Professor Mark Chambers

Background Human, animal, and environmental health are increasingly threatened by the emergence and spread of antibiotic resistance. Inappropriate use of antibiotic treatments commonly contributes to this threat, but it is also becoming apparent that multiple, interconnected environmental factors can play a significant role. Thus, a One Health approach is required for a comprehensive understanding of the environmental dimensions of antibiotic resistance and inform science-based decisions and actions. The broad and multidisciplinary nature of the problem poses several open questions drawing upon a wide heterogeneous range of studies. Objective This study seeks to collect and catalogue the evidence of the potential effects of environmental factors on the abundance or detection of antibiotic resistance determinants in the outdoor environment, i.e., antibiotic resistant bacteria and mobile genetic elements carrying antibiotic resistance genes, and the effect on those caused by local environmental conditions of either natural or anthropogenic origin. Methods Here, we describe the protocol for a systematic evidence map to address this, which will be performed in adherence to best practice guidelines. We will search the literature from 1990 to present, using the following electronic databases: MEDLINE, Embase, and the Web of Science Core Collection as well as the grey literature. We shall include full-text, scientific articles published in English. Reviewers will work in pairs to screen title, abstract and keywords first and then full-text documents. Data extraction will adhere to a code book purposely designed. Risk of bias assessment will not be conducted as part of this SEM. We will combine tables, graphs, and other suitable visualisation techniques to compile a database i) of studies investigating the factors associated with the prevalence of antibiotic resistance in the environment and ii) map the distribution, network, cross-disciplinarity, impact and trends in the literature.

This study assessed the elemental status of cross-bred dairy cows in small holder farms in Sri Lanka, with the aim to establish the elemental baseline and identify possible deficiencies. For this purpose, 458 milk, hair, serum and whole blood samples were collected from 120 cows in four regions of Northern and Northwestern Sri Lanka, (namely Vavaniya, Mannar, Jaffna and Kurunegala). Farmers also provided a total of 257 samples of feed, which included local fodder as well as 79 supplement materials. The concentrations of As, Ca, Cd, Co, Cr, Cu, Fe, I, K, Mg, Mn, Mo, Na, Ni, Pb, Se, V and Zn were determined by inductively coupled plasma mass spectrometry (ICP-MS). Evaluation of the data revealed that all cows in this study could be considered deficient in I and Co (18.6-78.5 mu g L-1 I and 0.06-0.65 mu g L-1 Co, in blood serum) when compared with deficiency upper boundary levels of 0.70 mu g L-1 Co and 50 mu g L-1 I. Poor correlations were found between the composition of milk or blood with hair, which suggests that hair is not a good indicator of mineral status. Most local fodders meet dietary requirements, with Sarana grass offering the greatest nutritional profile. Principal component analysis (PCA) was used to assess differences in the elemental composition of the diverse types of feed, as well as regional variability, revealing clear differences between forage, concentrates and nutritional supplements, with the latter showing higher concentrations of non-essential or even toxic elements, such as Cd and Pb.

A fingerprint offers a convenient, noninvasive sampling matrix for monitoring therapeutic drug use. However, a barrier to widespread adoption of fingerprint sampling is the fact that the sample volume is uncontrolled. Fingerprint samples (n = 140) were collected from patients receiving the antibiotic isoniazid as part of their treatment, as well as from a drug-naive control group (n = 50). The fingerprint samples were analyzed for isoniazid (INH) and acetylisoniazid (AcINH), using liquid chromatography high-resolution mass spectrometry. The data set was analyzed retrospectively for metabolites known to be present in eccrine sweat. INH or AcINH was detected in 89% of the fingerprints collected from patients and in 0% of the fingerprints collected from the control group. Metabolites lysine, ornithine, pyroglutamic acid, and taurine were concurrently detected alongside INH/AcINH and were used to determine whether the fingerprint sample was sufficient for testing. Given a sufficient sample volume, the fingerprint test for INH use has sensitivity, specificity, and accuracy of 100%. Normalization to taurine was found to reduce intradonor variability. Fingerprints are a novel and noninvasive approach to monitor INH therapy. Metabolites can be used as internal markers to demonstrate a sufficient sample volume for testing and reduce intradonor variability.

Purpose: Simulation-based medical education has changed the teaching of clinical practice skills, with scenario-based simulations being particularly effective in supporting learning in veterinary medicine. In this study, we explore the efficacy of simulation education to teach infection prevention and control (IPC) as part of Antimicrobial Stewardship (AMS) teaching for early years clinical veterinary medicine undergraduates. Methods: The intervention was designed as a 30-minute workshop with a simulation and script delivered online for 130 students as a part of hybrid teaching within the undergraduate curriculum. Learning outcome measures were compared between an intervention group and waitlist-control group using one-way between-groups analysis of covariance tests. Results: Significant differences between groups were found for outcomes measures related to short-term knowledge gain and confidence in IPC and AMS in small animal clinical practice. However, lateral knowledge transfer to large animal species clinical practice showed no significant differences. Student feedback indicated that the intervention was an enjoyable and engaging way to learn AMS. Conclusions: The intervention provided short-term knowledge gain in IPC protocols and enhanced procedural skills via active learning and motivation to learn in large groups of students. Future improvements would be to include large animal clinical scenario discussions and evaluate longer-term knowledge gain.

Genetically modified Vibrio cholerae strain 638 (biotype El Tor, serotype Ogawa) has previously been shown to be immunogenic in animal models and in human trials. Our objective in the work reported herein was to describe the process development methods for the production of the 638 attenuated cholera vaccine. Cell seed bank, culture of biomass, lyophilization and final formulation were processes were developed. The results show kinetics of culture that fulfils a logistical model. The microbiological properties, colonizing capability, immunogenicity and non-toxigenicity of the final product were indistinguishable from the properties of the working seed lot. We conclude that the non-reactogenic, immunogenic and protective strain 638 is robust and can withstand the fermentation processes required for large-scale production of a vaccine.

This book is a systematic, comprehensive treatise of bovine tuberculosis and the bacterium, Mycobacterium bovis, which causes it. Bovine tuberculosis remains a major cause of economic loss in cattle industries worldwide. There is no one book which treats the various aspects of the problem in a systematic way.

•Ensuring adherence and sufficient exposure to anti-tuberculosis medications is essential for successful treatment.•Finger sweat offers a non-invasive alternative to monitor patients.•Isoniazid can be detected in finger sweat for 1–6 h following controlled administration of the drug.•Normalisation to creatinine can be used to account for differences in sample volume and was shown to increase correlation to serum. Insufficient exposure and poor compliance with anti-tuberculosis (TB) medications are risk factors for treatment failure and the development of drug resistance. Measurement of drugs in biological samples, such as blood and saliva, can be used to assess adherence and make dose adjustments by therapeutic drug monitoring (TDM). Finger sweat testing is a convenient and non-invasive method to monitor patients. To assess the feasibility of finger sweat testing for medication adherence and as a semi-quantitative tool for TDM analysis. Ten patients provided finger sweat, blood and saliva samples following a controlled dose of isoniazid. Samples were analysed by liquid chromatography-mass spectrometry. Isoniazid can be detected in finger sweat 1–6 h following administration at typically prescribed dosages. The normalisation of isoniazid to creatinine increases the correlation between finger sweat and serum isoniazid concentration and provides a means to account for inconsistent sample volumes. We describe the time-course measurement of isoniazid (or drug-to-creatinine ratio) in finger sweat compared to the pharmacokinetic profile in blood for the first time. This technique, adaptable for other drugs, could reduce the burden on clinics and improve patient experience. [Display omitted]

Antimicrobial resistance is of growing concern in human and animal health. The aim of this study was to raise awareness and perception of risk of infection-related behaviours during routine preparation for veterinary surgery. We took a multi-disciplinary and multi-method approach to 'make visible, the invisible' by illustrating how microbial contamination can be spread during the preparation process for surgical procedures. The design-led visualization approach enhanced inter-disciplinary team and workshop participant contributions during the co-development of an innovative digital tool to support training for veterinary practitioners and students. After experiencing the intervention, 92% of 51 participants agreed to change their behaviour and stated an intention to implement an infection control behaviour that aligned with training objectives. The 3D graphics enhanced the delivery of training content by making difficult and abstract contamination concepts easy to understand. A similar approach could be taken for human health applications.

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.

Protection of cattle against bovine tuberculosis by vaccination could be an important control strategy in countries where there is persistent Mycobacterium bovis infection in wildlife and in developing countries where it is not economical to implement a tuberculin test and slaughter control program. The main aim of such a vaccination strategy would be to reduce transmission of infection by reducing the lung pathology caused by infection and preventing seeding of the organism to organs from which M. bovis could be excreted. Recent reports of successful DNA vaccination against Mycobacterium tuberculosis in small-animal models have suggested that DNA vaccines act by reducing lung pathology without sensitizing animals to tuberculin testing. We therefore evaluated the ability of vaccines consisting of DNA encoding the mycobacterial antigens MPB83 and 85A to reduce lung pathology and prevent hematogenous spread in guinea pigs challenged with a low dose of aerosolized M. bovis . Vaccination with MPB83 DNA reduced the severity of pulmonary lesions, as assessed by histopathology, and resembled M. bovis BCG vaccination in this respect. However, unlike BCG vaccination, MPB83 DNA vaccination did not protect challenged guinea pigs from hematogenous spread of organisms to the spleen. In contrast, vaccination with antigen 85A DNA, a promising DNA vaccine for human tuberculosis, had no measurable protective effect against infection with M. bovis.

Tuberculosis caused by infection with Mycobacterium tuberculosis or M. bovis remains one of the most important infectious diseases of man and animals, and continues to inflict a huge cost in both health and financial terms. The current vaccine, BCG demonstrates variable efficacy and so a more robust vaccine strategy to either replace or supplement BCG is required. We have utilised a DNA prime-BCG boost strategy in a murine M. bovis challenge model using a cocktail of 3 DNA vaccines (encoding Hsp65, Hsp70 and Apa) followed by BCG. Controls were inoculated with vector DNA only, coding DNA only, BCG only or vector DNA followed by BCG boost. Analysis of immune responses by ELISpot prior to challenge, revealed that the coding DNA/BCG prime boost resulted in an increased frequency of antigen-specific IFNγ producing cells compared to the other regimes. When spleen cell cytokine production to BCG antigens was analysed, significantly more IFNγ and IL-12 was seen in those groups primed with DNA (coding or vector) prior to BCG than those receiving BCG alone. Analysis of bacterial counts revealed that DNA priming followed by BCG boost further improved the protective immunity induced by BCG alone. Surprisingly, inoculation with vector DNA was as efficacious as the coding DNA in enhancing BCG protection. Taken together these results indicate that whilst the coding DNA vaccines induce antigen specific responses, treatment with the vector DNA is sufficient for the increase in protective immunity over that induced by BCG, suggesting that the vector DNA may be acting as a non-specific adjuvant for BCG immunization.

Tuberculosis remains one of the most significant diseases of humans and animals. The only currently available vaccine against this disease is a live, attenuated vaccine, bacillus Calmette-Guérin (BCG), which was originally derived from Mycobacterium bovis and despite its variable efficacy is the most widely administered vaccine in the world. With the advent of the human immunodeficiency virus-AIDS pandemic concern has been raised over the safety of BCG. Moreover, since BCG sensitizes vaccinated individuals to the tuberculin test, vaccination with BCG prevents diagnosis of infection in vaccinated individuals. Recently, auxotrophic strains of BCG have been generated by insertional mutagenesis which have been shown to be safer than the parent BCG strain following administration to mice with severe combined immunodeficiency disease. These strains have also been shown to give comparable protection against intravenous and intratracheal challenge of BALB/c mice with M. tuberculosis relative to conventional BCG. Here we report that one of these mutants, a leucine auxotroph of BCG, conferred significant protection of the lungs and spleens of guinea pigs infected with M. bovis and protection of the spleens of guinea pigs infected with M. tuberculosis in the absence of a cutaneous hypersensitivity reaction to tuberculin. Therefore, protective immunity to tuberculosis may, at least in part, be achieved without sensitization to the tuberculin skin test. These results indicate that it may be possible to develop a new generation of vaccines based on BCG that are protective, are safe for use in the immunocompromised, and do not preclude the use of the tuberculin skin test in both humans and animals.

