Detection of Mycobacterium bovis infection and production of interleukin-2 by in vitro stimulation of badger lymphocytes

The Eurasian badger (Meles meles) is considered to be an important wildlife reservoir for Mycobacterium bovis infection of cattle in Ireland and in GB. However, rapid diagnosis of tuberculosis in live badgers has been constrained through a lack of suitable immuno-diagnostic reagents for detection of M. bovis-infected animals. To date, there have been no reports of cytokine activity in badgers that might be associated with specific immune responses to M. bovis infection. In this study, nine badgers were removed from an area with a persistent tuberculosis problem in cattle herds and tuberculosis was confirmed in four of the animals by "post-mortem" examination and M. bovis culture. In preliminary investigations of interleukin-2 (IL-2) activity, we were able to demonstrate that lymphoblasts prepared from badger peripheral blood mononuclear cells (PBMCs) proliferated when cultured in the presence of human recombinant IL-2 (HrIL-2). Supernatants derived from purified protein derivative of tuberculin (PPD-bovine) stimulated PBMC cultures also induced blastogenesis of badger-derived lymphoblasts. The results demonstrate that badger lymphocytes are responsive to HrIL-2 and that PPD-bovine stimulation of badger PBMC results in production of bio-active IL-2.     

Laboratory study of Mycobacterium bovis infection in badgers and calves

Two experiments with badgers infected with Mycobacterium bovis are described. In the first, badgers were infected by intravenous inoculation of a bovine isolate of M bovis. The course of the disease in these and its spread to healthy badgers and calves was monitored by clinical, immunological and bacteriological means. In the second experiment a group of naturally infected badgers were observed for a period of up to four years. They were found to excrete M bovis in their faeces for periods of between 165 and 1305 days before they died of tuberculosis or were killed. M bovis was also shed in the urine. The badgers in both experiments were examined regularly and blood samples were taken for complement fixation tests. Faeces, urine, pus and sputum were also collected for cultural and biological tests and the badgers were skin tested using Weybridge bovine and avian tuberculin. The skin tests were uniformly negative while the complement fixation test were positive in some infected badgers but gave very variable results. Only the isolation of M bovis gave a definite diagnosis of tuberculosis in the living badger but a number of badgers which were found to have tuberculosis at post mortem were not detected while alive by this method. Environmental samples from the yards, including badger faeces, soil, hay, scrapings from feeding bowls and water were regularly examined for the presence of M bovis but apart from faeces only one water sample was positive, indicating that the organism did not persist for long in the environment. In both experiments calves developed sensitivity to bovine tuberculin after six months' exposure to infected badgers. The experiments further demonstrate the potential of a badger population to become endemically infected with M bovis and to act as a source of infection for cattle.     

Immunological responses following experimental endobronchial infection of badgers (Meles meles) with different doses of Mycobacterium bovis

The Eurasian badger (Meles meles) is a wildlife reservoir for Mycobacterium bovis infection in Ireland and Great Britain and has been implicated in the transmission of tuberculosis to cattle. Vaccination of badgers is an option that could be used as part of a strategy to control the disease. In this study we used an endobronchial infection procedure to inoculate groups of badgers with three different doses (3x10(3), 2x10(2) and <10 Colony Forming Units (CFUs)) of M. bovis. After 17 weeks the disease status of each animal was determined by post-mortem pathology and culture for M. bovis. Each of the inoculum doses resulted in establishment of infection in the badgers. The cell-mediated immune (CMI) responses were measured by lymphocyte transformation assay (LTA) of peripheral blood mononuclear cells (PBMCs) cultured with bovine tuberculin (PPD-B). In each infected group the CMI responses increased with a kinetic profile corresponding to the delivered dose and the post-mortem pathology. The serological responses were measured by ELISA and a multi-antigen print immunoassay (MAPIA) in order to investigate any changes in the antigenic repertoire associated with different infective doses. In contrast to the CMI responses, the ELISA and MAPIA showed that the recognition of antigens by the badgers was intermittent and not strongly influenced by the dose of M. bovis.     

Immunological responses of Eurasian badgers (Meles meles) vaccinated with Mycobacterium bovis BCG (bacillus calmette guerin)

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

A lymphocyte transformation assay for the detection of Mycobacterium bovis infection in the Eurasian badger (Meles meles).

