Is moving from targeted culling to BCG-vaccination of badgers (Meles meles) associated with an unacceptable increased incidence of cattle herd tuberculosis in the Republic of Ireland? A practical non-inferiority wildlife intervention study in the Republic of Ireland (2011-2017).

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

Survival of Mycobacterium bovis BCG oral vaccine during transit through a dynamic in vitro model simulating the upper gastrointestinal tract of badgers.

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.

Immunological responses of European badgers (Meles Meles) to infection with Mycobacterium bovis.

Mycobacterium bovis is the main cause of bovine tuberculosis and its eradication is proving difficult in many countries because of wildlife reservoirs, including European badgers (Meles meles) in the UK Ireland. Following the development of badger specific immunological reagents, many studies have shown that some aspects of the cellular and serological immune responses of badgers to virulent M. bovis and the attenuated M. bovis BCG (Bacille of Calmette and Guérin) strain are similar to those seen in other animal hosts infected with M. bovis. However, badgers also appear to have developed specific immunological responses to M. bovis infection which may be associated with mild inflammatory responses. Badgers may therefore represent an interesting natural host for M. bovis that can provide a more thorough understanding of efficient immunological responses to tuberculosis.

Assessment of the safety of Bacillus Calmette-Guérin vaccine administered orally to badgers (Meles meles).

European badgers (Meles meles) are a wildlife reservoir for Mycobacterium bovis (M. bovis) in parts of England, Wales and Ireland, constituting a potential source of tuberculosis (TB) infection for cattle. Vaccination of badgers against TB is one of the tools available for helping reduce the prevalence of bovine TB in badgers, made possible by the licensing in 2010 of Bacillus Calmette-Guérin (BCG) vaccine for intramuscular administration to badgers (BadgerBCG). However, practical limitations associated with administering an injected vaccine to wild animals make an oral, bait-delivered form of the vaccine highly desirable. Evaluation of the safety of oral BCG to badgers and the environment is a mandatory step on the road to licensing an oral vaccine. This study had the following objectives: (a) to determine whether adverse effects followed the oral administration of BCG vaccine to badgers; (b) to measure the quantity and frequency of BCG excreted in the faeces of vaccinated badgers; and (c) to assess whether there was evidence of the vaccine spreading to unvaccinated, 'sentinel' badgers sharing the same environment as vaccinated animals. We report here that the oral administration per badger of ≥6.4 × 109 cfu BCG, followed 14 days later by a single oral dose of ≥6.4 × 107 cfu BCG caused no adverse physical effects and did not affect the social behaviour and feeding habits of the vaccinated animals. BCG was cultured from the faeces of two of nine vaccinated animals (372 cfu/g and 996 cfu/g, respectively) approximately 48 h after the higher dose of BCG was administered and by one of the nine vaccinated animal (80 cfu/g) approximately 24 h after receiving the lower dose of BCG. We found no evidence for the transmission of BCG to unvaccinated, sentinel, badgers housed with the vaccinated animals despite the occasional excretion of BCG in faeces.

Quantitative interferon-gamma responses predict future disease progression in badgers naturally infected with Mycobacterium bovis.

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.

Badger macrophages fail to produce nitric oxide, a key anti-mycobacterial effector molecule.

The European badger is recognised as a wildlife reservoir for bovine tuberculosis (bTB); the control of which is complex, costly and controversial. Despite the importance of badgers in bTB and the well-documented role for macrophages as anti-mycobacterial effector cells, badger macrophage (bdMφ) responses remain uncharacterised. Here, we demonstrate that bdMφ fail to produce nitric oxide (NO) or upregulate inducible nitric oxide synthase (iNOS) mRNA following Toll-like receptor (TLR) agonist treatment. BdMφ also failed to make NO after stimulation with recombinant badger interferon gamma (bdIFNγ) or a combination of bdIFNγ and lipopolysaccharide. Exposure of bdMφ to TLR agonists and/or bdIFNγ resulted in upregulated cytokine (IL1ß, IL6, IL12 and TNFα) mRNA levels indicating that these critical pathways were otherwise intact. Although stimulation with most TLR agonists resulted in strong cytokine mRNA responses, weaker responses were evident after exposure to TLR9 agonists, potentially due to very low expression of TLR9 in bdMφ. Both NO and TLR9 are important elements of innate immunity to mycobacteria, and these features of bdMφ biology would impair their capacity to resist bTB infection. These findings have significant implications for the development of bTB management strategies, and support the use of vaccination to reduce bTB infection in badgers.

Testing of a palatable bait and compatible vaccine carrier for the oral vaccination of European badgers (Meles meles) against tuberculosis.

