Biomedical subjects

R Glyn Hewinson

Publications and source records attributed to R Glyn Hewinson.

At least 19 recordsLinked to original sources

PubMed2026

Uncovering the genomic landscape of Mycobacterium bovis in Wales.

Bovine tuberculosis (bTB), caused by the bacterium Mycobacterium bovis, is one of the most pressing animal health issues in Wales today. It negatively impacts cattle health, affects profitability and trade, and can decimate years of genetic improvement towards desirable production traits. It also imposes substantial financial, social, and psychological burdens on farming communities. Eradication of bTB requires an understanding of local transmission pathways to target effective disease-control interventions. In this study, we characterised the genomic diversity of M. bovis across Wales by analysing the genome sequence of 379 M. bovis isolates obtained from culture-positive animals in Wales in 2021. Analyses uncovered three prevalent clusters that are geographically distinct. A further three clusters containing fewer isolates were also geographically separated, two of which had particularly large SNP distances from most other Welsh isolates, suggesting independent introductions of M. bovis strains that are not endemic to Wales. Fine-scale and epidemiologically relevant genetic structuring was identified within the six main clusters, indicating region-specific evolution, which can drive local disease dynamics. Finally, SNPs were identified in coding genes that have the potential for important advantageous physiological consequences that may impact host-pathogen interactions and necessitate further investigation.

Animals↗

PubMed2006

Culling and cattle controls influence tuberculosis risk for badgers.

Human and livestock diseases can be difficult to control where infection persists in wildlife populations. In Britain, European badgers (Meles meles) are implicated in transmitting Mycobacterium bovis, the causative agent of bovine tuberculosis (TB), to cattle. Badger culling has therefore been a component of British TB control policy for many years. However, large-scale field trials have recently shown that badger culling has the capacity to cause both increases and decreases in cattle TB incidence. Here, we show that repeated badger culling in the same area is associated with increasing prevalence of M. bovis infection in badgers, especially where landscape features allow badgers from neighboring land to recolonize culled areas. This impact on prevalence in badgers might reduce the beneficial effects of culling on cattle TB incidence, and could contribute to the detrimental effects that have been observed. Additionally, we show that suspension of cattle TB controls during a nationwide epidemic of foot and mouth disease, which substantially delayed removal of TB-affected cattle, was associated with a widespread increase in the prevalence of M. bovis infection in badgers. This pattern suggests that infection may be transmitted from cattle to badgers, as well as vice versa. Clearly, disease control measures aimed at either host species may have unintended consequences for transmission, both within and between species. Our findings highlight the need for policymakers to consider multiple transmission routes when managing multihost pathogens.

Animals↗

PubMed2006

Development of cattle TB vaccines in the UK.

In 1996, an independent scientific committee chaired by Professor John Krebs, tasked to review the problem of bovine tuberculosis (TB) in GB, concluded that vaccination of cattle offered the best long-term solution for controlling the disease in the National Herd. This view has been re-affirmed recently in the House of Commons Environment, Food and Rural Affairs Committee's report on Bovine TB (2004) and by the findings of the Independent Scientific Group Vaccine Scoping Sub-committee. Significant progress in developing TB vaccines for cattle has been made over the last 5 years. Specifically: (i) DNA or protein subunit vaccines used in combination with BCG have been shown to give superior protection against experimental challenge in cattle than BCG (heterologous prime-boost); (ii) prototype reagents that allow discrimination between vaccinated and infected animals have been developed; and (iii) and correlates of disease severity have been identified that can predict the success or failure of vaccination. These significant advances are detailed in this review with a summary of future directions that TB vaccine development for cattle is likely to take.

Animals↗

PubMed2006

Bottlenecks and broomsticks: the molecular evolution of Mycobacterium bovis.

Mycobacterium bovis is the cause of tuberculosis in cattle and is a member of the Mycobacterium tuberculosis complex. In contrast to many other pathogenic bacterial species, there is little evidence for the transfer and recombination of genes between cells. The clonality of this group of organisms indicates that the population structure is dominated by reductions in diversity, caused either by population bottlenecks or selective sweeps as entire chromosomes become fixed in the population. We describe how these forces have shaped not only the phylogeny of this group but also, at a very local level, the population structure of Mycobacterium bovis in the British Isles. We also discuss the practical implications of applying this knowledge to understanding the spread of infection and the development of improved vaccines and diagnostic tests.