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

Cloning and sequencing of badger (Meles meles) interferon gamma and its detection in badger lymphocytes

The European badger (Meles meles) has been identified as a reservoir for Mycobacterium bovis and is implicated in the maintenance and transmission of tuberculosis in cattle. There is a need for a sensitive test of M. bovis infection in badgers and the current serodiagnostic test used for this purpose has low sensitivity. As observed for other species, assay of interferon-gamma (IFNgamma) produced in response to M. bovis antigens is a more sensitive test of tuberculosis. With this objective in sight, we report the first step in the development of an ELISA for badger IFNgamma. The badger IFNgamma gene was cloned and sequenced and used to generate a specific polyclonal antibody to the cytokine. The gene sequence demonstrated regions that were conserved within the IFNgamma genes of other mammals. The badger sequence was most similar to the canine, showing similar structural organisation of the gene and 88% amino acid identity. Rabbits were immunised with DNA encoding badger IFNgamma and the resulting polyclonal antiserum demonstrated specificity for canine IFNgamma by immunoblot of a commercial recombinant canine IFNgamma. The antiserum was used to detect intracellular badger IFNgamma by flow cytometry analysis of badger lymphocytes stimulated with mitogen.     

Managing public demand for badger rehabilitation in an area of England with endemic tuberculosis

Badgers are a popular and protected species in England, despite their association with tuberculosis (Mycobacterium bovis infection) in cattle. Casualty badgers are commonly presented to veterinarians and wildlife rescue centres following injury, as a result of disease, or as orphans. Strict policies are adopted for their rehabilitation and release, with respect to the prevention of spread of tuberculosis, these policies differ between adult badgers and badger cubs. Adult badger casualties are not normally tested for M. bovis infection prior to release, but are instead kept in isolation and released back where found. A study of casualty adult badgers found 10% to be positive on a single serological test. These animals had a variety of clinical signs that had resulted in none of them being released back to the wild. Badger cubs are serologically tested for evidence of M. bovis infection on three occasions during rearing; 13% were found to test positive. Positive animals were examined at post-mortem and cultures made for M. bovis; 12.5% of serologically positive animals were found to be culture positive. Alternative test methods and zoonotic risks are considered.     

Tuberculosis in cattle herds are sentinels for Mycobacterium bovis infection in European badgers (Meles meles): the Irish Greenfield Study

In Ireland badgers are removed in response to tuberculosis (TB) breakdowns in cattle herds (focal culling). Prevalence studies, conducted using a detailed post mortem and bacteriological examination, showed that 36-50% of badgers were infected with Mycobacterium bovis. Focal culling forms part of the medium term national strategy for the control of bovine TB in cattle and is based on the premise that badgers in areas with herd breakdowns have a higher prevalence of infection than the badger population at large. However, the hypothesis that cattle can be used as sentinels for infection in the badger population has never been formally tested. In this study we tested the hypothesis by determining the infection prevalence in badgers in areas where there had been historically, a consistently low prevalence of infection in cattle. Low cattle TB prevalence areas were defined as those herds with ≤ 2 standard reactors in the annual round of skin testing over the preceding 5 years (Greenfield sites). Using GIS, and adjusting for variation in land use, previous culling and cattle density, 198 Greenfield sites were identified and surveyed, and 138 areas with badger setts or signs of badger activity were identified. A single badger was removed from 87 sites and all were examined using detailed post mortem and bacteriological procedures. A prevalence of M. bovis infection of 14.9% was found in the Greenfield site badgers. This prevalence was significantly lower (P<0.001) than in badgers removed during focal culling (36.6%). The results validate the use of cattle as sentinels for TB in badgers and support the medium term national strategy for the control of bovine TB. The geographic variation in M. bovis infection prevalence in the Irish badger populations will be used when devising strategies for the incorporation of badger vaccination into the long term bovine TB control programme.     

