Environmental monitoring of Mycobacterium bovis in badger feces and badger sett soil by real-time PCR, as confirmed by immunofluorescence, immunocapture, and cultivation

Real-time PCR was used to detect and quantify Mycobacterium bovis cells in naturally infected soil and badger faeces. Immunomagnetic capture, immunofluorescence and selective culture confirmed species identification and cell viability. These techniques will prove useful for monitoring M. bovis in the environment and for elucidating transmission routes between wildlife and cattle

Bovine tuberculosis in Swedish farmed deer

Bovine tuberculosis (BTB) was introduced into Swedish farmed deer herds in 1987. Epidemiological investigations showed that 10 deer herds had become infected (July 1994) and a common source of infection, a consignment of 168 imported farmed fallow deer, was identified (I). As trace-back of all imported and in-contact deer was not possible, a control program, based on tuberculin testing, was implemented in July 1994. As Sweden has been free from BTB since 1958, few practising veterinarians had experience in tuberculin testing. In this test, result relies on the skill, experience and conscientiousness of the testing veterinarian. Deficiencies in performing the test may adversely affect the test results and thereby compromise a control program. Quality indicators may identify possible deficiencies in testing procedures. For that purpose, reference values for measured skin fold thickness (prior to injection of the tuberculin) were established (II) suggested to be used mainly by less experienced veterinarians to identify unexpected measurements. Furthermore, the within-veterinarian variation of the measured skin fold thickness was estimated by fitting general linear models to data (skin fold measurements) (III). The mean square error was used as an estimator of the within-veterinarian variation. Using this method, four (6%) veterinarians were considered to have unexpectedly large variation in measurements. In certain large extensive deer farms, where mustering of all animals was difficult, meat inspection was suggested as an alternative to tuberculin testing. The efficiency of such a control was estimated in paper IV and V. A Reed Frost model was fitted to data from seven BTB-infected deer herds and the spread of infection was estimated (< 0.6 effective contacts per deer and year) (IV). These results were used to model the efficiency of meat inspection in an average extensive Swedish deer herd. Given a 20% annual slaughter and meat inspection, the model predicted that BTB would be either detected or eliminated in most herds (90%) 15 years after introduction of one infected deer. In 2003, an alternative control for BTB in extensive Swedish deer herds, based on the results of paper V, was implemented

Flow cytometric detection of gamma interferon can effectively discriminate Mycobacterium bovis BCG-vaccinated cattle from M. bovis-infected cattle

Mycobacterium bovis is the causative agent of bovine tuberculosis, a disease that is increasing in incidence in United Kingdom cattle herds. In addition to increasing economic losses, the rise in bovine tuberculosis poses a human health risk. There is an urgent requirement for effective strategies for disease eradication; this will likely involve vaccination in conjunction with current test and slaughter policies. A policy involving vaccination would require an accurate diagnosis of M. bovis-infected animals and the potential to distinguish these animals from vaccinates. Currently used diagnostic tests, the skin test and gamma interferon (IFN-γ) blood test, have a sensitivity of up to 95%. A further complication is that M. bovis BCG-vaccinated animals are also scored positive by these tests. We tested the hypothesis that the quantification of IFN-γ-producing lymphocytes by flow cytometric analysis of intracellular IFN-γ expression would provide a more accurate discrimination of M. bovis-infected animals from BCG vaccinates. Significant numbers of IFN-γ-expressing CD4(+) T cells were detected following culture of heparinized blood from M. bovis-infected animals, but not from BCG vaccinates, with purified protein derived from M. bovis (PPD-B) or live mycobacteria. Only 1 of 17 BCG-vaccinated animals had a significant number of CD4(+) T lymphocytes expressing IFN-γ, compared with 21/22 M. bovis-infected animals. This assay could allow an accurate diagnosis of M. bovis and allow the discrimination of BCG-vaccinated cattle from infected cattle

Impact of external sources of infection on the dynamics of bovine tuberculosis in modelled badger populations

