife reservoirs would improve disease management and eradication efforts. Use of volatile organic compound analysis in breath and fecal samples is being developed and optimized as a means to detect disease in humans and animals. In this study we demonstrate that VOCs present in fecal samples can be used to discriminate between non-vaccinated and BCG-vaccinated cattle prior to and after Mycobacterium bovis challenge.

The oral vaccination of wild badgers (Meles meles) with live Bacillus Calmette-Guérin (BCG) is one of the tools being considered for the control of bovine tuberculosis (caused by Mycobacterium bovis) in the UK. The design of a product for oral vaccination requires that numerous, and often competing, conditions are met. These include the need for a highly palatable, but physically stable bait that will meet regulatory requirements, and one which is also compatible with the vaccine formulation; in this case live BCG. In collaboration with two commercial bait companies we have developed a highly attractive and palatable bait recipe designed specifically for European badgers (Meles meles) that meets these requirements. The palatability of different batches of bait was evaluated against a standardised palatable control bait using captive badgers. The physical properties of the bait are described e.g. firmness and colour. The microbial load in the bait was assessed against European and US Pharmacopoeias. The bait was combined with an edible vaccine carrier made of hydrogenated peanut oil in which BCG vaccine was stable during bait manufacture and cold storage, demonstrating <0.5 log 10 reduction in titre after 117weeks' storage at -20°C. BCG stability in bait was also evaluated at +4°C and under simulated environmental conditions (20°C, 98% Relative Humidity; RH). Finally, iophenoxic acid biomarkers were utilised as a surrogate for the BCG vaccine, to test variants of the vaccine-bait design for their ability to deliver biomarker to the gastrointestinal tract of individual animals. These data provide the first detailed description of a bait-vaccine delivery system developed specifically for the oral vaccination of badgers against Mycobacterium bovis using live BCG., (Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.)

In the UK and the Republic of Ireland, the European badger (Meles meles) is a maintenance host for Mycobacterium bovis, and may transmit the infection to cattle causing bovine tuberculosis (TB). Vaccination of badgers using an injectable Bacillus Calmette-Guerin (BCG) vaccine is undertaken in some areas of the UK with the intention of interrupting this transmission, and vaccination research is underway in Ireland. An oral badger TB vaccine is also under development. We investigated the behaviour of badgers and non-target wildlife species towards three candidate baits being considered for delivering BCG to badgers orally. Bait preference was investigated by recording removal rates of baits and through the use of video surveillance at 16 badger setts. We found high variation in rates of bait removal by badgers among setts but no significant differences in removal rates among bait types or in preference behaviour from video footage. Variation in bait removal among setts correlated with the number of nights on which badgers were seen at the sett, with most baits being removed where badgers were seen on >50% of nights during the ten-day study period. Relatively few baits were removed at setts with low levels of recorded badger activity. Monitoring badger activity prior to bait deployment may therefore be useful in increasing bait uptake and vaccine coverage. Bait removal by badgers increased over the ten-day study period, suggesting initial neophobic behaviour at some setts and that a period of 'pre-feeding' may be required prior to vaccine deployment. Our results indicate that all three candidate baits are attractive to badgers. Removal of baits by non-target wildlife species was generally low, but varied among bait types, with smaller baits in packaging less likely to be removed. Enclosing baits in packaging is likely to deter non-target species, although in some cases non-target species did remove up to 13% of packaged baits., (Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.)

Oral-delivery Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine in a lipid matrix has been shown to confer protection against M. bovis infection and reduce the severity of tuberculosis (TB) when fed to brushtail possums (Trichosurus vulpecula), the major wildlife vector of bovine TB in New Zealand. Here we demonstrate the feasibility of aerial delivery of this live vaccine in bait form to an M. bovis-infected wild possum population, and subsequently assess vaccine uptake and field efficacy. Pre-trial studies indicated a resident possum population at very low density (<0.6 possums/ha) at the field site, with a 5.1% prevalence of macroscopic TB lesions. Pilot studies indicated that flavoured lipid matrix baits in weather-proof sachets could be successfully sown aerially via helicopter and were palatable to, and likely to be consumed by, a majority of wild possums under free-choice conditions. Subsequently, sachet-held lipid baits containing live BCG vaccine were sown at 3 baits/ha over a 1360 ha area, equating to >5 baits available per possum. Blood sampling conducted two months later provided some evidence of vaccine uptake. A necropsy survey conducted one year later identified a lower prevalence of culture-confirmed M. bovis infection and/or gross TB lesions among adult possums in vaccinated areas (1.1% prevalence; 95% CI, 0-3.3%, n = 92) than in unvaccinated areas (5.6%; 0.7-10.5%, n = 89); P = 0.098. Although not statistically different, the 81% efficacy in protecting possums against natural infection calculated from these data is within the range of previous estimates of vaccine efficacy in trials where BCG vaccine was delivered manually. We conclude that, with further straightforward refinement to improve free-choice uptake, aerial delivery of oral BCG vaccine is likely to be effective in controlling TB in wild possums. We briefly discuss contexts in which this could potentially become an important complementary tool in achieving national eradication of TB from New Zealand wildlife., Competing Interests: Two of the authors (FA & BB) are named co-inventors of the vaccine that was being trialed in this study. This does not alter these authors’ or any of their co-authors’, adherence to PLOS ONE policies on sharing data and materials.