Mycobacterium bovis infection in badger cubs: Re-assessing the evidence for maternally derived immunological protection from advanced disease

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

Experimental tuberculosis in the European badger (Meles meles) after endobronchial inoculation of Mycobacterium bovis: I. Pathology and bacteriology

The aim was to develop an endobronchial infection procedure for the study of Mycobacterium bovis infection in badgers. The badgers were anaesthetised and a cannula was passed per os to the tracheal bifurcation. When in place 1 ml of M. bovis suspension was inoculated. Three concentrations of M. bovis suspension were used; <10 colony forming units (cfu), approximately 10(2) cfu and approximately 3 x 10(3) cfu. The badgers were examined at three weekly intervals for clinical signs of disease and a tracheal aspirate was collected at each examination. The badgers were euthanased 17 weeks post infection (pi) and at the post mortem examination a wide range of tissues were examined for gross and histopathological lesions of tuberculosis and cultured for M. bovis. A sample of bronchial alveolar lavage (BAL) fluid was collected at post mortem for culture. At post mortem examination 17 weeks after infection, gross and histopathological lesions of tuberculosis were observed in all badgers inoculated with the high and medium dose and 1/3 inoculated with the low dose. M. bovis was recovered from all inoculated badgers. Infection in the high dose group was more widely disseminated than in the other groups. The number of sites with gross and histopathological lesions increased with increasing dose of M. bovis. All tracheal aspirates were negative on culture and only one BAL, collected from a badger of the high dose group, was positive on culture. No clinical signs due to the experimental infection were observed. The endobronchial route of inoculation is an effective route for establishing experimental infection, and could be used for studies of tuberculosis pathogenesis, immunology of M. bovis infection in badgers and for challenging badgers in vaccine protection studies. Badgers appeared to be very susceptible to infection by this procedure even with a dose of < 10 cfu but appear to control and limit the resulting infection.     

Geographical association between the genotype of bovine tuberculosis in found dead badgers and in cattle herds

In a survey, 457 badgers that had been found dead in Wales were postmortem-examined, and samples were examined by histology and by extended culture (for up to 12 weeks). Mycobacterium bovis was cultured from 55 badgers (12.0 per cent), and the histology typical of M bovis infection was seen in a further six (1.3 per cent). The prevalence in badgers in each of 10 geographical areas varied between 0 and 26 per cent (P<0.001), and was associated with the incidence of confirmed M bovis infection in cattle herds in the same areas (P<0.01). In northern Wales, bTB was rare in both hosts. An infected badger was 12.3 times more likely to be within 5 km of a confirmed cattle bTB breakdown than an uninfected badger. The M bovis isolates from badgers belonged to one of four genotypes defined by spoligotype and variable number tandem repeat type. These genotypes were also found in 290 concurrent confirmed herd breakdowns, and tended to be similar to the genotypes in badgers in the same geographical areas. When badgers and cattle no more than 30 km apart were compared, the genotype diversity was greater in cattle than in badgers (P=0.016), suggesting that the movement of cattle plays a greater part in the spatial distribution of M bovis than the movement of badgers.     

The effect of varying levels of population control on the prevalence of tuberculosis in badgers in Ireland

We examined the effect of varying levels of badger population control on the prevalence of Mycobacterium bovis infection in badgers in four counties of Ireland. In the 'Removal' and 'Buffer' areas, proactive culling was conducted to substantially reduce and subsequently maintain badger populations at a low level for five years. In the 'Reference' areas, localised reactive culling was conducted in association with herd breakdowns. The infection status of badgers was determined using bacteriology. A total of 2696 badgers were recruited into the study, and 19.0% were found to be infected with M. bovis. The two population control strategies had differing effects on the subsequent prevalence of tuberculosis in badger populations. Proactive culling led to a long term decrease in the prevalence of tuberculosis in the re-emergent populations. Although there was an overall decline in the disease prevalence, no consistent trend in disease prevalence as a result of reactive culling was observed.     

Spatial relationship between Mycobacterium bovis strains in cattle and badgers in four areas in Ireland

We investigated whether strains (restriction fragment length polymorphism, RFLP-types) of Mycobacterium bovis isolated from badgers and from cattle clustered among and within four areas in Ireland. The spatial scan test and nearest-neighbor analysis were used as the spatial cluster-detection techniques. In addition, for each of the major strains, associations between the distance to badger setts and the "centroid" of the cattle farm were assessed in a logistic model. Overall, between September 1997 and May 2000, 316 and 287 M. bovis samples, from badgers and cattle, respectively, were strain-typed. The distribution of strains in badgers, and separately in cattle, differed among areas. Within each of the four large areas, badgers and cattle tended to have similar strains; this is consistent with the sharing of M. bovis strains within an area. In more detailed within-area analyses, some spatial clusters of M. bovis strains were detected, separately, in both cattle and badgers. Almost half of the infected badger setts with a specific strain were located outside of the "detected" clusters. There was no association between the number of infected badgers with a specific M. bovis strain within 2 or 5 km distances to cattle herds, and the risk of the same strain in cattle. We speculate about the dynamic nature of badger movements, as an explanation for the absence of more clusters of most of the strains of M. bovis isolated from badgers, and its impact on trying to study transmission of M. bovis between cattle and badger.     