Increase of antimicrobial resistance (AMR) is a global threat to health. The AMR profile of bacteria isolated from domesticated animals and free-ranging wildlife has been studied, but there are relatively few studies of bacteria isolated from captive wild animals. Understanding the dynamics of AMR in different populations is key to minimizing emergence of resistance and to preserve the efficacy of antimicrobials. In this study, fecal samples were collected from 17 species of healthy ungulates from a zoological collection in southeast England, which yielded 39 and 55 spp. isolates for further analysis. Antibiotic sensitivity was investigated using agar disk diffusion. isolates were resistant to a range of antibiotics, with resistance to ampicillin being the most common (28%). All isolates were susceptible to apramycin, enrofloxacin, chloramphenicol, and florfenicol. None tested positive for extended-spectrum beta-lactamase or AmpC activity. Seven of 39 (18%) isolates were resistant to three or more antibiotic classes. The isolates were further analyzed using multilocus sequence typing, which identified four pairs of identical sequence type isolates and 27 diverse strains. The spp. isolates were resistant to a range of antibiotics, with resistance to cefpodoxime seen in 95% of isolates. All spp. isolates were susceptible to ampicillin, gentamicin, chloramphenicol, and vancomycin. This study identified multidrug-resistant phenotypes in enterobacterial isolates that were like those commonly found in domestic ungulates. There was no apparent spatial clustering of the resistance profiles within the zoo. Review of the medical records of individual animals showed no direct relation to the AMR profiles observed. Observed resistance to antibiotics rarely or never used may have been due to coselection or directly acquired from other sources.

Background Oral vaccination with Mycobacterium bovis Bacille of Calmette and Guerin (BCG) has provided protection against M. bovis to badgers both experimentally and in the field. There is also evidence suggesting that the persistence of live BCG within the host is important for maintaining protection against TB. Here we investigated the capacity of badger inductive mucosal sites to absorb and maintain live BCG. The targeted mucosae were the oropharyngeal cavity (tonsils and sublingual area) and the small intestine (ileum). Results We showed that significant quantities of live BCG persisted within badger in tissues of vaccinated badgers for at least 8 weeks following oral vaccination with only very mild pathological features and induced the circulation of IFNγ-producing mononuclear cells. The uptake of live BCG by tonsils and drainage to retro-pharyngeal lymph nodes was repeatable in the animal group vaccinated by oropharyngeal instillation whereas those vaccinated directly in the ileum displayed a lower frequency of BCG detection in the enteric wall or draining mesenteric lymph nodes. No faecal excretion of live BCG was observed, including when BCG was delivered directly in the ileum. Conclusions The apparent local loss of BCG viability suggests an unfavorable gastro-enteric environment for BCG in badgers, which should be taken in consideration when developing an oral vaccine for use in this species.

The Mycobacterium tuberculosis complex (MTBC) is a group of bacteria that cause tuberculosis (TB) in diverse hosts, including captive and free-ranging wildlife species. There is significant research interest in developing immunodiagnostic tests for TB that are both rapid and reliable, to underpin disease surveillance and control. The aim of this study was to carry out an updated review of diagnostics for TB in non-bovid species with a focus predominantly on those based on measurement of immunity. A search was carried out to identify relevant papers meeting a pre-defined set of inclusion criteria. Forty-one papers were identified from this search, from which only twenty papers contained data to measure and compare diagnostic performance using diagnostic odds ratio. The diagnostic tests from each study were ranked based on sensitivity, specificity, and diagnostic odds ratio to define high performing tests. High sensitivity and specificity values across a range of species were reported for a new antigenic target, P22 complex, demonstrating it to be a reliable and accurate antigenic target. Since the last review of this kind was undertaken, the immunodiagnosis of TB in meerkats and African wild dogs was reported for the first time. Suid species showed the most consistent immunological responses and highlight a potential dichotomy between humoral and cellular immune responses.

Specific antituberculous resistance appears to be induced following inoculation of live but not dead BCG. This dependence on BCG viability may explain the diverse responses in terms of protective immunity in clinical trials of BCG conducted worldwide over the last thirty years. In order to dissect out simple parameters which may differentiate a protective from a non-protective response, we have developed a murine in vivo model of experimental BCG infection to study the immune response in draining lymph nodes following footpad inoculation with either live or killed BCG preparations. In this model, live but not heat-killed BCG efficiently migrate to the draining lymph nodes and stimulate the early accumulation of mononuclear cells. In addition, live and heat-killed BCG stimulate different responses in terms of the level of expression of interferon-gamma, inducible nitric oxide (iNOS), as well as macrophage and dendritic cell markers in the draining lymph nodes. This divergent in vivo response was reproduced in vitro when pure macrophage cultures were infected with BCG and responded differently to live and dead preparations, producing significant levels of TNF and reactive nitrogen intermediates only when infected with live BCG. Taken together, these observations suggest that the differences encountered in vivo may be related to the ability of live BCG to migrate to local lymph node, where they cause the accumulation of cells expressing protective cytokines and therefore inducing an efficient immune response. These findings may have important implications for the design of new anti-tuberculosis vaccines.

ollection of finger sweat is explored here as a rapid and convenient way of monitoring patient adherence to antipsychotic drugs. Finger sweat samples (n = 426) collected from patients receiving treatment with clozapine, quetiapine and olanzapine were analysed by liquid chromatography mass spectrometry, including a subgroup of patients with paired plasma samples. Finger sweat samples were also analysed from a negative control group and patients who had handled antipsychotic medication only. The finger sweat test (based on the detection of parent drug in one donated sample) was 100% effective in monitoring adherence within commonly prescribed dosing ranges. In comparison to participants who handled the medication only, the test could distinguish between contact and administration through monitoring of the drug metabolite, or the level of parent drug. Additionally, in a subgroup of patients prescribed clozapine, a statistically significant correlation was observed between the mass of parent drug in finger sweat and plasma concentration. The finger sweat technology shows promise as a dignified, noninvasive method to monitor treatment adherence in patients taking antipsychotics.

The diagnosis and control of Mycobacterium bovis infection (bovine tuberculosis: TB) continues to present huge challenges to the British cattle industry. A clearer understanding of the magnitude and duration of immune response to M. bovis infection in the European badger (Meles meles) – a wildlife maintenance host – may assist with the future development of diagnostic tests, and vaccination and disease management strategies. Here, we analyse 5280 diagnostic test results from 550 live wild badgers from a naturally-infected population to investigate whether one diagnostic test (a gamma interferon release [IFNγ] assay, n = 550 tests) could be used to predict future positive results on two other tests for the same disease (a serological test [n = 2342 tests] and mycobacterial culture [n = 2388 tests]) and hence act as an indicator of likely bacterial excretion or disease progression. Badgers with the highest IFNγ optical density (OD) values were most likely to subsequently test positive on both serological and culture tests, and this effect was detectable for up to 24 months after the IFNγ test. Furthermore, the higher the original IFNγ OD value, the greater the chance that a badger would subsequently test positive using serology. Relationships between IFNγ titres and mycobacterial culture results from different types of clinical sample suggest that the route of infection may affect the magnitude of immune response in badgers. These findings identify further value in the IFNγ test as a useful research tool, as it may help us to target studies at animals and groups that are most likely to succumb to more progressive disease.

In wildlife disease management there are few diseases for which vaccination is a viable option. The human vaccine BCG has been used for the control of bovine tuberculosis in badgers since 2010 and is expected to increase. Understanding the long-term effects of repeated vaccination campaigns on disease prevalence is vital, but modelling thus far has generally assumed that a vaccine provides perfect protection to a proportion of the population, and that animals exposed to a repeated vaccination have a second independent chance of becoming protected. We held a workshop with experts in the field to obtain consensus over the main pathways for partial protection in the badger, and then simulated these using an established model. The available data supported the possibility that some individuals receive no benefit from the BCG vaccine, others may result in a delayed disease progression and in the remaining animals, vaccine protected the individual from any onward transmission. Simulating these pathways using different levels of overall efficacy demonstrated that partial protection leads to a reduced effect of vaccination, but in all of the identified scenarios it was still possible to eradicate disease in an isolated population with no disease introduction. We also identify those potential vaccination failures that require further investigation to determine which of our proposed pathways is the more likely.

© 2015, Springer-Verlag Berlin Heidelberg. In the UK and Republic of Ireland, the European badger Meles meles is considered a maintenance host for bTB and is involved in transmission of infection to cattle. A badger vaccine delivered in an oral bait is currently under development as part of an ongoing effort to reduce levels of disease in the badger population. An oral vaccine would likely be deployed in close vicinity to badger burrows (setts), such that bait will most likely be taken by the target species. However, a range of nontarget species may also occur close to badger setts, and some may potentially interfere with or consume baits. In this study, we used surveillance cameras to record the presence of nontarget species at 16 badger setts involved in a bait deployment study in southwest England. We recorded significant levels of nontarget species activity close to badger setts. The most commonly observed species were small rodents, which were observed at all setts, and in some cases accounted for >90 % of nontarget species observations. A total of 11 other nontarget species were also observed, indicating that a broad range of species may potentially come into contact with vaccine baits deployed at badger setts. Although the majority of these species were not observed interacting directly with baits, small rodents and squirrels were observed eating baits in a number of instances. In addition, monitoring of bait disappearance at 24 setts indicated that small rodents may take >30 % of bait deployed at some setts. The implications for the deployment of an oral vaccine for badgers are discussed.

Needle-free methods of vaccination may allow rapid, simple and safe vaccination of large populations. Oral vaccination is the best established method but faces the hurdle of oral tolerance to the vaccine antigen. Skin-based transcutaneous immunization (TCI) offers an alternative needle-free route of vaccination that is able to induce protective immunity without the problem of oral tolerance. Helicobacter pylori is an important human pathogen associated with a number of gastrointestinal disorders, including gastritis, peptic ulcers and gastric tumors. Conventional treatments involving the use of antibiotics have a number of limitations and the development of an effective vaccine is the best long-term treatment option. A variety of experimental vaccines to Helicobacter have been reported. The paper reviewed here combines the approach of TCI with the use of a novel lipid antigen delivery system, hitherto only used for oral vaccination, to evaluate the potential for TCI for a simple vaccination strategy against Helicobacter and potentially other disease-causing organisms.

Between October 2005 and May 2006, a total of 727 badgers found dead in Wales were reported, and 550 were delivered to the Regional Laboratories of the Veterinary Laboratories Agency (VLA). Of the 459 carcasses suitable for examination, 55 were deemed to be infected with Mycobacterium bovis on the basis of culture, spoligotyping, and variable-number tandem repeat typing. Acid-fast bacteria were observed histologically in a further six badgers, but these bacteria were not confirmed as M. bovis by culture. A rapid serological test (BrockTB Stat-Pak) performed on thoracic blood showed a sensitivity of 35% and a specificity of 99%. Presence of M. bovis infection was 45 times more likely to be confirmed postmortem by culture in BrockTB Stat-Pak-reactive animals than in seronegative ones. Using visible carcass lesions as a marker of bovine tuberculosis (bTB) infection had a similar sensitivity (38%) but was significantly less specific (84%) than serology. The overall accuracy of the antibody detection was 93% ( 346 correct results from 374 tests), whereas the accuracy of regarding visible lesions as a marker for bTB infection was 78% ( 354 correct from 453 carcasses examined). Culture remains the gold standard method for detecting M. bovis infection in badgers. However, where resources are limited and/or an instant result is preferred, the BrockTB Stat-Pak could be used in field surveillance efforts to identify animals which should be examined further by only submitting test-negative animals to more detailed postmortem examination and culture.