Boosting BCG using heterologous prime-boost represents a promising strategy for improved tuberculosis (TB) vaccines, and adenovirus (Ad) delivery is established as an efficacious boosting vehicle. Although studies demonstrate that intranasal administration of Ad boost to BCG offers optimal protection, this is not currently possible in cattle. Using Ad vaccine expressing the mycobacterial antigen TB10.4 (BCG/Ad-TB10.4), we demonstrate, parenteral boost of BCG immunised mice to induce specific CD8(+) IFN-γ producing T cells via synergistic priming of new epitopes. This induces significant improvement in pulmonary protection against Mycobacterium bovis over that provided by BCG when assessed in a standard 4week challenge model. However, in a stringent, year-long survival study, BCG/Ad-TB10.4 did not improve outcome over BCG, which we suggest may be due to the lack of additional memory cells (IL-2(+)) induced by boosting. These data indicate BCG-prime/parenteral-Ad-TB10.4-boost to be a promising candidate, but also highlight the need for further understanding of the mechanisms of T cell priming and associated memory using Ad delivery systems. That we were able to generate significant improvement in pulmonary protection above BCG with parenteral, rather than mucosal administration of boost vaccine is critical; suggesting that the generation of effective mucosal immunity is possible, without the risks and challenges of mucosal administration, but that further work to specifically enhance sustained protective immunity is required., (Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.)

Tuberculosis (TB) remains a global pandemic, in both animals and man, and novel vaccines are urgently required. Heterologous prime-boost of BCG represents a promising strategy for improved TB vaccines, with respiratory delivery the most efficacious to date. Such an approach may be an ideal vaccination strategy against bovine TB (bTB), but respiratory vaccination presents a technical challenge in cattle. Inert bacterial spores represent an attractive vaccine vehicle. Therefore we evaluated whether parenterally administered spores are efficacious when used as a BCG boost in a murine model of immunity against Mycobacterium bovis. Here we report the use of heat-killed, TB10.4 adsorbed, Bacillus subtilis spores delivered via subcutaneous injection to boost immunity primed by BCG. We demonstrate that this approach improves the immunogenicity of BCG. Interestingly, this associated with substantial boosting of IL-17 responses; considered to be important in protective immunity against TB. These data demonstrate that parenteral delivery of spores represents a promising vaccine vehicle for boosting BCG, and identifies potential for optimisation for use as a vaccine for bovine TB., (Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.)

Bovine tuberculosis remains a major economic and animal welfare concern worldwide. Cattle vaccination is being considered as part of control strategies. This approach, used alongside conventional control policies, also requires the development of vaccine-compatible diagnostic assays to distinguish vaccinated from infected animals (DIVA). We discuss progress made on optimizing the only potentially available vaccine, bacille Calmette Guérin (BCG), and on strategies to improve BCG efficacy. We also describe recent advances in DIVA development based on the detection of host cellular immune responses by blood-testing or skin-testing approaches. Finally, to accelerate vaccine development, definition of host biomarkers that provide meaningful stage-gating criteria to select vaccine candidates for further testing is highly desirable. Some progress has also been made in this area of research, and we summarize studies that defined either markers predicting vaccine success or markers that correlate with disease stage or severity.