Animals↗

PubMed2006

Metabolic fingerprints of Mycobacterium bovis cluster with molecular type: implications for genotype-phenotype links.

Mycobacterium bovis is the causative agent of bovine tuberculosis. Various genetic typing techniques have been used to trace the reservoirs of infection; however, they have limited success in population genetics and outbreak studies. Fourier-transform infrared spectroscopy (FT-IR) is a rapid phenotypic typing technique, which may be used to generate a metabolic fingerprinting and is increasingly used to characterize bacteria. When coupled with multivariate cluster analysis, this powerful combination has sufficient resolving power to discriminate bacteria down to subspecies level; however, to date this method has not been used in the differentiation of mycobacteria. Multiple isolates of the ten major spoligotypes in the UK, recovered from different geographical locations, were analysed using FT-IR. Hierarchical cluster analysis of the spectra showed that the isolates could be differentiated according to their spoligotypes. Six of the spoligotype FT-IR clusters were very homogeneous and all isolates were recovered together. However, the remaining four groups displayed a more heterogeneous phenotype, which may reflect greater variation than previously suspected within these groups. Included in the ten spoligotypes are the two most dominant isolates in the UK, designated types 9 and 17. Whilst type 17 showed a highly conserved phenotype as judged by FT-IR, type 9 showed a very heterogeneous metabolic profile and isolates were recovered throughout the dendrogram. This variation in type 9 is reflected in the high degree of diversity observed by variable number tandem repeats (VNTR) analysis, underlining the exquisite resolving power of FT-IR.

Animals↗

PubMed2006

Immune responses induced in cattle by vaccination with a recombinant adenovirus expressing Mycobacterial antigen 85A and Mycobacterium bovis BCG.

Cattle were vaccinated with an adenovirus expressing the mycobacterial antigen 85A (rAd85A), with Mycobacterium bovis BCG followed by rAd85A heterologous boosting, or with rAd85A followed by BCG boosting. BCG/rAd85A resulted in the highest direct gamma interferon responses. Cultured enzyme-linked immunospot assay analysis demonstrated that memory responses were induced by all three protocols but were strongest after BCG/rAd85A and rAd85A/BCG vaccination.

Adenoviridae↗

PubMed2006

Molecular analysis of human and bovine tubercle bacilli from a local setting in Nigeria.

To establish a molecular epidemiological baseline for the strains causing tuberculosis in Nigeria, a survey of isolates from humans and cattle was carried out. Spoligotyping and variable-number tandem-repeat analysis revealed that the majority of tuberculosis disease in humans in Ibadan, southwestern Nigeria, is caused by a single, closely related group of Mycobacterium tuberculosis strains. Using deletion typing, we show that approximately 13% of the disease in humans in this sample was caused by strains of Mycobacterium africanum and Mycobacterium bovis rather than M. tuberculosis. Molecular analysis of strains of M. bovis recovered from Nigerian cattle show that they form a group of closely related strains that show similarity to strains from neighboring Cameroon. Surprisingly, the strains of M. bovis recovered from humans do not match the molecular type of the cattle strains, and possible reasons for this are discussed. This is the first molecular analysis of M. tuberculosis complex strains circulating among humans and cattle in Nigeria, the results of which have significant implications for disease control.

Animals↗

PubMed2005

Recent advances in our knowledge of Mycobacterium bovis: a feeling for the organism.