© 2015 The Author(s) Infection by the intracellular bacterial pathogen Mycobacterium tuberculosis (Mtb) is a major cause of morbidity and mortality worldwide. Slow progress has been made in lessening the impact of tuberculosis (TB) on human health, especially in parts of the world where Mtb is endemic. Due to the complexity of TB disease, there is still an urgent need to improve diagnosis, prevention, and treatment strategies to control global spread of disease. Active research targeting avenues to prevent infection or transmission through vaccination, to diagnose asymptomatic carriers of Mtb, and to improve antimicrobial drug treatment responses is ongoing. However, this research is hampered by a relatively poor understanding of the pathogenesis of early infection and the factors that contribute to host susceptibility, protection, and the development of active disease. There is increasing interest in the development of adjunctive therapy that will aid the host in responding to Mtb infection appropriately thereby improving the effectiveness of current and future drug treatments. In this review, we summarize what is known about the host response to Mtb infection in humans and animal models and highlight potential therapeutic targets involved in TB granuloma formation and resolution. Strategies designed to shift the balance of TB granuloma formation toward protective rather than destructive processes are discussed based on our current knowledge. These therapeutic strategies are based on the assumption that granuloma formation, although thought to prevent the spread of the tubercle bacillus within and between individuals contributes to manifestations of active TB disease in human patients when left unchecked. This effect of granuloma formation favors the spread of infection and impairs antimicrobial drug treatment. By gaining a better understanding of the mechanisms by which Mtb infection contributes to irreversible tissue damage, down regulates protective immune responses, and delays tissue healing, new treatment strategies can be rationally designed. Granuloma-targeted therapy is advantageous because it allows for the repurpose of existing drugs used to treat other communicable and non-communicable diseases as adjunctive therapies combined with existing and future anti-TB drugs. Thus, the development of adjunctive, granuloma-targeted therapy, like other host-directed therapies, may benefit from the availability of approved drugs to aid in treatment and prevention of TB. In this review, we have attempted to summarize the results of published studies in the context of new innovative approaches to host-directed therapy that need to be more thoroughly explored in pre-clinical animal studies and in human clinical trials.

In the UK there has been a sharp rise in the incidence of bovine tuberculosis since the early 1990s and the badger has been identified as an important wildlife reservoir for this infection. Infected badgers can excrete Mycobacterium bovis, putting other badgers and cattle at risk of becoming infected. Vaccination has been proposed as an approach to reducing the excretion of M. bovis by tuberculous badgers. In order to evaluate the efficacy of a badger vaccine it will be necessary to accurately determine the number of badgers excreting M. bovis without removing them for post-mortem evaluation. The existing live tests for tuberculosis in the badger (culture, indirect ELISA, Western blot) have not been assessed for their ability to detect badgers excreting M. bovis. Over the past 18 years, badgers from 31 social groups have been trapped and sampled in a study area of the Cotswold escarpment. We have examined the serological responses of 128 badgers trapped between 1985 and 1998 from social groups where M. bovis infection was endemic. These responses were compared with culture from faeces, urine, tracheal aspirates and bite wound swabs taken from these animals while alive. ELISA was found to be more sensitive than Western blot in detecting badgers excreting M. bovis. The majority of culture-positive badgers excreted M. bovis intermittently over the period of study. As a result, there was only a 27.5% chance of sampling a badger for culture when it was excreting M. bovis. In contrast, a positive ELISA result correctly predicted 68.2% of badgers with a history of excreting M. bovis. In the absence of alternative live tests for the badger, the Brock Test indirect ELISA appears to be more valuable than culture for measuring the effect of vaccination on reducing the number of badgers at risk of transmitting tuberculosis.

The current tuberculin test and slaughter strategy for the control of bovine tuberculosis in cattle has failed to prevent a sharp rise in cases over recent years, especially in the south-west of England. A recent scientific review has concluded that the development of a cattle vaccine holds the best prospect for tuberculosis control in British herds. In order to continue with test and slaughter-based control strategies, the development of TB vaccines that do not compromise the specificity of the tuberculin skin test are required. This report describes results of cattle vaccination experiments with TB DNA vaccines expressing the mycobacterial antigens MPB70, MPB83, and Ag85A and constitutes the first published vaccination study with DNA vaccines undertaken in a target host species. All calves vaccinated with the MPB83 expressing plasmid demonstrated potent cellular immune responses, characterised by CD4 + T cells producing interferon-gamma as well as humoral immunity characterised by IgG1 biased specific antibodies. Vaccination with MPB70 was less effective with immune responses only observed in half of the vaccinated animals, while vaccination with Ag85A did not result in vaccine-induced immune responses. Intramuscular vaccination was found to stimulate stronger cellular responses than intradermal immunisation. Significantly, the specificity of tuberculin skin testing was not compromised by DNA vaccination since none of the vaccinated calves showed positive skin test reactivity.

The prevalence, distribution and pathology related to infection with Mycobacterium bovis and other mycobacteria were determined in trapped (n=36) and road-killed (n=121) badgers in Spain from 2006 to 2010. The prevalence of M. bovis based on bacteriological culture from road-killed badgers was 8/121 (6.6%) and from trapped badgers was 0/36 (0%). Tuberculosis/M. bovis infection was evident in 15/121 (12.4%) road-killed badgers when bacteriology and histopathology were combined. Mycobacterium avium complex was isolated by culture from the tracheal aspirate of 1/36 (2.8%) trapped badgers and from tissue pools from 8/121 (6.6%) road-killed badgers.

The pathology, frequency and diagnostic implications of 'no visible lesion' (NVL) tuberculosis (Tb), i. e. infection with Mycobacterium bovis in the absence of macroscopic lesions, are described in a wide taxonomic range of wildlife hosts. Information collected and evaluated on the definition and occurrence of NVL Tb, histopathological characteristics, post-mortem techniques to detect minimal lesions, and diagnostic difficulties revealed most Tb-infected individuals with NVL had minute tuberculous lesions, which were difficult to see by eye. Acid-fast organisms (AFO) were sometimes detected in the lesions. Ideally, mycobacterial culture of pools of lymph nodes and/or oropharyngeal tonsils is necessary for the accurate diagnosis of Tb in the absence of macroscopic lesions. At a very minimum, the diagnostic methods applied for studying the prevalence of Tb in the population should be clearly described, to allow comparison between studies.

Bovine tuberculosis (TB) in Great Britain adversely affects animal health and welfare and is a cause of considerable economic loss. The situation is exacerbated by European badgers (Meles meles) acting as a wildlife source of recurrent Mycobacterium bovis infection to cattle. Vaccination of badgers against TB is a possible means to reduce and control bovine TB. The delivery of vaccine in oral bait holds the best prospect for vaccinating badgers over a wide geographical area. There are practical limitations over the volume and concentration of Bacillus of Calmette and Guérin (BCG) that can be prepared for inclusion in bait. The production of BCG in a bioreactor may overcome these issues. We evaluated the efficacy of oral, bioreactor-grown BCG against experimental TB in badgers. We demonstrated repeatable protection through the direct administration of at least 2.0 × 108 colony forming units of BCG to the oral cavity, whereas vaccination via voluntary consumption of bait containing the same preparation of BCG did not result in demonstrable protection at the group-level, although a minority of badgers consuming bait showed immunological responses and protection after challenge equivalent to badgers receiving oral vaccine by direct administration. The need to deliver oral BCG in the context of a palatable and environmentally robust bait appears to introduce such variation in BCG delivery to sites of immune induction in the badger as to render experimental studies variable and inconsistent.

The epithelial lining of the lung is often the first point of interaction between the host and inhaled pathogens, allergens and medications. Epithelial cells are therefore the main focus of studies which aim to shed light on host-pathogen interactions, to dissect the mechanisms of local host immunity and study toxicology. If these studies are not to be conducted exclusively in vivo, it is imperative that in vitro models are developed with a high in vitro- in vivo correlation. We describe here a co-culture bilayer model of the bovine alveolus, designed to overcome some of the limitations encountered with mono-culture and live animal models. Our system includes bovine pulmonary arterial endothelial cells (BPAECs) seeded onto a permeable membrane in 24 well Transwell format. The BPAECs are overlaid with immortalised bovine alveolar type II epithelial cells and the bilayer cultured at air-liquid interface for 14 days before use; in our case to study host-mycobacterial interactions. Characterisation of novel cell lines and the bilayer model have provided compelling evidence that immortalised bovine alveolar type II cells are an authentic substitute for primary alveolar type II cells and their culture as a bilayer in conjunction with BPAECs provides a physiologically relevant in vitro model of the bovine alveolus.   The bilayer model may be used to study dynamic intracellular and extracellular host-pathogen interactions, using proteomics, genomics, live cell imaging, in-cell ELISA and confocal microscopy. The model presented in this article enables other researchers to establish an in vitro model of the bovine alveolus that is easy to set up, malleable and serves as a comparable alternative to in vivo models, whilst allowing study of early host-pathogen interactions, currently not feasible in vivo. The model therefore achieves one of the 3Rs objectives in that it replaces the use of animals in research of bovine respiratory diseases.

Background: Many investigations into the determinants of hand hygiene (HH) behaviour have explored only individual predictors or were designed according to arguably overly simplistic models of behaviour. Consequently, important influences on HH behaviour, including habit and emotion, are sometimes neglected. This study is the first to employ the Theory of Interpersonal Behaviour as a comprehensive model for understanding the determinants of HH behaviour. Method: A self-report questionnaire was conducted with staff from two large UK veterinary referral practices. Participants (n = 75) reported their HH behaviour and responded to statements rating the importance of social norms, self-protection, patient protection, time pressures, access to equipment, habit and disgust, to their HH behaviour. Results: Regression analysis showed that, overall, determinants explained 46% of variance (p 

Mycobacterium abscessus complex (MABC) is an important pathogen of immunocompromised patients. Accurate and rapid determination of MABC at the subspecies level is vital for optimal antibiotic therapy. Here we have used comparative genomics to design MABC subspecies-specific PCR assays. Analysis of single nucleotide polymorphisms and core genome multilocus sequence typing showed clustering of genomes into three distinct clusters representing the MABC subspecies M. abscessus, M. bolletii and M. massiliense. Pangenome analysis of 318 MABC genomes from the three subspecies allowed for the identification of 15 MABC subspecies-specific genes. In silico testing of primer sets against 1,663 publicly available MABC genomes and 66 other closely related Mycobacterium genomes showed that all assays had >97% sensitivity and >98% specificity. Subsequent experimental validation of two subspecies-specific genes each showed the PCR assays worked well in individual and multiplex format with no false-positivity with 5 other mycobacteria of clinical importance. In conclusion, we have developed a rapid, accurate, multiplex PCR-assay for discriminating MABC subspecies that could improve their detection, diagnosis and inform correct treatment choice.