The incidence of bovine tuberculosis (bTB) in the GB has been increasing since the 1980s. Immunisation, alongside current control measures, has been proposed as a sustainable measure to control bTB. Immunisation with Mycobacterium bovis bacillus Calmette-Guerin (BCG) has been shown to protect against bTB. Furthermore, much experimental data indicates that pulmonary local immunity is important for protection against respiratory infections including Mycobacterium tuberculosis and that pulmonary immunisation is highly effective. Here, we evaluated protection against M. bovis, the main causative agent of bTB, conferred by BCG delivered subcutaneously, endobronchially or by the new strategy of simultaneous immunisation by both routes. We also tested simultaneous subcutaneous immunisation with BCG and endobronchial delivery of a recombinant type 5 adenovirus expressing mycobacterial antigen 85A. There was significantly reduced visible pathology in animals receiving the simultaneous BCG/BCG or BCG/Ad85 treatment compared to naïve controls. Furthermore, there were significantly fewer advanced microscopic granulomata in animals receiving BCG/Ad85A compared to naive controls. Thus, combining local and systemic immunisation limits the development of pathology, which in turn could decrease bTB transmission.

White-tailed deer (Odocoileus virginianus) serve as a reservoir for bovine tuberculosis, caused by Mycobacterium bovis, and can be a source of infection in cattle. Vaccination with M. bovis Bacille Calmette Guerin (BCG) is being considered for management of bovine tuberculosis in deer. Presently, no method exists to non-invasively monitor the presence of bovine tuberculosis in deer. In this study, volatile organic compound profiles of BCG-vaccinated and non-vaccinated deer, before and after experimental challenge with M. bovis strain 95-1315, were generated using solid phase microextraction fiber head-space sampling over suspended fecal pellets with analysis by gas chromatography/mass spectrometry. Chromatograms were processed using XCMS Online to characterize ion variation among treatment groups. The principal component scores resulting from significant (α = 0.05) ion responses were used to build linear discriminant analysis models. The sensitivity and specificity of these models were used to evaluate the feasibility of using this analytical approach to distinguish within group comparisons between pre- and post-M. bovis challenge: non-vaccinated male or female deer, BCG-vaccinated male deer, and the mixed gender non-vaccinated deer data. Seventeen compounds were identified in this analysis. The peak areas for these compounds were used to build a linear discriminant classification model based on principal component analysis scores to evaluate the feasibility of discriminating between fecal samples from M. bovis challenged deer, irrespective of vaccination status. The model best representing the data had a sensitivity of 78.6% and a specificity of 91.4%. The fecal head-space sampling approach presented in this pilot study provides a non-invasive method to discriminate between M. bovis challenged deer and BCG-vaccinated deer. Additionally, the technique may prove invaluable for BCG efficacy studies with free-ranging deer as well as for use as a non-invasive monitoring system for the detection of tuberculosis in captive deer and other livestock.

Background: BCG vaccination is recommended at birth in low-income countries, but vaccination is often delayed. Often 20-dose vials of BCG are not opened unless at least ten children are present for vaccination ("restricted vial-opening policy"). BCG coverage is usually reported as 12-month coverage, not disclosing the delay in vaccination. Several studies show that BCG at birth lowers neonatal mortality. We assessed BCG coverage at different ages and explored reasons for delay in BCG vaccination in rural Guinea-Bissau., Methods: Bandim Health Project (BHP) runs a health and demographic surveillance system covering women and their children in 182 randomly selected village clusters in rural Guinea-Bissau. BCG coverage was assessed for children born in 2010, when the restricted vial-opening policy was universally implemented, and in 2012-2013, where BHP provided BCG to all children at monthly visits in selected intervention regions. Factors associated with delayed BCG vaccination were evaluated using logistic regression models. Coverage between intervention and control regions were evaluated in log-binomial regression models providing prevalence ratios., Results: Among 3951 children born in 2010, vaccination status was assessed for 84%. BCG coverage by 1 week of age was 11%, 38% by 1 month, and 92% by 12 months. If BCG had been given at first contact with the health system, 1-week coverage would have been 35% and 1-month coverage 54%. When monthly visits were introduced in intervention regions, 1-month coverage was higher in intervention regions (88%) than in control regions (51%), the prevalence ratio being 1.74 (1.53-2.00). Several factors, including socioeconomic factors, were associated with delayed BCG vaccination in the 2010-birth cohort. When BCG was available at monthly visits these factors were no longer associated with delayed BCG vaccination, only region of residence was associated with delayed BCG vaccination., Conclusion: BCG coverage during the first months of life is low in Guinea-Bissau. Providing BCG at monthly vaccination visits removes the risk factors associated with delayed BCG vaccination.