Background The persistence of bovine TB (bTB) in various countries throughout the world is enhanced by the existence of wildlife hosts for the infection. In Britain and Ireland, the principal wildlife host for bTB is the badger (Meles meles). The objective of our study was to examine the dynamics of bTB in badgers in relation to both badger-derived infection from within the population and externally-derived, trickle-type, infection, such as could occur from other species or environmental sources, using a spatial stochastic simulation model. Results The presence of external sources of infection can increase mean prevalence and reduce the threshold group size for disease persistence. Above the threshold equilibrium group size of 6–8 individuals predicted by the model for bTB persistence in badgers based on internal infection alone, external sources of infection have relatively little impact on the persistence or level of disease. However, within a critical range of group sizes just below this threshold level, external infection becomes much more important in determining disease dynamics. Within this critical range, external infection increases the ratio of intra- to inter-group infections due to the greater probability of external infections entering fully-susceptible groups. The effect is to enable bTB persistence and increase bTB prevalence in badger populations which would not be able to maintain bTB based on internal infection alone. Conclusions External sources of bTB infection can contribute to the persistence of bTB in badger populations. In high-density badger populations, internal badger-derived infections occur at a sufficient rate that the additional effect of external sources in exacerbating disease is minimal. However, in lower-density populations, external sources of infection are much more important in enhancing bTB prevalence and persistence. In such circumstances, it is particularly important that control strategies to reduce bTB in badgers include efforts to minimise such external sources of infection

Reference genome and comparative genome analysis for the WHO reference strain for Mycobacterium bovis BCG Danish, the present tuberculosis vaccine

Background: Mycobacterium bovis bacillus Calmette-Guerin (M. bovis BCG) is the only vaccine available against tuberculosis (TB). In an effort to standardize the vaccine production, three substrains, i.e. BCG Danish 1331, Tokyo 172-1 and Russia BCG-1 were established as the WHO reference strains. Both for BCG Tokyo 172-1 as Russia BCG-1, reference genomes exist, not for BCG Danish. In this study, we set out to determine the completely assembled genome sequence for BCG Danish and to establish a workflow for genome characterization of engineering-derived vaccine candidate strains.ResultsBy combining second (Illumina) and third (PacBio) generation sequencing in an integrated genome analysis workflow for BCG, we could construct the completely assembled genome sequence of BCG Danish 1331 (07/270) (and an engineered derivative that is studied as an improved vaccine candidate, a SapM KO), including the resolution of the analytically challenging long duplication regions. We report the presence of a DU1-like duplication in BCG Danish 1331, while this tandem duplication was previously thought to be exclusively restricted to BCG Pasteur. Furthermore, comparative genome analyses of publicly available data for BCG substrains showed the absence of a DU1 in certain BCG Pasteur substrains and the presence of a DU1-like duplication in some BCG China substrains. By integrating publicly available data, we provide an update to the genome features of the commonly used BCG strains. Conclusions: We demonstrate how this analysis workflow enables the resolution of genome duplications and of the genome of engineered derivatives of the BCG Danish vaccine strain. The BCG Danish WHO reference genome will serve as a reference for future engineered strains and the established workflow can be used to enhance BCG vaccine standardization

Social group size affects Mycobacterium bovis infection in European badgers (Meles meles)

1. In most social animals, the prevalence of directly transmitted pathogens increases in larger groups and at higher population densities. Such patterns are predicted by models of Mycobacterium bovis infection in European badgers (Meles meles). 2. We investigated the relationship between badger abundance and M. bovis prevalence, using data on 2696 adult badgers in 10 populations sampled at the start of the Randomized Badger Culling Trial. 3. M. bovis prevalence was consistently higher at low badger densities and in small social groups. M. bovis prevalence was also higher among badgers whose genetic profiles suggested that they had immigrated into their assigned social groups. 4. The association between high M. bovis prevalence and small badger group size appeared not to have been caused by previous small-scale culling in study areas, which had been suspended, on average, 5 years before the start of the current study. 5. The observed pattern of prevalence might occur through badgers in smaller groups interacting more frequently with members of neighbouring groups; detailed behavioural data are needed to test this hypothesis. Likewise, longitudinal data are needed to determine whether the size of infected groups might be suppressed by disease-related mortality. 6. Although M. bovis prevalence was lower at high population densities, the absolute number of infected badgers was higher. However, this does not necessarily mean that the risk of M. bovis transmission to cattle is highest at high badger densities, since transmission risk depends on badger behaviour as well as on badger density