Significant and rapid progress has been made in our knowledge and understanding of Mycobacterium bovis since the last international M. bovis conference 5 years ago. Much of this progress has been underpinned by the completion of the genome sequence. This important milestone has catalysed research into the development of a number of improved tools with which to combat bovine tuberculosis. In this article we will review recent progress made in the development of these tools and in our understanding of the organism, its evolution and spread. Comparison of the genome sequence with those of other members of the Mycobacterium tuberculosis complex has enabled insights into the evolution of M. bovis. This analysis also indicates that the M. tuberculosis complex have the propensity to adapt to new host species. The use of high throughput molecular typing methods has revealed that the recent bovine tuberculosis epidemic in Great Britain is being driven by a number of clonal expansions, which cannot be explained by random mutation and drift alone. Completion of a number of mycobacterial genome sequences has allowed the development of antigen mining techniques that rapidly identify M. bovis-specific genes. These can then be used as reagents in the gamma interferon assay to increase the specificity of the assay and also to discriminate between Bacillus of Calmette and Guérin (BCG) vaccinated animals and those infected with M. bovis. In the longer term, comparisons between the genomes of M. bovis and BCG will allow insight into how BCG became attenuated following serial passage on artificial growth media and reveal clues into how to improve the vaccine efficacy of BCG.

Animals↗

PubMed2005

Protective efficacy induced by Mycobacterium bovis bacille Calmette-Guèrin can be augmented in an antigen independent manner by use of non-coding plasmid DNA.

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

Animals↗

PubMed2005

Ecotypes of the Mycobacterium tuberculosis complex.

A phylogeny of the Mycobacterium tuberculosis complex has recently shown that the animal-adapted strains are found in a single lineage marked by the deletion of chromosomal region 9 (RD9) [Brosch et al., 2002. A new evolutionary scenario for the Mycobacterium tuberculosis complex. Proc. Natl Acad. Sci. USA 99 (6), 3684-3689]. We have obtained the spoligotype patterns of the RD9 deleted strains used to generate this new evolutionary scenario and we show that the presence of spoligotype spacers 3, 9, 16, 39, and 40-43 is phylogenetically informative in this lineage. We have used the phylogenetically informative spoligotype spacers to screen a database of spoligotype patterns and have identified further members of a group of strains apparently host-adapted to antelopes. The presence of the spoligotype spacers is congruent with the phylogeny generated by chromosomal deletions, suggesting that recombination is rare or absent between strains of this lineage. The phylogenetically informative spacers, in concert with the previously identified single nucleotide mutations and chromosomal deletions, can be used to identify a series of clades in the RD9 deleted lineage each with a separate host preference. Finally, we discuss the application of the ecotype concept to this series of clades and suggest that the M. tuberculosis complex may best be described as a series of host-adapted ecotypes.

Animals↗

PubMed2005

Synthetic peptide vaccination in cattle: induction of strong cellular immune responses against peptides derived from the Mycobacterium bovis antigen Rv3019c.

Fully synthetic peptide vaccines possess attractive cost and safety attributes. However, peptide vaccines that induce cell-mediated immunity require both selection of appropriate peptides and the development of adjuvant formulations supporting the induction of cellular immunity. An adjuvant formulation composed of emulsigen and the synthetic CpG motif containing ODN2007 was tested in cattle for its ability to induce cellular immunity after peptide vaccination, and compared to Rv3019c DNA vaccination. Peptides from the protective Mycobacterium bovis antigen Rv3019c were included into the vaccine on the basis of their frequent and strong recognition by T cells from M. bovis infected or BCG vaccinated cattle. Following peptide vaccination, strong IFN-gamma and proliferative T cell responses were observed. Proliferative, but no significant IFN-gamma responses were induced by DNA vaccination. Peptide vaccination boosted responses primed by DNA vaccination. In conclusion, emulsigen and CpG motif containing ODN constitute a promising adjuvant formulation to deliver peptides to veterinary species.

Animals↗

PubMed2005

Frequency of IFN-gamma producing cells correlates with adjuvant enhancement of bacille Calmette-Guèrin induced protection against Mycobacterium bovis.

Tuberculosis caused by infection with Mycobacterium tuberculosis or Mycobacterium bovis remains one of the most important infectious diseases of man and animals. The current vaccine M. bovis Calmette-Guérin (BCG) demonstrates variable efficacy and so a more robust strategy to either replace, or more likely supplement it, is required. Prime-boost strategies where immunity induced by BCG is boosted by a second heterologous vaccine represent a promising avenue of research. We have evaluated the ability of a protein subunit vaccine using the antigen Rv3019c to either prime or boost immunity induced by BCG in a murine M. bovis challenge model. Despite the induction of anamnestic T cell responses, we report that antigen-independent immune stimulation with adjuvant in conjunction with BCG could enhance the level of protection induced by BCG alone. Importantly this improved protection correlated with pre-infection frequencies of ex vivo IFN-gamma producing cells in the spleen, providing a possible surrogate correlate of protection for future vaccination studies.