In both humans and animals, controversy exists concerning the duration of protection induced by BCG vaccine against tuberculosis (TB) and whether revaccination enhances protection. A long-term study was undertaken to determine whether BCG-vaccinated calves would be protected against challenge with Mycobacterium bovis 2½ years after vaccination and to determine the effect of revaccination after 2 years. Seventy-nine calves were divided into five groups (n = 15-17 calves/group) with four of the groups vaccinated subcutaneously with 105 CFU of BCG Danish at 2-4 weeks of age and the fifth group serving as non-vaccinated controls. Three of the four BCG-vaccinated groups were revaccinated 2 years after the initial vaccination. One BCG-vaccinated group was revaccinated with BCG. A second group was vaccinated subcutaneously with a TB protein vaccine consisting of biopolyester particles (Biobeads) displaying two mycobacterial proteins, ESAT-6 and Antigen 85A, mixed with an adjuvant. A third group was vaccinated with TB proteins from M. bovis culture filtrate, mixed with an adjuvant. Twenty-three weeks after the BCG revaccination, all animals were challenged endotracheally with virulent M. bovis and a further 13 weeks later, animals were killed and necropsied to determine protection against TB. The BCG-vaccinated animals produced positive tuberculin caudal fold intradermal (15 of 62 animals) and IFN-γ TB test responses (six of 62 animals) at 6 months after vaccination, but not at subsequent time-points compared to the non-vaccinated animals. Calves receiving a single vaccination with BCG vaccine 2½ years prior to challenge were not protected against TB, while those revaccinated with BCG 2 years after the initial vaccination displayed significant reductions in lung and pulmonary lymph node lesion scores compared to the non-vaccinated animals. In contrast, no reduction in lesion scores was observed in the animals revaccinated with the TB protein vaccines with their immune responses biased towards induction of antibody.

Bovine tuberculosis (TB) is a significant threat to the cattle industry in England and Wales. It is widely acknowledged that a combination of measures targeting both cattle and wildlife will be required to eradicate bovine TB or reduce its prevalence until European official freedom status is achieved. Vaccination of cattle and/or badgers could contribute to bovine TB control in Great Britain, although there are significant gaps in our knowledge regarding the impact that vaccination would actually have on bovine TB incidence. Laboratory studies have demonstrated that vaccination with BCG can reduce the progression and severity of TB in both badgers and cattle. This is encouraging in terms of the prospect of a sustained vaccination programme achieving reductions in disease prevalence; however, developing vaccines for tackling the problem of bovine TB is challenging, time-consuming and resource-intensive, as this review article sets out to explain., (British Veterinary Association.)

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 2×10(9) infectious units (iu) delivered intradermally conferred the most consistent and strongest responses of the different regimes tested., (Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.)

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., (Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.)

Mycobacterium bovis (M. bovis) causes major economy and public health problem in numerous countries. In Great Britain, despite the use of a test-and-slaughter strategy, the incidence of bovine tuberculosis (bTB) in cattle has steadily risen in recent years. One strategy being considered to reduce the burden of bTB in cattle is the development of an efficient vaccine. The only current potentially available vaccine against tuberculosis, live attenuated M. bovis bacille Calmette-Guérin (BCG), has demonstrated variable efficacy in both humans and cattle and the development of improved vaccination strategies for cattle is a research priority. In this study we assessed the immunogenicity in cattle of two recombinant BCG strains, namely BCG Pasteur Δzmp1::aph and BCG Danish Δzmp1. By applying a recently defined predictive immune-correlate of protection (T cell memory responses measured by cultured ELISPOT), we have compared these two recombinant BCG with wild-type BCG Danish SSI. Our results demonstrated that both strains induced superior T cell memory responses compared to wild-type BCG. These data provide support for the prioritisation of testing BCG Danish Δzmp1 in vaccination/M. bovis challenge studies to determine its protective efficacy., (Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.)