The oral vaccination of wild badgers (Meles meles) with live Bacillus Calmette-Guérin (BCG) is one of the tools being considered for the control of bovine tuberculosis (caused by Mycobacterium bovis) in the UK. The design of a product for oral vaccination requires that numerous, and often competing, conditions are met. These include the need for a highly palatable, but physically stable bait that will meet regulatory requirements, and one which is also compatible with the vaccine formulation; in this case live BCG. In collaboration with two commercial bait companies we have developed a highly attractive and palatable bait recipe designed specifically for European badgers (Meles meles) that meets these requirements. The palatability of different batches of bait was evaluated against a standardised palatable control bait using captive badgers. The physical properties of the bait are described e.g. firmness and colour. The microbial load in the bait was assessed against European and US Pharmacopoeias. The bait was combined with an edible vaccine carrier made of hydrogenated peanut oil in which BCG vaccine was stable during bait manufacture and cold storage, demonstrating <0.5 log10 reduction in titre after 117weeks' storage at -20°C. BCG stability in bait was also evaluated at +4°C and under simulated environmental conditions (20°C, 98% Relative Humidity; RH). Finally, iophenoxic acid biomarkers were utilised as a surrogate for the BCG vaccine, to test variants of the vaccine-bait design for their ability to deliver biomarker to the gastrointestinal tract of individual animals. These data provide the first detailed description of a bait-vaccine delivery system developed specifically for the oral vaccination of badgers against Mycobacterium bovis using live BCG.

No Compensatory Relationship between the Innate and Adaptive Immune System in Wild-Living European Badgers.

The innate immune system provides the primary vertebrate defence system against pathogen invasion, but it is energetically costly and can have immune pathological effects. A previous study in sticklebacks found that intermediate major histocompatibility complex (MHC) diversity correlated with a lower leukocyte coping capacity (LCC), compared to individuals with fewer, or many, MHC alleles. The organization of the MHC genes in mammals, however, differs to the highly duplicated MHC genes in sticklebacks by having far fewer loci. Using European badgers (Meles meles), we therefore investigated whether innate immune activity, estimated functionally as the ability of an individual's leukocytes to produce a respiratory burst, was influenced by MHC diversity. We also investigated whether LCC was influenced by factors such as age-class, sex, body condition, season, year, neutrophil and lymphocyte counts, and intensity of infection with five different pathogens. We found that LCC was not associated with specific MHC haplotypes, MHC alleles, or MHC diversity, indicating that the innate immune system did not compensate for the adaptive immune system even when there were susceptible MHC alleles/haplotypes, or when the MHC diversity was low. We also identified a seasonal and annual variation of LCC. This temporal variation of innate immunity was potentially due to physiological trade-offs or temporal variation in pathogen infections. The innate immunity, estimated as LCC, does not compensate for MHC diversity suggests that the immune system may function differently between vertebrates with different MHC organizations, with implications for the evolution of immune systems in different taxa.

Age-related declines in immune response in a wild mammal are unrelated to immune cell telomere length.

Senescence has been hypothesized to arise in part from age-related declines in immune performance, but the patterns and drivers of within-individual age-related changes in immunity remain virtually unexplored in natural populations. Here, using a long-term epidemiological study of wild European badgers (Meles meles), we (i) present evidence of a within-individual age-related decline in the response of a key immune-signalling cytokine, interferon-gamma (IFNγ), to ex vivo lymphocyte stimulation, and (ii) investigate three putative drivers of individual variation in the rate of this decline (sex, disease and immune cell telomere length; ICTL). That the within-individual rate of age-related decline markedly exceeded that at the population level suggests that individuals with weaker IFNγ responses are selectively lost from this population. IFNγ responses appeared to decrease with the progression of bovine tuberculosis infection (independent of age) and were weaker among males than females. However, neither sex nor disease influenced the rate of age-related decline in IFNγ response. Similarly, while ICTL also declines with age, variation in ICTL predicted neither among- nor within-individual variation in IFNγ response. Our findings provide evidence of within-individual age-related declines in immune performance in a wild mammal and highlight the likely complexity of the mechanisms that generate them.

Genetic variation of the MHC class II DRB genes in the Japanese weasel, Mustela itatsi, endemic to Japan, compared with the Siberian weasel, Mustela sibirica.

Major histocompatibility complex (MHC) genes encode proteins that play a critical role in vertebrate immune system and are highly polymorphic. To further understand the molecular evolution of the MHC genes, we compared MHC class II DRB genes between the Japanese weasel (Mustela itatsi), a species endemic to Japan, and the Siberian weasel (Mustela sibirica), a closely related species on the continent. We sequenced a 242-bp region of DRB exon 2, which encodes antigen-binding sites (ABS), and found 24 alleles from 31 M. itatsi individuals and 17 alleles from 21 M. sibirica individuals, including broadly distributed, species-specific and/or geographically restricted alleles. Our results suggest that pathogen-driven balancing selection have acted to maintain the diversity in the DRB genes. For predicted ABS, nonsynonymous substitutions exceeded synonymous substitutions, also indicating positive selection, which was not seen at non-ABS. In a Bayesian phylogenetic tree, two M. sibirica DRB alleles were basal to the rest of the sequences from mustelid species and may represent ancestral alleles. Trans-species polymorphism was evident between many mustelid DRB alleles, especially between M. itatsi and M. sibirica. These two Mustela species divided about 1.7 million years ago, but still share many MHC alleles, indicative of their close phylogenetic relationship.