Mycobacterium bovis is the causative agent of bovine tuberculosis and the predominant cause of zoonotic tuberculosis in people. Bovine tuberculosis occurs in farmed cattle but also in a variety of wild animals, which form a reservoir of infection. Although direct transmission of tuberculosis occurs between mammals, the low frequency of contact between different host species and abundant shedding of bacilli by infected animals suggests an infectious route via environmental contamination. Other intracellular pathogens that transmit via the environment deploy strategies to survive or exploit predation by environmental amoebae. To explore if M. bovis has this capability, we investigated its interactions with the soil and dung-dwelling amoeba, Dictyostelium discoideum. We demonstrated that M. bovis evades phagocytosis and destruction by D. discoideum and actively transits through the amoeba using the ESX-1 Type VII Secretion System as part of a programme of mechanisms, many of which have been co-opted as virulence factors in the mammalian host. This capacity of M. bovis to utilise an environmental stage between mammalian hosts may enhance its transmissibility. In addition, our data provide molecular evidence to support an evolutionary role for amoebae as training grounds for the pathogenic M. tuberculosis complex.

There are three basic approaches to managing diseases: directly reduce the reproductive rate of the pathogen, reduce host (or infected host) density, or manipulate the environment to reduce contact between diseased and susceptible animals. In this chapter we will look at the first of these approaches. Since disease transmission results from direct or indirect contact between infectious and susceptible individuals, there are two ways to target an infectious agent: either limit the number of susceptible individuals by vaccinating them, or treat infected individuals in order to reduce the duration or intensity of the infectious period and the number of infectious individuals present at any given time. The overall aim of this chapter is to consider the conditions under which vaccination and treatment may make a valuable contribution to the control of infectious diseases in wild mammal populations. Both field research and mathematical modelling approaches have been used to address this question. For vaccination, early mathematical models of infectious disease dynamics suggested a simple answer: vaccination is useful as soon as the rate of control ensures that a sufficient proportion of the population is immune for a sufficient period of time (Bailey 1957). At the individual level, this herd immunity means that any given infectious individual has a low probability of encountering a susceptible animal. If the disease is introduced into a vaccinated population, the mean number of secondary infections caused by each infected case will be lower than unity, thus preventing further outbreaks from occurring (R

Bacillus Calmette–Guérin (BCG) vaccine is the only licensed vaccine against tuberculosis (TB) in humans and animals. It is most commonly administered parenterally, but oral delivery is highly advantageous for the immunisation of cattle and wildlife hosts of TB in particular. Since BCG is susceptible to inactivation in the gut, vaccine formulations were prepared from suspensions of Eudragit L100 copolymer powder and BCG in phosphate-buffered saline (PBS), containing Tween® 80, with and without the addition of mannitol or trehalose. Samples were frozen at -20 °C, freeze-dried and the lyophilised powders were compressed to produce BCG–Eudragit matrices. Production of the dried powders resulted in a reduction in BCG viability. Substantial losses in viability occurred at the initial formulation stage and at the stage of powder compaction. Data indicated that the Eudragit matrix protected BCG against simulated gastric fluid (SGF). The matrices remained intact in SGF and dissolved completely in simulated intestinal fluid (SIF) within three hours. The inclusion of mannitol or trehalose in the matrix provided additional protection to BCG during freeze-drying. Control needs to be exercised over BCG aggregation, freeze-drying and powder compaction conditions to minimise physical damage of the bacterial cell wall and maximise the viability of oral BCG vaccines prepared by dry powder compaction.

Organ-on-chip technology has made it to the forefront of emerging technologies through its ability to better simulate the microenvironment of the target organ. This chapter will focus on lung-on-chip technology, beginning by introducing the concept of inhaled therapeutics and where absorption is targeted (upper or lower respiratory tract). The introduction will discuss the importance of lung-on-chip as a tool to study inhaled drug permeability, particularly noting the potential to monitor parameters such as physiological environment, dynamic mechanical and shear stress, transepithelial electrical resistance, and live imaging under perfusion. The authors provide and critically assess notable examples of both cell line and primary models, before discussing the importance of LOC advances and technical challenges that remain.

Aims: We sought to develop a new method that enables the assessment of the immune response of guinea pigs during TB vaccine evaluation studies, without the need to cull or anaesthetize animals. Method and Results: Guinea pigs were vaccinated with five different formulations of oral BCG. One week prior to challenge with Mycobacterium bovis, blood (50200 mu l) was taken from the ears of vaccinated subjects. Host RNA was isolated and amplified following antigenic restimulation of PBMCs for 24 h with 30 mu g of bovine PPD. The up- or down-regulation of gamma-interferon (IFN-gamma), a key cytokine involved in protection against tuberculosis, was assessed using real-time PCR. The relative expression of prechallenge IFN-gamma mRNA in the vaccinated groups (n = 5) correlated (P < 0.001) with protection against M. bovis challenge. Conclusion: We have demonstrated that it is possible to take blood samples and track IFN-gamma responses in guinea pigs that then go on to be exposed to M. bovis, thus providing prechallenge vaccine uptake information. Significance and Impact of the Study: This methodology will also be applicable for tracking the immune responses of vaccinated guinea pigs over time that then go on to be challenged with M. tuberculosis during human TB vaccine evaluation studies.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, represents a major animal health issue. In the United Kingdom and the Republic of Ireland, European badgers (Meles meles) have been shown to act as a reservoir of M. bovis infection, hindering the eradication of bTB in livestock. The availability of suitable diagnostic assays, particularly those that may be applied in a “trap-side” setting, would facilitate the implementation of a wider range of disease control strategies. Here we evaluate the Dual Path Platform (DPP) VetTB assay, a lateral-flow type test for detecting antibodies to M. bovis antigens (MPB83 and ESAT-6/CFP-10). Both serum and whole blood were evaluated as diagnostic samples. Additionally, two methods were evaluated for interpretation of test results (qualitative interpretation by eye and quantitative measurement using an optical reader). The antibody response to MPB83 detected by the DPP VetTB assay increased significantly following experimental M. bovis infection of badgers, whilst the response to ESAT-6/CFP-10 showed no significant change. In sera from TB-free captive and naturally M. bovis infected wild badgers the MPB83 response exhibited a sensitivity of 55 % by eye and quantitative reader (95 % CI: 40–71 and 38–71, respectively), with slightly lower specificity when read by eye (93 % compared to 98 %; 95 % CI: 85–100 and 90–100, respectively). In whole blood, the DPP VetTB assay MPB83 response exhibited a sensitivity of 65 % (95 % CI: 50–80) when interpreted by eye and 53 % (95 % CI: 36–69) using quantitative values, whilst the specificity was 94 % and 98 % respectively (95 % CI: 88–100 and 90–100). Comparison with contemporaneous diagnostic test results from putatively naturally infected and TB-free badgers demonstrated varying levels of agreement. Using sera from naturally M. bovis infected and TB-free badgers, with post mortem confirmation of disease status, the DPP VetTB assay exhibited a sensitivity of 60 % (95 % CI: 41–77) when interpreted using quantitative values (specificity 95 %; 95 % CI: 76–100), and 67 % (95 % CI: 50–84) when read by eye (specificity 95 %; 95 % CI: 86–100). Further work is required to robustly characterize the DPP VetTB assay’s performance in a wider selection of samples, and in the practical and epidemiological contexts in which it may be applied.

This datasheet on tuberculosis covers Identity, Overview, Associated Diseases, Pests or Pathogens, Distribution, Hosts/Species Affected, Diagnosis, Pathology, Epidemiology, Prevention/Control, Further Information.

Accurate detection of infection with Mycobacterium bovis in live badgers would enable targeted tuberculosis control. Practical challenges in sampling wild badger populations mean that diagnosis of infection at the group (rather than the individual) level is attractive. We modelled data spanning 7 years containing over 2000 sampling events from a population of wild badgers in southwest England to quantify the ability to correctly identify the infection status of badgers at the group level. We explored the effects of variations in: (1) trapping efficiency; (2) prevalence of M. bovis; (3) using three diagnostic tests singly and in combination with one another; and (4) the number of badgers required to test positive in order to classify groups as infected. No single test was able to reliably identify infected badger groups if 80% sensitive, at least 94% specific, and able to be performed rapidly in the field.

Antimicrobial-resistant bacteria are a growing global healthcare threat. Uptake of appropriate infection prevention and control (IPC) measures is heavily influenced by human risk perception, consequent behaviour and the ways humans and animals interact within the environment. Effective IPC communication and teaching tools are necessary to ensure that individuals’ understanding and behaviours are in line with scientific recommendations. This chapter describes a novel approach to developing an IPC training tool to raise the perception and understanding of risk of infection to animal patients during routine veterinary surgical procedures. The researchers ‘made the invisible visible’, revealing bacterial contamination sources and their spread during preparation for surgery via a dynamic 3-layer interactive virtual model of a veterinary practice based on real-world data on human, animal and bacterial interactions. They used a serious storytelling approach, visualisation, simple gamification techniques and a collaborative design approach to engage students, nurses and surgeons from the veterinary community in the co-development of the tool. Participants were invited to identify risky behaviours, direct and indirect sources of bacterial contamination, and were prompted to reflect on the potential consequences of poor or improved IPC measures on the patient outcome and residual bacterial contamination in the practice environment. The study was conducted over two phases. Phase 1 achieved proof-of-concept: at evaluation, 92% of 51 trial participants stated an intention to change their behaviour and to implement infection controls that aligned with training objectives. In Phase 2, the tool was enhanced, and software was developed to a beta-version to enable self-paced training on web-based and mobile platforms. The co-development and evaluation process, importance of end-user engagement throughout and findings are discussed.

The infection of both captive and free-ranging wildlife species with pathogenic mycobacteria (including Mycobacterium tuberculosis) poses a zoonotic risk and continues to cause challenges for the livestock industry, zoos and governments around the world. Central to the management and control of tuberculosis is timely and accurate diagnosis. In many cases, bacterial culture is insufficiently sensitive and confirmation of TB post-mortem is neither feasible nor desirable. In this context, there is still considerable research interest in, and need for, immunological methods for diagnosis. Reviews on this topic were published in 2005 and 2009, but since then veterinarians and other researchers have continued to evaluate immunodiagnostic approaches to TB. These include serological tests such as lateral-flow devices, and enzyme-linked immunosorbent assay (ELISA) and those based on evaluation of cell-mediated immunity, such as the tuberculin skin test and interferon-gamma release assay (IGRA). Since 2009, the range of publications on this topic has been extended to a number of new species, including South American camelids, black rhinoceros, lions and non-human primates. Therefore, it seemed appropriate to review the literature in the 3 years since 2009 and provide an overview of progress.

Background An oral vaccine is a potential tool to tackle the reservoir of Mycobacterium bovis in European badgers (Meles meles), which contributes to tuberculosis of cattle in the British Isles. Inferences about vaccine protection against experimental challenge with M. bovis depend on the measurement of tuberculosis. Assessment of tuberculosis in larger species, such as badgers, is typically based on the tuberculous lesions visible at post-mortem examination and histopathology. We have developed a robust scoring system for tuberculous lesions by combining several parallel measures, which we call the “disease burden score” (DBS). Methods Alternative scoring systems were compared within a regression analysis applied to observations from a total of 168 badgers from eight studies, including 107 badgers subjected to vaccination treatment and 61 non-vaccinated controls. The analysis included incidental observations that were recorded from each badger as potential covariate factors explaining some of the variation among animals sourced from the wild. Results DBS was found to be the most accurate and reliable of the scoring systems compared. By taking account of significant covariates affecting disease, application of the DBS reduced residual variance by 22.9%. A previously used measure, based on assessment of visible lesions, was suboptimal due to non-uniform variance that increased with expected value, although square root transformation addressed this issue. The covariate model fitted to DBS included sex (males had higher DBS), weight (negatively associated with DBS) and immunological evidence of prior exposure to Mycobacterium avium (positively associated with DBS). Conclusions We identified improved measures of tuberculous disease derived from data already collected. We also demonstrated that the proper scaling of measurements of disease in such models is necessary and can be determined empirically. The covariates which were most strongly associated with the severity of disease are important in experimental studies involving outbred animals with variable background.

