Leigh A. L. Corner

Mycobacterium bovis infection in the Eurasian badger (Meles meles): the disease, pathogenesis, epidemiology and control.

Eurasian badgers (Meles meles) are an important wildlife reservoir of tuberculosis (Mycobacterium bovis) infection in Ireland and the United Kingdom. As part of national programmes to control tuberculosis in livestock, considerable effort has been devoted to studying the disease in badgers and this has lead to a rapid increase in our knowledge of tuberculosis in this host. Tuberculosis in badgers is a chronic infection and in a naturally-infected population the severity of disease can vary widely, from latent infection (infection without clinical signs and no visible lesions) to severe disease with generalized pathology. The high prevalence of pulmonary infection strongly supports the lungs as the principal site of primary infection and that inhalation of infectious aerosol particles is the principal mode of transmission. However, other routes, including transmission via infected bite wounds, are known to occur. The ante-mortem diagnosis of infection is difficult to achieve, as clinical examination and immunological and bacteriological examination of clinical samples are insensitive diagnostic procedures. Because infection in the majority of badgers is latent, the gross post-mortem diagnosis is also insensitive. A definitive diagnosis can only be made by the isolation of M. bovis. However, to gain a high level of sensitivity in the bacteriological examination, a large number of tissues from each badger must be cultured and sensitive culture methods employed. The transmission and maintenance of M. bovis in badger populations are complex processes where many factors influence within-population prevalence and rates of transmission. Badger social structures and the longevity of infected animals make them an ideal maintenance host for M. bovis infection. Badgers are directly implicated in the transmission of infection to cattle and the inability to eradicate the disease from cattle is, in part, a consequence of the interactions between the two species. A detailed understanding and knowledge of the epidemiology and pathogenesis of the disease are recognized as fundamental for devising new strategies to control infection with a view to limiting interspecies transmission. Vaccination, in spite of formidable challenges, is seen as the best long-term strategy option and studies with captive badgers have shown that vaccination with M. bovis bacillus Calmette-Guérin (BCG) induces protection when delivered by a variety of routes. Continued research is required to develop effective technologies to control the disease both in badgers and cattle. A combination of strategies, which employ the optimal use and targeting of resources, is likely to make a significant contribution towards eradication of the disease.

Bacillus Calmette-Guérin vaccination reduces the severity and progression of tuberculosis in badgers

Control of bovine tuberculosis (TB) in cattle has proven particularly challenging where reservoirs of infection exist in wildlife populations. In Britain and Ireland, control is hampered by a reservoir of infection in Eurasian badgers (Meles meles). Badger culling has positive and negative effects on bovine TB in cattle and is difficult, costly and controversial. Here we show that Bacillus Calmette-Guérin (BCG) vaccination of captive badgers reduced the progression, severity and excretion of Mycobacterium bovis infection after experimental challenge. In a clinical field study, BCG vaccination of free-living badgers reduced the incidence of positive serological test results by 73.8 per cent. In common with other species, BCG did not appear to prevent infection of badgers subjected to experimental challenge, but did significantly reduce the overall disease burden. BCG vaccination of badgers could comprise an important component of a comprehensive programme of measures to control bovine TB in cattle.

Granuloma Encapsulation Is a Key Factor for Containing Tuberculosis Infection in Minipigs

A transthoracic infection involving a low dose of Mycobacterium tuberculosis has been used to establish a new model of infection in minipigs. The 20-week monitoring period showed a marked Th1 response and poor humoral response for the whole infection. A detailed histopathological analysis was performed after slicing the formalin-fixed whole lungs of each animal. All lesions were recorded and classified according to their microscopic aspect, their relationship with the intralobular connective network and their degree of maturity in order to obtain a dissemination ratio (DR) between recent and old lesions. CFU counts and evolution of the DR with time showed that the proposed model correlated with a contained infection, decreasing from week 9 onwards. These findings suggest that the infection induces an initial Th1 response, which is followed by local fibrosis and encapsulation of the granulomas, thereby decreasing the onset of new lesions. Two therapeutic strategies were applied in order to understand how they could influence the model. Thus, chemotherapy with isoniazid alone helped to decrease the total number of lesions, despite the increase in DR after week 9, with similar kinetics to those of the control group, whereas addition of a therapeutic M. tuberculosis fragment-based vaccine after chemotherapy increased the Th1 and humoral responses, as well as the number of lesions, but decreased the DR. By providing a local pulmonary structure similar to that in humans, the mini-pig model highlights new aspects that could be key to a better understanding tuberculosis infection control in humans.

Use of an Electronic Nose To Diagnose Mycobacterium bovis Infection in Badgers and Cattle

ABSTRACT It is estimated that more than 50 million cattle are infected with Mycobacterium bovis worldwide, resulting in severe economic losses. Current diagnosis of tuberculosis (TB) in cattle relies on tuberculin skin testing, and when combined with the slaughter of test-positive animals, it has significantly reduced the incidence of bovine TB. The failure to eradicate bovine TB in Great Britain has been attributed in part to a reservoir of the infection in badgers (Meles meles). Accurate and reliable diagnosis of infection is the cornerstone of TB control. Bacteriological diagnosis has these characteristics, but only with samples collected postmortem. Unlike significant wild animal reservoirs of M. bovis that are considered pests in other countries, such as the brushtail possum (Trichosurus vulpecula) in New Zealand, the badger and its sett are protected under United Kingdom legislation (The Protection of Badgers Act 1992). Therefore, an accurate in vitro test for badgers is needed urgently to determine the extent of the reservoir of infection cheaply and without destroying badgers. For cattle, a rapid on-farm test to complement the existing tests (the skin test and gamma interferon assay) would be highly desirable. To this end, we have investigated the potential of an electronic nose (EN) to diagnose infection of cattle or badgers with M. bovis, using a serum sample. Samples were obtained from both experimentally infected badgers and cattle, as well as naturally infected badgers. Without exception, the EN was able to discriminate infected animals from controls as early as 3 weeks after infection with M. bovis, the earliest time point examined postchallenge. The EN approach described here is a straightforward alternative to conventional methods of TB diagnosis, and it offers considerable potential as a sensitive, rapid, and cost-effective means of diagnosing M. bovis infection in cattle and badgers.