Tuberculosis infection in cattle and badgers in an area of Staffordshire

The recent history of tuberculosis infection in cattle in Staffordshire is described, showing how a problem area in the north of the county was identified. The subsequent culture of Mycobacterium bovis from badger faeces and a badger carcase is reported together with the operations to remove badgers from the area.     

A DNA vaccine encoding MPB83 from Mycobacterium bovis reduces M. bovis dissemination to the kidneys of mice and is expressed in primary cell cultures of the European badger (Meles meles)

Nucleic acid (DNA) vaccination against tuberculosis in the European badger (Meles meles) is one approach to addressing the escalating problem of bovine tuberculosis in Great Britain. The aim of vaccination is to reduce the burden of tuberculosis within the badger population and the shedding of Mycobacterium bovis to levels that would break the transmission of infection to cattle. To this end, the vaccine would be required to limit the amount of disseminated tuberculosis in the badger, especially dissemination to the kidney from where M. bovis can be shed in the urine. A promising candidate DNA vaccine encoding a 26 kDa major antigen (MPB83) of M. bovis was evaluated in a mouse model of disseminated M. bovis infection. Using the DNA vaccine, protection against infection of the kidney was found to be greater than that achieved with the current live vaccine, Bacille Calmette-Guerin (BCG). Kidney tissue and skeletal muscle from the badger was used to derive primary cell cultures in which to examine the expression of MPB83 following transfection with the DNA vaccine. Kidney cortex gave rise to a monotypic culture of epithelial cells whilst the muscle gave rise to a mixed culture of fibroblasts and myoblasts. During culture the myoblasts differentiated into multinucleated myotubes, verified by immunofluorescent detection of mammalian desmin. Successful expression of MPB83 by transfected epithelial and myotube cells was confirmed by immunofluorescence using a monoclonal antibody specific to the protein. These observations fulfil the early requirements for the development of a DNA vaccine for badger tuberculosis.     

Infection of Eurasian badgers (Meles meles) with Mycobacterium bovis and Mycobacterium avium complex in Spain

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.     

Antigen specific immunological responses of badgers (Meles meles) experimentally infected with Mycobacterium bovis

European badgers (Meles meles) are considered to be an important reservoir of infection for Mycobacterium bovis and are implicated in the transmission of tuberculosis to cattle in Ireland and Great Britain. Accurate tests are required for tuberculosis surveillance in badger populations and to provide a basis for the development of strategies, including vaccination, to reduce the incidence of the infection. In this study, we have developed an endobronchial M. bovis infection model in badgers in which we measured cell-mediated immune and serological responses for up to 24 weeks post-infection. Groups of badgers were subjected to necropsy at 6-week intervals and the gross lesion severity status compared with immune responses measured in blood samples taken throughout the course of the study. The panel of antigens included bovine and avian tuberculins (PPD) as well as single antigens, ESAT-6, CFP-10, MPB70, Rv3019c, Rv3873, Rv3878 and Rv3879, all known to be recognised by the immune system in other animal models of tuberculosis infection. Our results demonstrated that M. bovis infected badgers responded to specific antigens as early as 6 weeks post-infection, consistent with the presence of visible lesions. The data also revealed unique patterns of antigen recognition with high levels of PBMC proliferation in the presence of CFP-10 but low proliferation levels with ESAT-6. Using a multi-antigen print immunoassay (MAPIA), we were able to confirm that MPB83 is the dominant antigen recognised by serum antibodies in infected badgers.     