Adjuvants, Immunologic↗

PubMed2005

Structure and function of the complex formed by the tuberculosis virulence factors CFP-10 and ESAT-6.

The secreted Mycobacterium tuberculosis complex proteins CFP-10 and ESAT-6 have recently been shown to play an essential role in tuberculosis pathogenesis. We have determined the solution structure of the tight, 1:1 complex formed by CFP-10 and ESAT-6, and employed fluorescence microscopy to demonstrate specific binding of the complex to the surface of macrophage and monocyte cells. A striking feature of the complex is the long flexible arm formed by the C-terminus of CFP-10, which was found to be essential for binding to the surface of cells. The surface features of the CFP-10.ESAT-6 complex, together with observed binding to specific host cells, strongly suggest a key signalling role for the complex, in which binding to cell surface receptors leads to modulation of host cell behaviour to the advantage of the pathogen.

Amino Acid Sequence↗

PubMed2005

Effect of oral vaccination of cattle with lipid-formulated BCG on immune responses and protection against bovine tuberculosis.

Cattle were given Mycobacterium bovis bacillus Calmette-Guerin (BCG) in a lipid-based formulation via the oral route and tested for immune responses and protection against a challenge with virulent M. bovis. Calves were vaccinated by orally administering a pellet containing 10(8) colony forming units (CFU) of BCG, or 10 pellets containing a total of 10(9) CFU of BCG, whereas positive controls were injected subcutaneously with 10(6) CFU of BCG. All of the subcutaneously vaccinated calves produced positive responses in the caudal fold tuberculin skin test at 8 weeks after vaccination, whereas only 3/9 of the low dose and 6/10 of the high dose orally-vaccinated animals produced positive reactions. None of the animals produced positive reactions to the mycobacterial antigens, ESAT-6 and CFP10 in the interferon-gamma (IFN-gamma) test and only a total of four of the BCG-vaccinated animals produced positive responses in either the standard IFN-gamma or comparative cervical skin test. Oral administration of 10 pellets of lipid-formulated BCG to cattle induced a significant level of protection against bovine tuberculosis compared to that observed in non-vaccinated animals and this level was similar to that seen in the BCG subcutaneously vaccinated animals. Oral vaccination of BCG in a lipid-formulation to calves was shown to induce some positive tuberculin skin test reactions, but could also induce protection against bovine tuberculosis.

Adjuvants, Immunologic↗

PubMed2005

Protective immunity against Mycobacterium bovis induced by vaccination with Rv3109c--a member of the esat-6 gene family.

In a number of clinical studies the current TB vaccine, Mycobacterium bovis bacille Calmette-Guerin (BCG), has provided little or no protection against pulmonary tuberculosis in cattle and man. A new generation of vaccines is therefore required to replace or supplement BCG. Safety concerns surrounding a number of strategies make protein subunits an attractive approach. Moreover, novel prime-boost strategies based on primary immunisations with BCG are not only showing promise but also present a clear strategy for testing new TB vaccines in clinical studies. We report the evaluation of six protein vaccine candidates for their ability to induce protective immunity in a murine virulent M. bovis challenge model. One protein (Rv3019c) induced reproducibly significant protection in the spleen and lungs approaching that induced by BCG. Detailed analysis of antigen-specific T cell responses revealed that despite robust responses in the spleen and lungs of vaccinated mice, there was no correlation between these responses and the protective efficacy of the vaccine. Significantly, Rv3019c also stimulated IFN-gamma responses in PBMC from BCG vaccinated cattle, indicating its potential for use in a heterologous prime-boost strategy in conjunction with BCG in the target species.

Animals↗

PubMed2005

TB vaccines for the World.

