Professor Mark Chambers
Mark graduated in 1989 from the University of Bristol with a BSc (Hons) in Cell and Molecular Pathology and went on to gain a PhD from University of Cambridge in 1992 for his work on the immune responses to human papillomavirus type 16 in a mouse model. Then followed two post-doctoral positions at Imperial College London, working on viral and bacterial adhesins in recombinant hepatitis B core antigen particles and the rational attenuation of Mycobacterium tuberculosis and expression of cytokine genes in recombinant Mycobacterium bovis BCG. In 1996 he moved to the government Veterinary Laboratories Agency (now Animal and Plant Heath Agency, APHA) to undertake work on vaccines, pathology, small animal models, and diagnostics for animal tuberculosis. There he led a number of Defra projects in this area, most notably resulting in 2010 with the licensing of BadgerBCG - the first licensed vaccine against TB in any animal species.
Mark has published extensively in the area of human and animal tuberculosis and related disciplines. He is a former acting TB Science Lead and Head of the Bacteriology Department at APHA. In October 2013 he was appointed Professor of Veterinary Bacteriology at the University of Surrey and held a joint position with APHA until February 2020. In March 2020, he joined the University of Surrey full-time to take up a new Chair as Professor of Microbiology and Disease Intervention. He splits his time 50/50 between the School of Veterinary Medicine and the School of Biosciences & Medicine where he is also Head of Department of Microbial Sciences. The joint Schools appointment reflects his research interest in both animal and human health, with particular focus on the replacement of animals in research and reducing antimicrobial use and resistance.
Areas of specialism
University roles and responsibilities
- Head of Department of Microbial Sciences
- Academic Theme Lead for Infection and Immunity
- Member of the Steering Committee for the Comparative Pathology Clinical Academic Group.
- Member of the NASPA Sub-Committee.
- Member of the Board of Studies for Veterinary Microbiology MSc.
- Member of the University Biological Safety Committee.
Mark is interested in the development, application and evaluation of disease intervention strategies in their very broadest sense. This brings him into multi-disciplinary collaborations as diverse as electrical engineering, chemistry, physics, design research, psychology, and mathematical modelling.
He has a long-standing reputation in the area of vaccination of wildlife against TB and until recently was working at APHA to develop and license vaccines against TB for use in badgers and cattle.
He is committed to the 3Rs and seeks to build a research group at the University focussed on the development on in vitro multi-cell models with the intention of replacing the use of animals in disease research. He was Principle Investigator on an NC3Rs Principal Investigator Award to develop an in vitro tissue culture model of the cow lung and Co-Investigator on the NC3Rs Principal Investigator Award of Professor Graham Stewart to use Dictyostelium to study the genetic basis of Mycobacterium bovis intracellular infection. Both awards were part of the NC3Rs initiative to replace animal models of bovine tuberculosis.
Mark was Co-Investigator on the recent AMRSim project, an AHRC-funded research project of Professor Alastair MacDonald, Glasgow School of Art with Dr Kayleigh Wyles, School of Psychology to make the 'invisible, visible' by building a three-dimensional graphical simulator of the interior of a veterinary practice in which humans, animals, and bacteria interact, according to rules observed from real-life.
He is currently Principal Investigator along with Dr Diane Lee, Research Fellow, on an award from the Horserace Betting Levy Board that aims to develop a model of the equine upper airway, using a nasal brush sampling technique, as an alternative to the more invasive tracheobronchial biopsy or bronchial brushing.
He is also work package leader in the European Commission FED-AMR project, which belongs to the 2nd call for Research Projects of the One Health European Joint Programme (OHEJP). The project evaluates the relevance of horizontal antimicrobial resistance gene (ARG) transfer on free extracellular DNA (exDNA) over ecosystem boundaries relative to bacterial conjugation and is a reasearch consortium of 9 European countries.