BCG vaccination against tuberculosis in European badgers (Meles meles): a review

Tuberculosis (TB) is a significant animal health problem in many parts of the world, and reservoirs of infection in wild animals complicate disease control efforts in farmed livestock, particularly cattle. Badgers (Meles meles) are a significant wildlife reservoir of Mycobacterium bovis infection for cattle in the United Kingdom (UK) and Republic of Ireland (ROI). Vaccination of badgers using an M. bovis strain bacille Calmette-Guérin (BCG) vaccine could potentially be an option in the national TB eradication strategy. Wildlife vaccination has been used successfully for other diseases in wildlife species, and may have a role to play in reducing M. bovis transmission at the wildlife-livestock interface. Research to date has provided evidence that BCG is protective in badgers, and a parenteral badger BCG vaccine has been licensed in the UK. Further research is required to develop effective strategies for vaccine deployment and to determine the effect of badger vaccination on cattle TB incidence.     

Trial design to estimate the effect of vaccination on tuberculosis incidence in badgers

The principal wildlife reservoir of Mycobacterium bovis in Ireland is the European badger. Studies in the Republic of Ireland (RoI) have shown that badgers culled in association with cattle herd tuberculosis breakdowns (focal culling) have a higher prevalence of infection than the badger population at large. This observation is one rationale for the medium term national strategy of focal badger culling. A vaccination strategy for the control of bovine tuberculosis (bTB) in badgers is a preferred long-term option. The Bacillus Calmette-Guérin (BCG) vaccine has been shown to decrease disease severity in captive badgers under controlled conditions. As the vaccine has been tested in a controlled environment with precise information on infection pressure, it cannot be assumed a priori that the effects of vaccination are similar in the wild, where other environmental and/or ecological factors prevail. For this reason we have designed a vaccine field trial to assess the impact of vaccination on the incidence of TB infection in a wild badger population. The selected study area for the vaccine trial (approximately 755 square kilometers) is divided into three zones each of which has similar characteristics in terms of size, number of main badger setts, cattle herds, cattle and land classification type. Three vaccination levels (100%, 50% and 0%) will be allocated to the three zones in a way that a gradient of vaccination coverage North to South is achieved. The middle zone (zone B) will be vaccinated at a 50% coverage but zone A and C will be randomly allocated with 100% or 0% vaccination coverage. Vaccination within zone B will be done randomly at individual badger level. The objective of this paper is to describe the design of a field tuberculosis vaccination trial for badgers, the epidemiological methods that were used to design the trial and the subsequent data analysis. The analysis will enable us to quantify the magnitude of the observed vaccination effect on M. bovis transmission in badgers under field conditions and to improve our knowledge of the biological effects of vaccination on susceptibility and infectiousness.     

The safety and immunogenicity of Bacillus Calmette-Guérin (BCG) vaccine in European badgers (Meles meles)

European badgers (Meles meles) are a wildlife reservoir for Mycobacterium bovis (M. bovis) in Great Britain (GB) and the Republic of Ireland and therefore constitute a potential source of infection for cattle. Reduction of badger densities in the Republic of Ireland has resulted in an associated reduction in the risk of a herd break-down with bovine tuberculosis and a study to determine whether this is also the case in GB has been running since 1997. If badgers are a significant source of M. bovis infection for cattle, vaccinating badgers with Bacillus Calmette-Guérin (BCG) might prove to be a long term, cost-effective strategy for controlling bovine tuberculosis whilst preserving badger populations. As a first step towards BCG vaccination of wild badgers, it was necessary to demonstrate safety of the vaccine in captive badgers. Therefore, captive badgers were vaccinated with a commercial source of BCG that is already licensed for administration to humans in GB-BCG Danish SSI. Using a protocol prescribed by the Veterinary Medicines Directorate (VMD) of GB, badgers were vaccinated with two consecutive doses of BCG via either the subcutaneous (s.c.) or intra-muscular (i.m.) routes. The first dose was high, ranging from 16 to 22 x 10(7) colony-forming units (CFU), and was followed 15 weeks later by a lower dose in the range of 4-7 x 10(5)CFU. Local reaction at the site of injection and general responses (body temperature, haematology and blood serum chemistry), behaviour and excretion of BCG were monitored for 28 weeks from the time of the first vaccination. The only side-effect observed was the occurrence of localised swelling at the site of BCG injection that disappeared 48 days after i.m. vaccination but persisted longer in the group vaccinated by the s.c. route. Immunological responses were measured at regular intervals. Strong cellular responses were observed 13 days after the first vaccination, which persisted for 76 days. The lower dose induced a weaker and shorter-lived response.