Performance of an environmental test to detect Mycobacterium bovis infection in badger social groups

A study by Courtenay and others (2006) demonstrated that the probability of detecting Mycobacterium bovis by PCR in soil samples from the spoil heaps of main badger setts correlated with the prevalence of excretion (infectiousness) of captured badgers belonging to the social group. It has been proposed that such a test could be used to target badger culling to setts containing infectious animals (Anon 2007). This short communication discusses the issues surrounding this concept, with the intention of dispelling any misconceptions among relevant stakeholders (farmers, policy makers and conservationists)

Tuberculosis vaccine strain _Mycobacterium bovis_ BCG Russia is a natural _recA_ mutant

The current tuberculosis vaccine is a live vaccine derived from _Mycobacterium bovis_ and attenuated by serial _in vitro_ passaging. All vaccine substrains in use stem from one source, strain Bacille Calmette-Gu&#xe9;rin. However, they differ in regions of genomic deletions, antigen expression levels, immunogenicity, and protective efficacy. As a RecA phenotype increases genetic stability and may contribute restricting the ongoing evolution of the various BCG substrains, we aimed to inactivate _recA_ by allelic replacement in BCG vaccine strains representing different phylogenetic lineages (Pasteur, Frappier, Denmark, Russia). Homologous gene replacement was successful in three out of four strains. However, only illegitimate recombination was observed in BCG substrain Russia. Sequence analyses of _recA_ revealed that a single nucleotide insertion in the 5&#x27; part of _recA_ led to a translational frameshift with an early stop codon making BCG Russia a natural _recA_ mutant. At the protein level BCG Russia failed to express RecA. According to phylogenetic analyses BCG Russia is an ancient vaccine strain most closely related to the parental _M. bovis_. Our data suggest that _recA_ inactivation in BCG Russia occurred early and is in part responsible for its high degree of genomic stability, resulting in a substrain that has less genetic alterations than other vaccine substrains with respect to _M. bovis_ AF2122/97 wild type

Genomic analysis of Mycobacterium bovis and other members of the Mycobacterium tuberculosis complex by isoenzyme analysis and pulsed-field gel electrophoresis.

Initially, multilocus enzyme electrophoresis was used to examine genetic relationships among 63 isolates of Mycobacterium bovis and 13 other members of the M. tuberculosis complex. The isolates were divided into five electrophoretic types, with a mean genetic diversity of 0.1. The strains were genetically homogenous, indicating that members of the complex were closely related. This supported the suggestion that they should be considered as subspecies of a single species. Pulsed-field gel electrophoresis (PFGE) was then used to differentiate these isolates, as well as 59 additional isolates of M. bovis from different parts of the world. PFGE differentiated these strains into 63 patterns (53 patterns for M. bovis). Isolates of M. bovis from Western Australia (n = 46) were more homogenous than isolates from other regions. Eight strains were identified in that state, and one predominantly bovine strain was isolated from two human beings and a feral pig. Although M. bovis isolates from different parts of the world had distinct DNA patterns, some were very similar. PFGE is a highly discriminatory technique for epidemiological studies of bovine tuberculosis. For example, it allowed differentiation between isolates of M. bovis cultured from animals in separate outbreaks of tuberculosis, it suggested the transmission of infection between certain properties, and it demonstrated the existence of multiple infections with different strains at certain farms