In September 2003, Montreal hosted the first international conference on "TB Vaccines for the World". The timing of the conference was prescient. Two major contracts for screening TB vaccines, the NIH vaccine screening contract and the European Union Fifth Framework TB Vaccine Cluster were coming to an end as was a major 5 year international initiative to develop vaccines against bovine tuberculosis. For the first time the TB vaccine community was getting a glimpse of the most promising vaccine candidates identified using a number of different animal models. Moreover, the first Phase I human trial of a new vaccine for TB based on boosting BCG with an attenuated vaccinia virus expressing Ag85A of M. tuberculosis, MVA85A, had just begun. In addition, the Aeras Global TB Vaccine Foundation had just been launched to apply an industrial pipeline approach to drive vaccine candidates through to clinical trials. The aim of this special edition of Tuberculosis is to encapsulate this defining moment in the development of TB vaccines so that it may be used it as a yardstick against which future progress may be measured. This article provides an overview of the scope of this task.

Animals↗

PubMed2005

The pyruvate requirement of some members of the Mycobacterium tuberculosis complex is due to an inactive pyruvate kinase: implications for in vivo growth.

Through examination of one of the fundamental in vitro characteristics of Mycobacterium bovis--its requirement for pyruvate in glycerol medium--we have revealed a lesion in central metabolism that has profound implications for in vivo growth and nutrition. Not only is M. bovis unable to use glycerol as a sole carbon source but the lack of a functioning pyruvate kinase (PK) means that carbohydrates cannot be used to generate energy. This disruption in sugar catabolism is caused by a single nucleotide polymorphism in pykA, the gene which encodes PK, that substitutes glutamic acid residue 220 with an aspartic acid residue. Substitution of this highly conserved amino acid residue renders PK inactive and thus blocks the ATP generating roles of glycolysis and the pentose phosphate pathway. This mutation was found to occur in other members of the M. tuberculosis complex, namely M. microti and M. africanum. With carbohydrates unable to act as carbon sources, the importance of lipids and gluconeogenesis for growth in vivo becomes apparent. Complementation of M. bovis with the pykA gene from M. tuberculosis H37Rv restored growth on glycerol. Additionally, the presence of a functioning PK caused the colony morphology of the complemented strain to change from the characteristic dysgonic growth of M. bovis to eugonic growth, an appearance normally associated with M. tuberculosis. We also suggest that the glycerol-soaked potato slices used for the derivation of the M. bovis bacillus Calmette and Guérin (BCG) vaccine strain selected for an M. bovis PK+ mutant, a finding that explains the alteration in colony morphology noted during the derivation of BCG. In summary, the disruption of a key step in glycolysis divides the M. tuberculosis complex into two groups with distinct carbon source utilization.

Animals↗

PubMed2005

Minimum infective dose of Mycobacterium bovis in cattle.

The aim of this work was to determine the minimum infective dose of Mycobacterium bovis necessary to stimulate specific immune responses and generate pathology in cattle. Four groups of calves (20 animals) were infected by the intratracheal route with 1,000, 100, 10, or 1 CFU of M. bovis. Specific immune responses (gamma interferon [IFN-gamma] and interleukin-4 [IL-4] responses) to mycobacterial antigens were monitored throughout the study, and the responses to the tuberculin skin test were assessed at two times. Rigorous post mortem examinations were performed to determine the presence of pathology, and samples were taken for microbiological and histopathological confirmation of M. bovis infection. One-half of the animals infected with 1 CFU of M. bovis developed pulmonary pathology typical of bovine tuberculosis. No differences in the severity of pathology were observed for the different M. bovis doses. All animals that developed pathology were skin test positive and produced specific IFN-gamma and IL-4 responses. No differences in the sizes of the skin test reactions, the times taken to achieve a positive IFN-gamma result, or the levels of the IFN-gamma and IL-4 responses were observed for the different M. bovis doses, suggesting that diagnostic assays (tuberculin skin test and IFN-gamma test) can detect cattle soon after M. bovis infection regardless of the dose. This information should be useful in modeling the dynamics of bovine tuberculosis in cattle and in assessing the risk of transmission.

Animals↗