Vaccination is a key strategy for control of tuberculosis (TB), and considerable progress has been made in the past 5 years to develop improved vaccines for humans and animals, differentiate vaccinated animals from those infected with Mycobacterium bovis and deliver vaccines to wildlife. Studies have moved from testing vaccines in small animal models to clinical trials in humans and from experimental challenge studies in cattle and wildlife to evaluation of vaccines in the field. Candidate vaccines undergoing testing in humans include live mycobacterial vaccines to replace bacille Calmette Guérin (BCG), subunit vaccines (virus vector or protein) to boost BCG and therapeutic vaccines used as an adjunct to chemotherapy. In cattle, a number of diagnostic tests have been developed and successfully tested for differentiating infected from vaccinated animals, which will facilitate the use of BCG vaccine in cattle. Encouraging results have been obtained from recent field trials in cattle using BCG vaccine to protect against natural exposure to M. bovis. To date, no subunit TB vaccines have induced improved protection compared with that for BCG, but prime-boost combinations of BCG with DNA, protein or virus-vectored vaccines have induced better protection than BCG vaccine alone. Development of an oral bait BCG formulation has demonstrated the practicality of delivering TB vaccines to wildlife. Oral BCG preparations have induced protection against experimental challenge of M. bovis in possums, badgers, wild boar and white-tailed deer and against natural exposure to M. bovis in possums. Recent progress in TB vaccine development has provided much impetus for their future use., (© 2013 Blackwell Verlag GmbH.)

"Test-and-slaughter" has been successful in industrialized countries to control and eradicate tuberculosis from cattle; however, this strategy is too expensive for developing nations, where the prevalence is especially high. Vaccination with the Calmette-Guérin (BCG) strain has been shown to protect against the development of lesions in vaccinated animals: mouse, cattle and wildlife species. In this study, the immune response and the pathology of vaccinated (BCG-prime and BCG prime-CFP-boosted) and unvaccinated (controls) calves were evaluated under experimental settings. A 10(6) CFU dose of the BCG strain was inoculated subcutaneously on the neck to two groups of ten animas each. Thirty days after vaccination, one of the vaccinated groups was boosted with an M. bovis culture filtrate protein (CFP). Three months after vaccination, the three groups of animals were challenged with 5×10(5) CFU via intranasal by aerosol with a field strain of M. bovis. The immune response was monitored throughout the study. Protection was assessed based on immune response (IFN-g release) prechallenge, presence of visible lesions in lymph nodes and lungs at slaughter, and presence of bacilli in lymph nodes and lung samples in histological analysis. Vaccinated cattle, either with the BCG alone or with BCG and boosted with CFP showed higher IFN-g response, fewer lesions, and fewer bacilli per lesion than unvaccinated controls after challenge. Animals with low levels of IFN-g postvaccine-prechallenge showed more lesions than animals with high levels. Results from this study support the argument that vaccination could be incorporated into control programs to reduce the incidence of TB in cattle in countries with high prevalence.

Vaccination of cattle with a commercial human tuberculosis (TB) vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG) Danish, at a dose equivalent to 5 human doses of BCG has protected these animals against TB in field and experimental trials. There is interest in determining whether a 10-fold-lower dose could still protect cattle but not induce a tuberculin intradermal test response. Two groups of calves (n = 9/group) were vaccinated subcutaneously with a lyophilized BCG Danish vaccine containing either 0.5 (1 × 10(5) to 4 × 10(5) CFU) or 5 (1 × 10(6) to 4 × 10(6) CFU) human doses of BCG Danish, with an additional group of 10 calves serving as nonvaccinated controls. Fifteen weeks after vaccination, these animals were challenged intratracheally with 5 × 10(3) CFU of virulent M. bovis and another 15 weeks later were slaughtered and examined for the presence of tuberculous lesions. Vaccination of the calves with either 0.5 or 5 equivalent human doses of BCG Danish induced similar levels of protection against challenge with M. bovis, with both groups showing significant reductions in the pathological and microbiological parameters compared to those for the the control group (P < 0.05). Vaccination with either of the two BCG doses induced similar numbers of animals responding to the tuberculin intradermal test at 11 weeks postvaccination. Vaccination with a 0.5 equivalent human dose of a commercial lyophilized BCG vaccine can protect cattle against challenge with M. bovis.