In approaching the development of a veterinary vaccine, researchers must choose from a bewildering array of options that can be combined to enhance benefit. The choice and combination of options is not just driven by efficacy, but also consideration of the cost, practicality, and challenges faced in licensing the product. In this review we set out the different choices faced by veterinary vaccine developers, highlight some issues, and propose some pressing needs to be addressed.

Polyomaviruses infect a diverse range of mammalian and avian hosts, and are associated with a variety of symptoms. However, it is unknown whether the viruses are found in all mammalian families and the evolutionary history of the polyomaviruses is still unclear. Here, we report the discovery of a novel polyomavirus in the European badger (Meles meles), which to our knowledge represents the first polyomavirus to be characterized in the family Mustelidae, and within a European carnivoran. Although the virus was discovered serendipitously in the supernatant of a cell culture inoculated with badger material, we subsequently confirmed its presence in wild badgers. The European badger polyomavirus was tentatively named Meles meles polyomavirus 1 (MmelPyV1). The genome is 5187 bp long and encodes proteins typical of polyomaviruses. Phylogenetic analyses including all known polyomavirus genomes consistently group MmelPyV1 with California sea lion polyomavirus 1 across all regions of the genome. Further evolutionary analyses revealed phylogenetic discordance amongst polyomavirus genome regions, possibly arising from evolutionary rate heterogeneity, and a complex association between polyomavirus phylogeny and host taxonomic groups.

The aim of this study was to determine the reliability of lactic acid bacteria (LAB) as heterologous hosts for the expression of MPB70 and MPB83, two Mycobacterium bovis antigens that possess diagnostics and immunogenic properties, respectively. We therefore generated recombinant cells of Lactococcus lactis and Lactobacillus plantarum that carried hybrid genes encoding MPB70 and MPB83 fused to signal peptides that are specifically recognized by LAB. Only L. lactis was able to secrete MPB70 using the L. lactis signal peptide Usp45, and to produce MPB83 as an immunogenic membrane protein following its expression with the signal peptide of the L. plantarum lipoprotein prsA. Inactivated cells of MPB83-expressing L. lactis cultures enhanced NF-κB activation in macrophages. Our results show that L. lactis is a reliable host for the secretion and functional expression of antigens that are naturally produced by M. bovis, the causative agent of bovine tuberculosis (bTB). This represents the first step on a long process to establishing whether recombinant LAB could serve as a food-grade platform for potential diagnostic tools and/or vaccine interventions for use against bTB, a chronic disease that primarily affects cattle but also humans and a wide range of domestic and wild animals.

Bovine tuberculosis is one of the biggest challenges facing cattle farming in Great Britain. European badgers (Meles meles) are a reservoir host for the causal agent, Mycobacterium bovis. There have been significant recent advances in diagnostic testing for tuberculosis in humans, cattle and badgers, with the development of species-specific assays for interferon-γ (IFN-γ), an important cytokine in tuberculous infections. Using data collected from longitudinal studies of naturally infected wild badgers, we report that the magnitude of the IFN-γ response to M. bovis antigens at the disclosing test event was positively correlated with subsequent progression of disease to a seropositive or excreting state. In addition, we show that the magnitude of the IFN-γ response, despite fluctuation, declined with time after the disclosing event for all badgers, but remained significantly higher in those animals with evidence of disease progression. We discuss how our findings may be related to the immunopathogenesis of natural M. bovis infection in badgers.

Background: Bovine tuberculosis (bTB) caused by Mycobacterium bovis is the most serious endemic disease affecting livestock in the UK. The European badger (Meles meles) is the most important wildlife reservoir of bTB transmission to cattle, making eradication particularly difficult. In this respect, oral vaccination with the attenuated M. bovis vaccine Bacillus Calmette-Guerin (BCG) has been suggested as a wide-scale intervention to reduce bTB infection in badgers. However, experimental studies show variable protection. Among the possibilities for this variation is that the resident gut bacteria may influence the success of oral vaccination in badgers; either through competitive exclusion and/or inhibition, or via effects on the host immune system. In order to explore this possibility, we have tested whether typical gut commensals such as Lactic Acid Bacteria (LAB) have the capacity to impact on the viability and survival rate of BCG and to modulate the immune response to BCG using an in vitromodel. Results: Twelve LAB isolated from badger faeces displayed inhibitory activity to BCG that was species-dependent. Weissella had a bacteriostatic effect, whereas isolates of enterococci, lactobacilli and pediococci had a more bactericidal activity. Furthermore, BCG-induced activation of the pro-inflammatory transcription factor NF-κB in human THP-1 macrophages was modulated by LAB in a strain-dependent manner. Most pediococci enhanced NF-κB activation but one strain had the opposite effect. Interestingly, isolates of enterococci, lactobacilli and weissella had different effects as immunomodulators of BCG-induced macrophage responses as some had no significant influence on NF-κB activation, but others increased it significantly. Conclusions: Our in vitro results show that LAB isolated from badgers exhibit significant inhibitory activity against BCG and influence the immune activation mediated by BCG in a human macrophage assay. These findings suggest that gut commensal bacteria could play a role in influencing the outcome of oral BCG vaccination. Inactivated cells of LAB, or LAB that are bacteriostatic but have a synergistic immunostimulatory effect with BCG, could be potential adjuvants to be used for oral vaccination in badgers. Further work is needed to take into account the complex nature of the gut microbiome, specific immunity of the badger and the in vivo context.

The aim of this study was to compare protection by bacille Calmette-Guérin (BCG) vaccination against aerosol challenge with either Mycobacterium bovis or Mycobacterium tuberculosis in guinea pigs. Animals were challenged 5 weeks after vaccination with 10 or 100 lung lesion—forming units (lfu) of M. bovis or M. tuberculosis. Four weeks after challenge, numbers of lung lesions and counts of viable mycobacteria in spleens were high in saline-immunized animals. In contrast, BCG vaccination resulted in fewer lung lesions; after challenge with 10 and 100 lfu, the reduction was greater in animals infected with M. bovis (mean number of lesions, 1.17 and 31.2, respectively; P < .02) than in those infected with M. tuberculosis (mean number of lesions, 16.2 and 75.8, respectively). No mycobacteria were recovered from spleens of BCG-vaccinated animals after challenge with 10 lfu of M. bovis, whereas 4 of 6 animals had detectable spleen mycobacterial counts after challenge with M. tuberculosis. Collectively, the results suggest that BCG vaccination may confer greater protection against challenge with M. bovis than challenge with M. tuberculosis.

A scientific review of bovine tuberculosis in Great Britain has concluded that the development of a cattle vaccine holds the best prospect for long-term disease control. Recent reports of successful DNA vaccination against Mycobacterium tuberculosis in small animal models have raised the possibility of using a similar strategy to produce vaccines against Mycobacterium bovis infection in cattle. To test this possibility, BALB/c mice were immunized with DNA encoding the M. bovis antigen MPB83. The mice responded to vaccination with a mixed IgG1/IgG2a response to the antigen and were protected from intravenous challenge with virulent M. bovis to a similar extent as those vaccinated with bacille Calmette-Guérin. The immunogenicity of the DNA vaccine in cattle was tested, after having established that DNA encoding MPB83 was immunogenic and elicited protective immunity in mice. In these studies, vaccinated animals had strong proliferative responses to MPB83.

The gut microbiota play a key role in the health of animals and humans. However, the dynamic properties and stability of the microbiota are poorly understood. We propose a regression technique for parameter inference of a mechanistic model to describe the temporal dynamics of these microbial communities. The model could be used for measuring community resilience against external perturbing factors, such as antibiotic therapy.

Recent studies suggest that badgers may be a potential reservoir of Mycobacterium bovis infection for cattle in Northern Spain. The objective of this study was to investigate potential epidemiological links between cattle and badgers. Culture and molecular typing data were available for cattle culled during the national tuberculosis (TB) eradication campaigns between 2008 and 2012, as well as from 171 necropsied badgers and 60 live animals trapped and examined over the same time period. Mycobacterium tuberculosis complex strains were isolated from pooled tissues of 14 (8.2%) necropsied badgers, of which 11 were identified as M. bovis: six different spoligotypes of M. bovis were subsequently identified. In two geographical locations where these isolates were shared between cattle and badgers, infected cattle herds and badgers lived in close contact. Although it remains unclear if badgers are a maintenance or spill-over host of M. bovis in this setting, it would appear prudent to have precautionary measures in place to reduce contact between cattle and badgers.

Heterogeneity in the progression of disease amongst individual wild animals may impact on both pathogen and host dynamics at the population level, through differential effects on transmission, mortality and reproductive output. The role of the European badger (Meles meles) as a reservoir host for Mycobacterium bovis infection in the UK and Ireland has been the focus of intense research for many years. Here, we investigate life-history correlates of infection in a high-density undisturbed badger population naturally infected with M. bovis. We found no evidence of a significant impact of M. bovis infection on female reproductive activity or success, with evidence of reproduction continuing successfully for several years in the face of M. bovis excretion. We also found evidence to support the hypothesis that female badgers are more resilient to established M. bovis infection than male badgers, with longer survival times following the detection of bacterial excretion. We discuss the importance of infectious breeding females in the persistence of M. bovis in badger populations, and how our findings in male badgers are consistent with testosterone-induced immunosuppression. In addition, we found significant weight loss in badgers with evidence of disseminated infection, based on the culture of M. bovis from body systems other than the respiratory tract. For females, there was a gradual loss of weight as infection progressed, whereas males only experienced substantial weight loss when infection had progressed to the point of dissemination. We discuss how these differences may be explained in terms of resource allocation and physiological trade-offs.

European badgers are a wildlife reservoir of bovine tuberculosis in parts of Great Britain. Accurate diagnosis of tuberculosis in badgers is important for the development of strategies for the control of the disease. Sensitive serological tests for badger TB are needed for reasons such as cost and simplicity. Assay of mucosal IgA could be useful for diagnosing respiratory pathogens such as Mycobacterium bovis and for monitoring the response to mucosal vaccination. To develop an IgA assay, we purified secretory IgA from badger bile, identifying secretory component (SC), heavy chain (HC) and light chain (LC), at 66, 46 and 27 Kda, respectively, on the basis of size comparison with other species. Monoclonal antibodies (mAbs) were generated to purified IgA.We selected two for ELISA development. The detection limit of the IgA-specific mAbs was found to be approximately 20 ng/mL when titrated against purified badger bile. One monoclonal antibody specific for badger IgA was used to detect IgA in serum and tracheal aspirate with specificity to an immunodominant antigen of M. bovis. An M. bovis infection dose-dependent IgA response was observed in experimentally infected badgers. IgA was also detected by immunohistochemistry in the lungs of bTB-infected badgers. With further characterisation, these represent new reagents for the study of the IgA response in badgers.