Antimicrobial-resistance (AMR) is a major threat to human health. Increasing numbers of resistant infections are leading to many existing antibiotics becoming ineffective. AMR is a challenge in veterinary medicine and AMR bacteria are more likely to emerge and transmit AMR genes where there are higher microbial densities. In previous work, we developed and evaluated an innovative training intervention, supported by a new type of digital tool, for training veterinary practice staff in infection prevention and control (IPC). This comprised an interactive mode of presentation and delivery supported by a 3D graphical simulation tool recreating the interior of a vet practice and the interactions of humans, animals, and microbes, according to rules observed from real-life. Additional visual layers, able to be toggled on/off, enabled normally invisible bacteria to be 'seen' as they spread via contacts within the indoor environment, and allowed the effect of various IPC measures to be seen easily. By 'making visible the invisible' our intention is to reduce microbial contamination in small animal vet practices, hence reduce reliance on antibiotics, and contribute to decreasing the risk of AMR developing. Our aim is to now maximise the impact of this work by migrating our innovative software tool to platforms where it is fit for deployment into a variety of self-paced learning environments to support the on-going training of veterinary practitioners in IPC and extend this further to students undertaking their veterinary studies. The use of the tools we are developing could change the perception of risk of infection to positively influence behaviours to minimise the risk of infection and ultimately the reliance on antimicrobials. By engaging with students at the beginning of their training, and by revisiting the themes throughout their curriculum, we intend to embed best practice as a matter of habit in the next generation of veterinary practitioners. The current tool is a laptop-hosted digital animation designed to support a trainer delivering a short in-house workshop session. In 2019, our intervention was delivered to 51 vet staff during a trial within a large referral vet practice. Participants found the novel approach taken to discussing IPC increased their awareness and appreciation of the issues: comments such as 'very clear', 'informative' and 'showed easy steps on how to prevent infection' were typical of the feedback received. Our trial demonstrated that our prototype intervention was able to meet its training objectives within a 30-minute trainer-led session. However, we found potential barriers to its adoption, but also suggestions for improved flexibility of delivery. Consequently, the objective of this follow-on project is to translate our trainer-led, single device-based activity onto alternative platforms for more flexible, scalable and cost-efficient modes of delivery, specifically mobile-, tablet-, and web-based use. These would facilitate stand-alone, self-paced learning to be used for professional training and for use in educational settings, e.g. for vet students and nurses at different stages of their education. Common to all platforms will be the promotion of reflective practice, e.g. what is perceived as risky practice or behaviour and how to improve this? During its use, data on users' perception of risk and self-reporting of behaviour before and after receiving training would be captured. The ambition is that these stand-alone platforms would achieve the same objective within the 30-minute time-frame but it would also be designed to be flexible enough for more individual self-paced and refresher sessions and as the basis for expanded group teaching and training sessions and seminars. We envisage the tool being available to professional practitioners and veterinary students in mobile or tablet form, and as an online resource forming an innovative element of more extensive and comprehensive training packages.
Over the course of this project, the relevance of horizontal antimicrobial resistance gene (ARG) transfer on free extracellular DNA (exDNA) over ecosystem boundaries relative to bacterial conjugation will be evaluated. ExDNA is widely present in natural environments and sufficiently stable to constitute an important reservoir for ARGs. The dissemination of AMR on exDNA over ecosystem boundaries will be monitored under controlled but naturally occurring environmental conditions in an open-air agricultural research area: The Hydrology Open Air Laboratory (HOAL) in Petzenkirchen, Austria. ARG concentrations, diversity, dynamic variability, mobility and bacterial biodiversity will be determined in an annual longitudinal study covering a crop growing period, different fertilisation and land management techniques and various different – but interlinked – environmental compartments along the route: pig farm –> manure –> soil –> crop/food/feed –> ground/surface water –> pig farm (and other associated human compartments). The results obtained from HOAL will be compared with data retrieved from equivalent compartments in Northern, Eastern and Southern Europe. Movement of ARGs over ecosystem barriers will be inferred by sequence comparisons and construction of phylogenetic trees of ARGs and ARBs. The linkage between human and non-human reservoirs of AMR will be investigated exemplarily with C. difficile as ARG transfer platform over ecosystem boundaries and conjugation as means for HGT.
The prevailing selection pressure in each tested habitat during the longitudinal study will be determined by quantifying antimicrobials, herbicides and trace elements in the tested compartments. Environmental conditions which may induce or inhibit the expression of competence genes that are necessary to enable the uptake of free extracellular DNA by bacteria will be identified in soil microcosms and in a pig gut model. The impact of transformation relative to conjugation will be evaluated using Acinetobacter sp. (transformation) and C. difficile (conjugation) as model organisms in these experiments.
Experimental data obtained during the project will be used to feed and tune probabilistic modelling of the emergence of AMR in target bacteria and to delineate the relative contribution of transformation and conjugation to ARG acquisition in soil environments. Mechanistic models will address key questions regarding the spatio-temporal changes observed in microbial communities and shall support the identification of critical control points for intervention to reduce the spread of AMR from environmental sources
Better model of the upper airway to study equine asthma.
More research is required on equine asthma and better models of the upper airway are needed to do so. Currently to obtain lung cells for this research a bronchoscope is placed down the horse’s windpipe and into the lungs, requiring the horse to be sedated and anaesthetised. This causes irritation and requires up to 48 hours recovery time. It is difficult to justify this technique in healthy horses, although normal cells are needed for research. We shall develop an alternative using ‘nasal brushing’. It requires no recovery period and may not even require sedation; being used successfully on children. Cells taken from the horse’s nose using a brush instrument will be transported to the laboratory for growth and characterisation to validate the method. They will be used to study disease mechanisms and to explore novel therapies for both mild and severe equine asthma, based on promising leads from human medicine.
Professor Alastair Macdonald, Glasgow School of Art - https://gtr.ukri.org/person/91A3FC97-9F21-4C04-BCBA-DDE2F709F536
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.
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.
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.
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.
© 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.
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.
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.
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.
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.
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
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 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.
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.
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.
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.
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.
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.
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.
© 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.
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.
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
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.
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.
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.
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.
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.
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.
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.
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.
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
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.
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.
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.
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.
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.
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.
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.
Orcid ID: orcid.org/0000-0002-8073-8069