Bovine tuberculosis (bTB) remains a problem on many dairy farms in Mexico, as well as a public health risk. We previously found a high frequency of Mycobacterium bovis DNA in colostrum from dairy cows using a nested PCR to detect mpb70. Since there are no reliable in vivo tests to determine the effectiveness of booster Mycobacterium bovis BCG vaccination against bTB, in this work we monitored M. bovis DNA in colostrum by using this nested PCR. In order to decrease the risk of adverse reactions in animals likely containing viable M. bovis, a single application of BCG and a subunit vaccine (EEP-1) formulated with M. bovis culture filtrate proteins (CFP) and a copolymer as the adjuvant was performed in tuberculin skin test-negative cattle (TST(-)), while TST reactor animals (TST(+)) received EEP-1 only. Booster immunization using EEP-1 was applied to both groups, 2 months after primary vaccination to whole herds and 12 months later to lactating cows. Colostrum samples were collected from 6 farms where the cows were vaccinated over a 12-month period postvaccination and, for comparison, from one control farm where the cows were not vaccinated with comparable bTB prevalence. We observed an inverse relationship between the frequency of M. bovis DNA detection and time postvaccination at the first (P < 0.001) and second (P < 0.0001) 6-month periods. Additionally, the concentration of gamma interferon (IFN-γ) was higher in mpb70 PCR-positive colostrum samples (P = 0.0003). These results suggest that M. bovis DNA frequency in colostrum could be a potentially useful biomarker for bTB vaccine efficacy on commercial dairy farms.

A prime-boost strategy conserving BCG is considered the most promising vaccine to control tuberculosis. A boost with a DNA vaccine containing the mycobacterial gene of a heat shock protein (pVAXhsp65) after BCG priming protected mice against experimental tuberculosis. However, anti-hsp65 immunity could worsen an autoimmune disease due to molecular mimicry. In this investigation, we evaluated the effect of a previous BCG or BCG/pVAXhsp65 immunization on experimental autoimmune encephalomyelitis (EAE) development. Female Lewis rats were immunized with BCG or BCG followed by pVAXhsp65 boosters. The animals underwent EAE induction and were daily evaluated for weight loss and clinical score. They were euthanized during recovery phase to assess immune response and inflammatory infiltration at the central nervous system. Previous immunization did not aggravate or accelerate clinical score or weight loss. In addition, this procedure clearly decreased inflammation in the brain. BCG immunization modulated the host immune response by triggering a significant reduction in IL-10 and IFN-γ levels induced by myelin basic protein. These data indicated that vaccination protocols with BCG or BCG followed by boosters with pVAXhsp65 did not trigger a deleterious effect on EAE evolution.

White-tailed deer (Odocoileus virginianus) experimentally infected with a virulent strain of Mycobacterium bovis have been shown to transmit the bacterium to other deer and cattle (Bos taurus) by sharing of pen waste and feed. The risk of transmission of M. bovis bacille Calmette-Guerin (BCG) vaccine from orally vaccinated white-tailed deer to other deer and cattle, however, is not well understood. In order to evaluate this risk, we orally vaccinated 14 white-tailed deer with 1×10(9) colony forming units BCG in lipid-formulated baits and housed them with nine non-vaccinated deer. Each day we exposed the same seven naïve cattle to pen space utilized by the deer to look for transmission between the two species. Before vaccination and every 60 days until the end of the study, we performed tuberculin skin testing on deer and cattle, as well as interferon-gamma testing in cattle, to detect cellular immune response to BCG exposure. At approximately 27 weeks all cattle and deer were euthanized and necropsied. None of the cattle converted on either caudal fold, comparative cervical tests, or interferon-gamma assay. None of the cattle were culture positive for BCG. Although there was immunological evidence that BCG transmission occurred from deer to deer, we were unable to detect immunological or microbiological evidence of transmission to cattle. This study suggests that the risk is likely to be low that BCG-vaccinated white-tailed deer would cause domestic cattle to react to the tuberculin skin test or interferon-gamma test through exposure to a BCG-contaminated environment.

Vaccination of neonatal calves with Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces a significant degree of protection against bovine tuberculosis, caused by infection with virulent M. 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 with M. 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 nonvaccinated M. 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 of M. bovis challenge 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.

Tuberculosis (TB) is a significant animal health problem in many parts of the world, and reservoirs of infection in wild animals complicate disease control efforts in farmed livestock, particularly cattle. Badgers (Meles meles) are a significant wildlife reservoir of Mycobacterium bovis infection for cattle in the United Kingdom (UK) and Republic of Ireland (ROI). Vaccination of badgers using an M. bovis strain bacille Calmette-Guérin (BCG) vaccine could potentially be an option in the national TB eradication strategy. Wildlife vaccination has been used successfully for other diseases in wildlife species, and may have a role to play in reducing M. bovis transmission at the wildlife-livestock interface. Research to date has provided evidence that BCG is protective in badgers, and a parenteral badger BCG vaccine has been licensed in the UK. Further research is required to develop effective strategies for vaccine deployment and to determine the effect of badger vaccination on cattle TB incidence., (Copyright © 2012 Elsevier Ltd. All rights reserved.)