A mass was identified within the left lateral lobe of the liver of a 10-year-old Eurasian badger (Meles meles). The mass was Friable and multilobulated, with blood-filled spaces between the lobules. Microscopically, the lesion consisted of sheets and trabeculae of neoplastic hepatocytes often forming cystic spaces containing erythrocytes, fibrin and necrotic debris. The histological appearance was consistent with hepatocellular carcinoma (HCC). Immunohistochemically, the neoplastic cells expressed cytokeratin 18 but riot von Willebrand factor. Multiple intranuclear (amphophilic or acidophilic) inclusion bodies were observed in hepatocytes at the junction between the tumour and normal hepatic tissue. HCCs have also been reported in other domestic and wild animals. As hepadnavirus infection has been associated with HCC in woodchucks, further histochemical and transmission electron microscopical Studies were performed; however, these demonstrated that the inclusions consisted of lipid droplets and not viral particles. To our knowledge, this is the first report of a naturally occurring HCC in a Eurasian badger. Crown Copyright (C) 2009 Published by Elsevier Ltd. All rights reserved.

Mycobacterium bovis Bacillus Calmette–Guérin (M. bovis BCG) was generated over a century ago for protection against Mycobacterium tuberculosis (Mtb) and is one the oldest vaccines still in use. The BCG vaccine is currently produced using a pellicle growth method, which is a complex and lengthy process that has been challenging to standardise. Fermentation for BCG vaccine production would reduce the complexity associated with pellicle growth and increase batch to batch reproducibility. This more standardised growth lends itself to quantification of the total number of bacilli in the BCG vaccine by alternative approaches, such as flow cytometry, which can also provide information about the metabolic status of the bacterial population. The aim of the work reported here was to determine which batch fermentation conditions and storage conditions give the most favourable outcomes in terms of the yield and stability of live M. bovis BCG Danish bacilli. We compared different media and assessed growth over time in culture, using total viable counts, total bacterial counts, and turbidity throughout culture. We applied fluorescent viability dyes and flow cytometry to measure real-time within-culture viability. Culture samples were stored in different cryoprotectants at different temperatures to assess the effect of these combined conditions on bacterial titres. Roisin’s minimal medium and Middlebrook 7H9 medium gave comparable, high titres in fermenters. Flow cytometry proved to be a useful tool for enumeration of total bacterial counts and in the assessment of within-culture cell viability and cell death. Of the cryoprotectants evaluated, 5% (v/v) DMSO showed the most significant positive effect on survival and reduced the negative effects of low temperature storage on M. bovis BCG Danish viability. In conclusion, we have shown a reproducible, more standardised approach for the production, evaluation, and storage of high titre, viable, BCG vaccine.

Alveolar type II (ATII) cells play a key role as part of the distal lung epithelium, including roles in the innate immune response and as self-renewing progenitors to replace alveolar type I (ATI) cells during regeneration of the alveolar epithelium. Their secretion of surfactant protein helps to maintain homeostasis in the distal lung and exert protective, antimicrobial properties. Despite the cell’s crucial roles, they remain difficult to study, in part due to inefficient and expensive isolation methods, a propensity to differentiate into alveolar type I cells in culture and susceptibility to fibroblast overgrowth from primary isolations. Published methods of isolation often require specialist technology, negatively impacting the development of in vitro models of disease, including bovine tuberculosis (BTB), a serious re-emerging disease in both animals and humans worldwide. We present here a simple and cost effective method that may be utilised in the generation of bovine primary ATII cells. These exhibit an ATII phenotype in 2D and 3D culture in our studies and are conducive to further study of the role of ATII cells in bovine respiratory diseases.

Human papillomaviruses (HPVs) are strictly intraepithelial pathogens: in the natural productive infection they induce benign epithelial proliferations of mucocutaneous surfaces, some of which may progress to malignancy. Benign HPV-induced lesions are chronic persistent growths; high levels of viral antigen are expressed in the apparent absence of a host immune response suggesting that these viruses have evolved efficient mechanisms of immune evasion. Cell-mediated responses are central in the pathogenesis of HPV and regression of both cutaneous and genital warts histologically resembles a delayed-type hypersensitivity response (DTH). The antigen(s) in the wart against which this response is initiated are not known but in an experimental murine model DTH responses to the E6 and E7 proteins of HPV-16 can be elicited when viral antigen is presented via the epithelial route. Priming with low levels of viral antigen in this model induces non-responsiveness and the loss of DTH. In HPV-associated cancers the E6/E7 genes are expressed and an antibody response to the proteins is found in at least 50% of cases indicating that these oncoproteins are potential targets for immunotherapy.

We describe epidemiological trends in Mycobacterium bovis infection in an undisturbed wild badger (Meles meles) population. Data were derived from the capture, clinical sampling and serological testing of 1803 badgers over 9945 capture events spanning 24 years. Incidence and prevalence increased over time, exhibiting no simple relationship with host density. Potential explanations are presented for a marked increase in the frequency of positive serological test results. Transmission rates (R0) estimated from empirical data were consistent with modelled estimates and robust to changes in test sensitivity and the spatial extent of the population at risk. The risk of a positive culture or serological test result increased with badger age, and varied seasonally. Evidence consistent with progressive disease was found in cubs. This study demonstrates the value of long-term data and the repeated application of imperfect diagnostic tests as indices of infection to reveal epidemiological trends in M. bovis infection in badgers.

Vaccine hesitancy does not only concern human vaccines but incorporates One Health policies also; including vaccination of cattle and badgers as part of the government's bovine tuberculosis eradication strategy for England. Both digital and social media can propagate healthcare misinformation and thus affect vaccine policy support. The use of social media monitoring to understand real-time public perceptions of One Health policies is crucial to identify misinformation and address public concerns appropriately to achieve successful policy implementation. Digital and social media data surrounding two government announcements regarding the bovine tuberculosis eradication strategy for England were collected and screened using the Meltwater media monitoring platform. Communication patterns were studied using InfraNodus. Twitter analysis was conducted to identify key influencers, public engagement, and trending communications. Online social media activity increased rapidly after each announcement. Initially, badger culling took primary public concern and major influencers were identified. Cattle vaccination dominated discussion after the second announcement, with public perception being influenced by increased online activity from news sites, animal welfare charities, governmental bodies, and medical professionals. The greatest ambiguity towards the strategy was detected within farming communities, with the main disparity existing between cattle vaccination and badger culling opinions. Social media monitoring has potential use in surveying public perception of government policy, both prior to, and after implementation to identify and address areas of miscommunication and misinformation to improve public support for One Health policies.

Background: In parts of Great Britain and Ireland, Eurasian badgers (Meles meles) constitute a reservoir of Mycobacterium bovis infection and a potential source of infection for cattle. In vitro diagnostic tests for live badgers are an important component of strategies to control TB in this species. Immunological tests have been developed for badgers, although little is known about the influence of the age of the animal on test performance. To address this, we evaluated the performance of three immunological tests for badgers with respect to the age of the animal: the Brock Test and BrockTB STAT-PAK (R) serological tests and the recently developed interferon-gamma enzyme immunoassay (IFN gamma EIA). Data published elsewhere suggested that seropositivity was associated with more progressive forms of TB in the badger. To gain further evidence for this, we used longitudinal data from a well-studied population of badgers to test for an association between the sensitivity of the Brock Test and the duration of TB infection. Results: Sensitivity of the two serological tests was approximately 54% for both cubs and adults. Sensitivity of the IFN gamma EIA was lower in cubs (57%) compared with adults (85%) when a common cut-off value was used to define test positivity. Taking data from the cubs alone, the IFN gamma EIA cut-off value could be adjusted to increase the sensitivity to 71% with no loss in specificity. As a general observation, specificity of all tests was higher in cubs, although only significantly so in the case of the Brock Test. Using logistic regression analysis to adjust for age, sensitivity of the Brock Test was significantly lower at first culture positive event (58%), but increased to >80% as infection progressed. Conclusion: These data suggest that serodiagnosis could be a valuable tool for detecting a higher proportion of badgers with the greatest probability of transmitting infection. The age category of the badger appeared to exert little influence on the performance of the serological tests. Although data were only available for the IFN gamma EIA in a small number of cubs, reduced sensitivity of the test in these individuals suggests a lower cut-off may be needed when testing younger animals.

Sulphur is essential for some of the most vital biological activities such as translation initiation and redox maintenance, and genes involved in sulphur metabolism have been implicated in virulence. Mycobacterium tuberculosis has three predicted genes for the prototrophic acquisition of sulphur as sulphate: cysA, part of an ABC transporter, and cysA2 and A3, SseC sulphotransferases. Screening for amino acid auxotrophs of Mycobacterium bovis BCG, obtained by transposon mutagenesis, was used to select methionine auxotrophs requiring a sulphur-containing amino acid for growth. We have characterized one of these auxotrophs as being disrupted in cysA. Both the cysA mutant and a previously identified mutant in an upstream gene, subI, were functionally characterized as being completely unable to take up sulphate. Complementation of the cysA mutant with the wild-type gene from M. tuberculosis restored prototrophy and the ability to take up sulphate with the functional characteristics of an ABC transporter. Hence, it appears that this is the sole locus encoding inorganic sulphur transport in the M. tuberculosis complex.

Wildlife is a global source of endemic and emerging infectious diseases. The control of tuberculosis (TB) in cattle in Britain and Ireland is hindered by persistent infection in wild badgers (Meles meles). Vaccination with Bacillus Calmette-Guerin (BCG) has been shown to reduce the severity and progression of experimentally induced TB in captive badgers. Analysis of data from a four-year clinical field study, conducted at the social group level, suggested a similar, direct protective effect of BCG in a wild badger population. Here we present new evidence from the same study identifying both a direct beneficial effect of vaccination in individual badgers and an indirect protective effect in unvaccinated cubs. We show that intramuscular injection of BCG reduced by 76% (Odds ratio = 0.24, 95% confidence interval (CI) 0.11-0.52) the risk of free-living vaccinated individuals testing positive to a diagnostic test combination to detect progressive infection. A more sensitive panel of tests for the detection of infection per se identified a reduction of 54% (Odds ratio = 0.46, 95% CI 0.26-0.88) in the risk of a positive result following vaccination. In addition, we show the risk of unvaccinated badger cubs, but not adults, testing positive to an even more sensitive panel of diagnostic tests decreased significantly as the proportion of vaccinated individuals in their social group increased (Odds ratio = 0.08, 95% CI 0.01-0.76; P = 0.03). When more than a third of their social group had been vaccinated, the risk to unvaccinated cubs was reduced by 79% (Odds ratio = 0.21, 95% CI 0.05-0.81; P = 0.02).

The Eurasian badger ( Meles meles) is a significant wildlife reservoir of Mycobacterium bovis in Great Britain. Improved control strategies against the disease in badgers require the development of diagnostic tests and vaccines. Here, we report the development of a comparative lymphocyte transformation assay (LTA) using bovine and avian tuberculin as antigen to detect cell-mediated responses in M. bovis-infected badgers. In a pilot study, the performance of this assay was compared with the existing indirect ELISA assay for the detection of tuberculous badgers. The sensitivity of the Comparative LTA was 87.5% compared with 62.5% for the indirect ELISA whereas the ELISA test gave a greater specificity (100% compared with 84.6% for the comparative LTA). Preliminary evidence suggests that for the comparative LTA, the blood may be stored overnight prior to testing and that this procedure might improve the specificity of the assay without compromising the sensitivity.