Association of quantitative interferon-γ responses with the progression of naturally acquired Mycobacterium bovis infection in wild European badgers (Meles meles).

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.

Mortality trajectory analysis reveals the drivers of sex-specific epidemiology in natural wildlife-disease interactions.

In animal populations, males are commonly more susceptible to disease-induced mortality than females. However, three competing mechanisms can cause this sex bias: weak males may simultaneously be more prone to exposure to infection and mortality; being 'male' may be an imperfect proxy for the underlying driver of disease-induced mortality; or males may experience increased severity of disease-induced effects compared with females. Here, we infer the drivers of sex-specific epidemiology by decomposing fixed mortality rates into mortality trajectories and comparing their parameters. We applied Bayesian survival trajectory analysis to a 22-year longitudinal study of a population of badgers (Meles meles) naturally infected with bovine tuberculosis (bTB). At the point of infection, infected male and female badgers had equal mortality risk, refuting the hypothesis that acquisition of infection occurs in males with coincidentally high mortality. Males and females exhibited similar levels of heterogeneity in mortality risk, refuting the hypothesis that maleness is only a proxy for disease susceptibility. Instead, sex differences were caused by a more rapid increase in male mortality rates following infection. Males are indeed more susceptible to bTB, probably due to immunological differences between the sexes. We recommend this mortality trajectory approach for the study of infection in animal populations.

Badger responses to small-scale culling may compromise targeted control of bovine tuberculosis.

Where wildlife disease requires management, culling is frequently considered but not always effective. In the British Isles, control of cattle tuberculosis (TB) is hindered by infection in wild badger (Meles meles) populations. Large-scale badger culling can reduce the incidence of confirmed cattle TB, but these benefits are undermined by culling-induced changes in badger behavior (termed perturbation), which can increase transmission among badgers and from badgers to cattle. Test-vaccinate/remove (TVR) is a novel approach that entails testing individual badgers for infection, vaccinating test-negative animals, and killing test-positive animals. Imperfect capture success, diagnostic sensitivity, and vaccine effectiveness mean that TVR would be expected to leave some infected and some susceptible badgers in the population. Existing simulation models predict that TVR could reduce cattle TB if such small-scale culling causes no perturbation, but could increase cattle TB if considerable perturbation occurs. Using data from a long-term study, we show that past small-scale culling was significantly associated with four metrics of perturbation in badgers: expanded ranging, more frequent immigration, lower genetic relatedness, and elevated prevalence of Mycobacterium bovis, the causative agent of TB. Though we could not reject the hypothesis that culling up to three badgers per social group might avoid perturbation, we also could not reject the hypothesis that killing a single badger prompted detectable perturbation. When considered alongside existing model predictions, our findings suggest that implementation of TVR, scheduled for 2014, risks exacerbating the TB problem rather than controlling it. Ongoing illegal badger culling is likewise expected to increase cattle TB risks.

Control strategies for wildlife tuberculosis in Ireland.

The principal domestic maintenance host for Mycobacterium bovis is infected cattle. In countries where comprehensive surveillance schemes have been applied, tuberculosis rarely affects an animal to the extent that it presents with clinical disease. In the latter stages of an eradication campaign, the aim is to maintain the disease-free status of clear herds and eliminate foci of infection in herds as well as restricting movement of infected animals from these herds, other than to slaughter. However, the eradication of tuberculosis from cattle herds may be compromised if infected wildlife species, such as Eurasian badgers (Meles meles), share the same environment and contribute to transmission of infection. The options for dealing with tuberculosis in the wildlife reservoir hosts are limited to segregation of domestic animals from the wildlife, culling of the wildlife host or vaccination. Options are further limited by conservation and social reasons, particularly where culling is concerned. In Ireland and the UK, vaccination of badgers against M. bovis, if successfully employed, could directly facilitate the completion of bovine tuberculosis eradication. Programmes of research into vaccination of badgers are being undertaken in both countries, and there is clear evidence that vaccination induces protection. Vaccine trials in captive badgers have established that the M. bovis bacille Calmette-Guérin (BCG) vaccine can induce a protective response that limits the distribution and severity of tuberculosis disease following experimental challenge. In Ireland, a large-scale field trial of oral BCG vaccination is being conducted to measure the protection generated in wild badgers subjected to natural transmission of infection and to estimate vaccine efficacy. The results will provide a framework for the development and implementation of a national strategy to address the disease in badger populations and if successful will remove this major impediment to tuberculosis eradication from cattle.

Overview of vaccination trials for control of tuberculosis in cattle, wildlife and humans.

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

BCG vaccination reduces risk of tuberculosis infection in vaccinated badgers and unvaccinated badger cubs.

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-Guérin (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).