The European badger (Meles meles) is a reservoir host of Mycobacterium bovis and responsible for a proportion of the tuberculosis (TB) cases seen in cattle in the United Kingdom and Republic of Ireland. An injectable preparation of the bacillus Calmette-Guérin (BCG) vaccine is licensed for use in badgers in the UK and its use forms part of the bovine TB eradication plans of England and Wales. However, there are practical limitations to the widespread application of an injectable vaccine for badgers and a research priority is the development of an oral vaccine deliverable to badgers in bait. Previous studies reported the successful vaccination of badgers with oral preparations of 108 colony forming units (CFU) of both Pasteur and Danish strains of BCG contained within a lipid matrix composed of triglycerides of fatty acids. Protection against TB in these studies was expressed as a reduction in the number and apparent progression of visible lesions, and reductions in the bacterial load and dissemination of infection. To reduce the cost of an oral vaccine and reduce the potential for environmental contamination with BCG, it is necessary to define the minimal efficacious dose of oral BCG for badgers. The objectives of the two studies reported here were to compare the efficacy of BCG Danish strain in a lipid matrix with unformulated BCG given orally, and to evaluate the efficacy of BCG Danish in a lipid matrix at a ten-fold lower dose than previously evaluated in badgers. In the first study, both BCG unformulated and in a lipid matrix reduced the number and apparent progression of visible lesions and the dissemination of infection from the lung. In the second study, vaccination with BCG in the lipid matrix at a ten-fold lower dose produced a similar outcome, but with greater intra-group variability than seen with the higher dose in the first study. Further research is needed before we are able to recommend a final dose of BCG for oral vaccination of badgers against TB or to know whether oral vaccination of wild badgers with BCG will significantly reduce transmission of the disease.

Elemental and molecular imaging play a crucial role in understanding disease pathogenesis. To accurately correlate elemental and molecular markers, it is desirable to perform sequential elemental and molecular imaging on a single tissue section. However, very little is known about the impact of performing these measurements in sequence. In this work, we highlight some of the challenges and successes associated with performing elemental mapping in sequence with mass spectrometry imaging. Specifically, the feasibility of molecular mapping using the mass spectrometry imaging (MSI) techniques matrix assisted laser desorption ionisation (MALDI) and desorption electrospray ionisation (DESI) in sequence with the elemental mapping technique particle induced X-ray emission (PIXE) is explored. Challenges for integration include substrate compatibility, as well as delocalisation and spectral changes. We demonstrate that whilst sequential imaging comes with some compromises, sequential DESI-PIXE imaging is sufficient to correlate sulphur, iron and lipid markers in a single tissue section at the 50-micrometre scale.

Subunit vaccines against tuberculosis show promise but require administration with adjuvants to stimulate relevant immune responses for protection. Guinea pigs are the model of choice for evaluating protective immunity to aerogenic challenge with virulent mycobacteria, but few studies have been undertaken to identify suitable adjuvants for vaccine screening in this species. Here, we compare the efficacy of several adjuvants to induce T cell responses to culture filtrate protein in guinea pigs. We report that of several adjuvants tested, the most promising was CpG ODN formulated in an aqueous emulsion. This adjuvant induced type 1 T cell responses equivalent to that of FIA, as measured by delayed-type hypersensitivity reactions (DTH), antigen-specific T cell proliferation and antigen-specific IgG1 and IgG2 responses. These data demonstrate the potential for CpG motif based adjuvants for use in TB vaccine screening in guinea pigs, and other diseases where a type 1 T cell response is required.

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

Wildlife species, such as the badger ( Meles meles), may act as maintenance hosts for Mycobacterium bovis and contribute to the spread and persistence of tuberculosis in associated cattle populations. Targeted vaccination of badgers against tuberculosis is an option that, if successfully employed, could directly facilitate the advancement of bovine tuberculosis eradication in affected areas. In this study, the immunological responses of a group of badgers vaccinated subcutaneously with low doses of Mycobacterium bovis bacillus calmette guerin (BCG) were measured in vitro and compared with non-vaccinated control animals over a period of 42 weeks. Peripheral blood mononuclear cells (PBMC) from badgers which had received repeated booster injections of BCG proliferated in response to culture with PPD-bovine (purified protein derivative of tuberculin). The proliferation was significantly greater than that seen in the non-vaccinated control group. In contrast, the proliferative response of PBMC from vaccinated badgers to PPD-avian declined relative to the control group. These results demonstrate that repeated vaccination of badgers with M. bovis BCG induced a population of T-lymphocytes responsive to specific antigens in PPD-bovine. Throughout the course of the study, the sera from all animals were tested (BrockTest) by an enzyme-linked immunosorbent assay (ELISA) system for the presence of antibodies to MPB83, a serodominant antigen whose expression is high in M. bovis, but very low in BCG (Pasteur). No animals at any stage showed seroconversion to the antigen, consistent with the tuberculosis-free status of the badgers under study.

Nucleic acid (DNA) vaccination against tuberculosis in the European badger (Meles meles) is one approach to addressing the escalating problem of bovine tuberculosis in Great Britain. The aim of vaccination is to reduce the burden of tuberculosis within the badger population and the shedding of Mycobacterium bovis to levels that would break the transmission of infection to cattle. To this end, the vaccine would be required to limit the amount of disseminated tuberculosis in the badger, especially dissemination to the kidney from where M bovis can be shed in the urine. A promising candidate DNA vaccine encoding a 26kDa major antigen (MPB 83) of M bovis was evaluated in a mouse model of disseminated M bovis infection. Using the DNA vaccine, protection against infection of the kidney was found to be greater than that achieved with the current live vaccine, Bacille Calmette-Guerı́n (BCG). Kidney tissue and skeletal muscle from the badger was used to derive primary cell cultures in which to examine the expression ofMPB 83 following transfection with the DNA vaccine. Kidney cortex gave rise to a monotypic culture of epithelial cells whilst the muscle gave rise to a mixed culture of fibroblasts and myoblasts. During culture the myoblasts differentiated into multinucleated myotubes, verified by immunofluorescent detection of mammalian desmin. Successful expression of MPB 83 by transfected epithelial and myotube cells was confirmed by immunofluorescence using a monoclonal antibody specific to the protein. These observations fulfil the early requirements for the development of a DNA vaccine for badger tuberculosis.

Alveolar type II (ATII) cells play a key role as part of the distal lung epithelium, including in the innate immune response and as self‐renewing progenitors to replace alveolar type I (ATI) cells during epithelial regeneration. Their secretion of surfactant protein helps maintain homeostasis and exerts protective, antimicrobial properties. ATII cells remain difficult to study, partly due to inefficient and expensive isolation methods, a propensity to differentiate into ATI cells, and susceptibility to fibroblast contamination. Published methods of isolation often require specialized technology, negatively impacting the development of in vitro models of disease, including bovine tuberculosis. Presented here is a simple and cost‐effective method for generation of bovine primary ATII cells. These cells exhibit an ATII phenotype in 2D and 3D culture and are conducive to further study of the role of ATII cells in bovine respiratory diseases.

The deployment of baits containing vaccines or toxins has been used successfully in the management of wildlife populations, including for disease control. Optimisation of deployment strategies seeks to maximise uptake by the targeted population whilst ensuring cost effectiveness. Tuberculosis (TB) caused by infection with Mycobacterium bovis affects a broad range of mammalian hosts across the globe, including cattle, wildlife and humans. The control of TB in cattle in the UK and Republic of Ireland is hampered by persistent infection in European badgers (Meles meles). The present study aimed to determine the best strategy for maximising uptake of an oral vaccine by wild badgers, using a surrogate novel bait deployed at 40 badger social groups. Baits contained a blood-borne biomarker (Iophenoxic Acid, IPA) in order to measure consumption in badgers subsequently cage trapped at targeted setts. Evidence for the consumption of bait was found in 83% (199/240) of captured badgers. The probability that badgers had consumed at least one bait (IPA >10 μg ml-1) was significantly higher following deployment in spring than in summer. Lower uptake amongst social groups where more badgers were captured, suggested competition for baits. The probability of bait consumption was significantly higher at groups where main and outlier setts were provided with baits than at those where outliers were present but not baited. Badgers captured 10–14 days post bait feeding had significantly higher levels of bait uptake compared to those caught 24–28 days later. Uptake rates did not vary significantly in relation to badger age and whether bait was placed above ground or down setts. This study suggests that high levels of bait uptake can be achieved in wild badger populations and identifies factors influencing the potential success of different deployment strategies. The implications for the development of an oral badger vaccine are discussed.

The behaviour of certain infected individuals within socially structured populations can have a disproportionately large effect on the spatio-temporal distribution of infection. Endemic infection with Mycobacterium bovis in European badgers (Meles meles) in Great Britain and Ireland is an important source of bovine tuberculosis in cattle. Here we quantify the risk of infection in badger cubs in a high-density wild badger population, in relation to the infection status of resident adults. Over a 24-year period, we observed variation in the risk of cub infection, with those born into groups with resident infectious breeding females being over four times as likely to be detected excreting M. bovis than cubs from groups where there was no evidence of infection in adults. We discuss how our findings relate to the persistence of infection at both social group and population level, and the potential implications for disease control strategies.

Sexually transmitted diseases (STDs) can be important drivers of population dynamics because of their negative effects on reproduction. However, screening for STDs, especially in wildlife populations, is widely neglected. Using the promiscuous, polygynandrous European badger (Meles meles) as a model, we investigated the presence and prevalence of herpesviruses (HVs) in a wild, high-density population and assessed potential differences in somatic fitness and female reproductive condition between infected and uninfected individuals. We collected n=98 genital swabs from 71 females (51 adults and 20 cubs) and 27 males (26 adults and 1 cub) during spring and summer 2015. Using a PCR specific for a mustelid α-HV, all genital-swab samples tested negative. In a panherpes PCR, a γ-HV was found in 55% (54/98; 39 adults and 15 cubs), identified as mustelid gammaherpesvirus 1 (MusGHV-1) using DNA sequencing. This contrasts with the results of a previous study, which reported MusGHV-1 in 98% (354/361) of blood samples taken from 218 badgers in the same population using PCR. The detection of MusHV-1 in the female reproductive tract strongly indicates the potential for a horizontal and, likely also a vertical, route of transmission. Our results suggest a potential linkage of genital HVs and impaired future reproductive success in females, but because reproductive failure can have many reasons in badgers, the causative link of this negative relationship remains to be investigated.

BACKGROUND/AIMSA number of subjective methods have been used to quantify the extent of the cutaneous delayed-type hypersensitivity (DTH) reaction. However, because of their subjective nature, significant differences in measurements may be seen between individual observers or laboratories unless thorough training is given to each observer. METHODSObjective measurement of the DTH reaction using a hand-held spectrophotometer is described. Guinea pigs were primed using inoculation with Mycobacterium bovis Bacille Calmette-Guerin and challenged five weeks later in the shaved flank with three doses of bovine purified protein derivative. The extent of the ensuing DTH reaction was measured 24 and 48 h later. Spectrophotometric measurement of the reaction site was compared with a control region of skin on each animal and expressed as the change within a standard colour space. Data obtained with the spectrophotometer was compared with the subjective measurement of the area of the DTH reaction by an experienced operator. RESULTSThe measurements obtained with the spectrophotometer correlated very closely with conventional measurement of the reaction area by a trained operator. The reaction size in square mm and changes along the red/green colour axis was correlated most strongly. CONCLUSIONSpectrophotometric measurement of the DTH reaction had advantages over conventional measuring techniques in terms of speed, reproducibility and reduced operator to operator variation. We conclude that the cutaneous DTH reaction may be simply and objectively quantified with the use of a hand-held spectrophotometer.

Attempts to eradicate tuberculosis from cattle and farmed deer in some countries have been frustrated by the existence of wildlife reservoirs of Mycobacterium bovis infection. Possum control programmes in New Zealand using poisons have shown clearly that the brushtail possum is an important source of infection for cattle and farmed deer, and the sum of evidence strongly suggests that badgers serve as a source of infection for cattle in the UK. Bovine tuberculosis can only be eradicated from these countries by controlling M. bovis infection in both wildlife and domestic animals. The most promising options for control of M. bovis infection in wildlife in the longer term include the development of a tuberculosis vaccine for wildlife and a strategy for biological control of possums. The aim of this review is to address the problems and approaches involved in the control of wildlife tuberculosis from an immunological perspective.

Bacillus Calmette-Guérin (BCG) vaccine is the only licensed vaccine against tuberculosis (TB) in humans and animals. It is most commonly administered parenterally but oral delivery is highly advantageous for immunisation of cattle and wildlife hosts of TB in particular. Since BCG is susceptible to inactivation in the gut, vaccine formulations were prepared from suspensions of Eudragit L100 copolymer powder and BCG in PBS, containing Tween 80, with and without the addition of mannitol or trehalose. Samples were frozen at -20oC, freeze-dried and the lyophilised powders were compressed to produce BCG-Eudragit matrices. Production of the dried powders resulted in a reduction in BCG viability. Substantial losses in viability occurred at the initial formulation stage and at the stage of powder compaction. Data indicated that the Eudragit matrix protected BCG against simulated gastric fluid (SGF). The matrices remained intact in SGF and dissolved completely in SIF within three hours. The inclusion of mannitol or trehalose in the matrix provided additional protection to BCG during freeze-drying. Control needs to be exercised over BCG aggregation, freeze-drying and powder compaction conditions to minimise physical damage of the bacterial cell wall and maximise the viability of oral BCG vaccines prepared by dry powder compaction.

The Eurasian badger (Meles meles) is a significant source of bovine tuberculosis in cattle in the UK and Ireland. Protection from infectious diseases, arising from maternal antibody transfer, is a well-established immunological phenomenon in mammals. In a previous study of wild badgers, transient serological responses in cubs were taken as evidence of maternal antibody transfer, and it was speculated this conferred protection from subsequent mycobacterial excretion following acquisition of tuberculosis. However successful defence against mycobacterial infections is likely to be dominated by a cell-mediated response. Using a substantially larger dataset from the same badger population, we revisited the hypothesis of maternally derived protection. Whilst we found a significant association between transient serological responses and absence of subsequent Mycobacterium bovis excretion, the likelihood of detection of such responses was not significantly associated either with badger age, or with infection in the breeding females within a cub's natal group. We concluded that although maternal antibody transfer in badgers almost certainly occurs, transient serological responses represent an invalid proxy, and the reduced likelihood of M. bovis excretion associated with transient responses was more likely to be due to the lower sensitivity of the Brock ELISA test in detecting badgers with less advanced disease.

Diagnostic tests for some conditions affecting cattle, such as tuberculosis, are often expensive and required over a prolonged period, so that the diagnostic tests involve more than one visit by a qualified vet. An alternative rapid and non-invasive diagnostic-test would be desirable. One possibility is the use of breath testing, which has been shown to have diagnostic potential in humans. The development of a device for taking a representative breath sample from a bovine animal is described. Six devices using different configurations were assessed, over three separate testing days, for their ability to take a representative breath sample which does not cause undue stress to the animal and for the ease of operator use. The main factor affecting the sample integrity was dead space, however temperature also played a role. The best samples causing the least stress to animals were taken using a nostril sampler. The nostril samplers were then used to take breath samples from cattle with and without tuberculosis which were then analysed using selected ion flow tube mass spectrometry and gas chromatography-mass spectrometry to demonstrate proof-of-principle. (c) 2012 IAgrE. Published by Elsevier Ltd. All rights reserved.

Bovine tuberculosis (bTB), a zoonosis mainly caused by Mycobacterium bovis has severe socio-economic consequences and impact on animal health. Host-pathogen interactions during M. bovis infection are poorly understood, especially early events which are difficult to follow in vivo. This study describes the utilisation of an in vitro co-culture model, comprising immortalised bovine alveolar type II (BATII) epithelial cells and bovine pulmonary arterial endothelial cells (BPAECs). When cultured at air-liquid interface, it was possible to follow the migration of live M. bovis Bacille Calmette-Guerin (BCG) and to observe interactions with each cell type, alongside cytokine release. Infection with BCG was shown to exert a detrimental effect primarily upon epithelial cells, with corresponding increases in IL8, TNF alpha, IL22 and IL17a cytokine release, quantified by ELISA. BCG infection increased expression of CD54, MHC Class I and II molecules in endothelial but not epithelial cells, which exhibited constitutive expression. The effect of peripheral blood mononuclear cell conditioned medium from vaccinated cattle upon apical-basolateral migration of BCG was examined by quantifying recovered BCG from the apical, membrane and basolateral fractions over time. The numbers of recovered BCG in each fraction were unaffected by the presence of PBMC conditioned medium, with no observable differences between vaccinated and naive animals.

Probiotics represent a non-invasive, environmentally-friendly alternative to reduce infectious diseases in wildlife species. Our aim was to evaluate the potential of typical gut commensals, such as lactic acid bacteria (LAB), as wildlife probiotics. The selected LAB were isolated from European badgers (Meles meles); a wildlife reservoir of bovine tuberculosis, and comprised four different genera: Enterococcus; Weissella; Pediococcus; and Lactobacillus. The enterococci displayed a phenotype and genotype that correlate with the production of antibacterial peptides and stimulation of antiviral responses. However, these isolates carry virulence and antibiotic resistance genes. Weissella showed some anti-mycobacterial activity due to their ability to produce lactate and ethanol. Interestingly, lactobacilli and pediococci modulated pro-inflammatory phagocytic responses that associate with protection against pathogens; and these responses agreed with the presence of immunomodulatory markers in their genomes. Although both lactobacilli and pediococci showed tolerance to antibiotics, this resistance was naturally acquired and almost all isolates possessed a strong phylogenetic relationship with isolates from food and healthy animals. Our results show that LAB display probiotic benefits that depend on the genera. Lactobacilli and pediococci are probably the most interesting candidates as probiotics against infectious diseases in wildlife because of their food-grade status and ability to modulate protective innate immune responses.

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.

One of the major transmission routes for the foodborne bacterial pathogen Campylobacter is undercooked poultry meat, contaminated from intestinal contents during processing. In broilers, Campylobacter can grow to very high densities in the caeca, and is often considered to be a commensal or an opportunistic pathogen in poultry. Reduction of caecal loads of Campylobacter may assist in lowering incidence rates of Campylobacter food poisoning. To achieve this, there needs to be a better understanding of the dynamics of Campylobacter colonisation in its natural niche, and the effect of the local microbiome on colonisation. Previous studies have shown that the microbiome differed between Campylobacter colonised and non–colonised chicken intestinal samples. To characterise the microbiome of Campylobacter-colonised broilers, caecal samples of 100 randomly selected birds from four farms were analysed using amplified 16S rRNA gene sequences. Bacterial taxonomic analysis indicated that inter-farm variation was greater than intra-farm variation. The two most common bacterial groups were Bacteroidetes and Firmicutes which were present in all samples and constituted 29.7 – 63.5% and 30.2 – 59.8% of the bacteria present, respectively. Campylobacter was cultured from all samples, ranging from 2 to 9 log10 CFU g-1. There was no clear link between Campylobacter counts and Firmicutes, Bacteroidetes or Tenericutes levels in the 16S rRNA Operational Taxonomic Unit (OTU)-based analysis of the caecal microbiome, but samples with high Campylobacter counts (> 9 log CFU g-1) contained increased levels of Enterobacteriaceae. A decrease in Lactobacillus abundance in chicken caeca was also associated with high Campylobacter loads. The reported associations with Lactobacillus and Enterobacteriaceae match changes in the intestinal microbiome of chickens and mice previously reported for Campylobacter infection, and raises the question about temporality and causation; as to whether increases in Campylobacter loads create conditions adverse to Lactobacilli and/or beneficial to Enterobacteriaceae, or that changes in Lactobacilli and Enterobacteriaceae levels created conditions beneficial for Campylobacter colonisation. If these changes can be controlled, this may open opportunities for modulation of chicken microbiota to reduce Campylobacter levels for improved food safety.

Vaccination of badgers by the subcutaneous, mucosal and oral routes with the Pasteur strain of Mycobacterium bovis bacille Calmette-Guérin (BCG) has resulted in significant protection against experimental infection with virulent M. bovis. However, as the BCG Danish strain is the only commercially licensed BCG vaccine for use in humans in the European Union it is the vaccine of choice for delivery to badger populations. As all oral vaccination studies in badgers were previously conducted using the BCG Pasteur strain, this study compared protection in badgers following oral vaccination with the Pasteur and the Danish strains. Groups of badgers were vaccinated orally with 10(8) colony forming units (CFU) BCG Danish 1331 (n = 7 badgers) or 10(8) CFU BCG Pasteur 1173P2 (n = 6). Another group (n = 8) served as non-vaccinated controls. At 12 weeks post-vaccination, the animals were challenged by the endobronchial route with 6 × 10(3) CFU M. bovis, and at 15 weeks post-infection, all of the badgers were euthanased. Vaccination with either BCG strain provided protection against challenge compared with controls. The vaccinated badgers had significantly fewer sites with gross pathology and significantly lower gross pathological severity scores, fewer sites with histological lesions and fewer sites of infection, significantly lower bacterial counts in the thoracic lymph node, and lower bacterial counts in the lungs than the control group. No differences were observed between either of the vaccine groups by any of the pathology and bacteriology measures. The ELISPOT analysis, measuring production of badger interferon - gamma (IFN-γ), was also similar across the vaccinated groups.

In developing an oral bait BCG vaccine against tuberculosis in badgers we wanted to understand the conditions of the gastrointestinal tract and their impact on vaccine viability. Conditions mimicking stomach and small-intestine caused substantial reduction in BCG viability. We performed in vivo experiments using a telemetric pH monitoring system and used the data to parameterise a dynamic in vitro system (TIM-1) of the stomach and small intestine. Some BCG died in the stomach compartment and through the duodenum and jejunum compartments. BCG survival in the stomach was greatest when bait was absent but by the time BCG reached the jejunum, BCG viability was not significantly affected by the presence of bait. Our data suggest that from a starting quantity of 2.85 ± 0.45 x 108 colony-forming units of BCG around 2 log10 may be killed before delivery to the intestinal lymphoid tissue. There are economic arguments for reducing the dose of BCG to vaccinate badgers orally. Our findings imply this could be achieved if we can protect BCG from the harsh environment of the stomach and duodenum. TIM-1 is a valuable, non-animal model with which to evaluate and optimise formulations to maximise BCG survival in the gastrointestinal tract.

Bovine tuberculosis (TB) continues to be an intractable problem in many countries, particularly where “test and slaughter” policies cannot be implemented or where wildlife reservoirs of Mycobacterium bovis infection serve as a recurrent source of infection for domestic livestock. Alternative control measures are urgently required and vaccination is a promising option. Although the M. bovis bacille Calmette-Guérin (BCG) vaccine has been used in humans for nearly a century, its use in animals has been limited, principally as protection against TB has been incomplete and vaccination may result in animals reacting in the tuberculin skin test. Valuable insights have been gained over the past 25 years to optimise protection induced by BCG vaccine in animals and in the development of tests to differentiate infected from vaccinated animals (DIVA). This review examines factors affecting the efficacy of BCG vaccine in cattle, recent field trials, use of DIVA tests and the effectiveness of BCG vaccine in other domestic livestock as well as in wildlife. Oral delivery of BCG vaccine to wildlife reservoirs of infection such as European badgers, brushtail possums, wild boar, and deer has been shown to induce protection against TB and could prove to be a practical means to vaccinate these species at scale. Testing of BCG vaccine in a wide range of animal species has indicated that it is safe and vaccination has the potential to be a valuable tool to assist in the control of TB in both domestic livestock and wildlife.