Oral vaccination of badgers (Meles meles) with BCG and protective immunity against endobronchial challenge with Mycobacterium bovis.

Eurasian badgers (Meles meles) are a reservoir host of Mycobacterium bovis and are implicated in the transmission of tuberculosis to cattle in Ireland and Great Britain. The development of a vaccine for use in badgers is considered a key element of any long-term sustainable campaign to eradicate the disease from livestock in both countries. The aim of this study was to investigate the protective response of badgers vaccinated orally with Bacille Calmette-Guérin (BCG) encapsulated in a lipid formulation, followed by experimental challenge with M. bovis. A group of badgers was vaccinated by inoculating the BCG-lipid mixture containing approximately 10(8)colony forming units (cfu) of BCG into the oesophagus. The control group was sham inoculated with the lipid formulation only. Thirteen weeks after vaccination all the badgers were challenged with approximately 10(4)cfu of M. bovis delivered by endobronchial inoculation. Blood samples were taken throughout the study and the cell mediated immune (CMI) responses in peripheral blood were monitored by the IFN-gamma ELISA and ELISPOT assay. At 17 weeks after infection all the badgers were examined post-mortem to assess the pathological and bacteriological responses to challenge. All badgers in both groups were found to be infected. However, a significant protective effect of BCG vaccination was measured as a decrease in the number and severity of gross lesions, lower bacterial load in the lungs, and fewer sites of infection. The analysis of immune responses showed that vaccination with BCG did not generate any detectable CMI immunological responses, however the levels of the responses increased in both groups following M. bovis infection. The results of the study showed that vaccination with oral BCG in the lipid formulation generated a protective effect in the badgers.

The prevalence and distribution of Mycobacterium bovis infection in European badgers (Meles meles) as determined by enhanced post mortem examination and bacteriological culture.

The accurate diagnosis of Mycobacterium bovis infection in badgers is key to understanding the epidemiology of tuberculosis in this species and has significant implications for devising strategies to limit spread of the disease. In this study, badgers (n=215) in the Republic of Ireland were examined at post mortem and tissues were collected from a range of anatomical locations and pooled into groups for bacterial culture of M. bovis. By assessing confirmed gross visible lesions (VL) alone, infection was detected in 12.1% of badgers. However, by including the results of all culture positive pooled samples, the overall infection prevalence increased significantly to 36.3%. Two-thirds (66.7%) of infected animals had no visible lesions (NVL). While the thoracic cavity (lungs and pulmonary lymph nodes) was found to be the most common site of infection, in a proportion of animals infection was absent from the lungs and draining lymph nodes and was confined to the lymph nodes of the carcase or the head. This may indicate an early extrapulmonary dissemination of infection or alternatively, in the case of the head lymph nodes, a secondary pathogenic pathway involving the lymphoid tissues of the upper respiratory tract (URT).

Vaccination of European badgers (Meles meles) with BCG by the subcutaneous and mucosal routes induces protective immunity against endobronchial challenge with Mycobacterium bovis

Mycobacterium bovis is endemic in badger (Meles meles) populations of Ireland and the United Kingdom and infected badgers are a potential source of infection for cattle. In domestic livestock tuberculosis causes economic losses from lost production and the costs associated with eradication programmes, and in addition there is a risk of zoonotic infection. Whereas culling is currently used to control tuberculous badger populations in Ireland, vaccination, if it were available, would be preferred. A study was undertaken to examine the protective responses of badgers vaccinated either by the subcutaneous or mucosal (intranasal and conjunctival) routes with bacille Calmette-Guérin (BCG), when challenged with M. bovis by the endobronchial route. Three groups of badgers were used. The first group (n=4) was vaccinated with approximately 5 x 10(5) colony forming units (cfu) of BCG by subcutaneous injection. In the second group (n=5) badgers were vaccinated via the mucosal route by instilling 1.0 x 10(5)cfu into each conjunctival sac and spraying 1.0 x 10(5)cfu into each nostril (final vaccine dose of 4 x 10(5)cfu). The control (n=5) badgers served as a non-vaccinated group. Twelve weeks post-vaccination all badgers in the three groups were challenged with approximately 10(4)cfu of M. bovis by endobronchial inoculation. At 12 weeks post-infection all badgers were examined post-mortem to assess the pathological and bacteriological responses to challenge. Gross and histological lesions of tuberculosis were seen in all challenged badgers and M. bovis was recovered from all challenged badgers. However, across six of the eight parameters used to measure disease severity, the infection in the vaccinated badgers was significantly less severe than in the control group. The BCG vaccine induced a significant protective effect in the badgers and the protective immunity was generated by subcutaneous and mucosal vaccination.

Mycobacterium bovis: A Model Pathogen at the Interface of Livestock, Wildlife, and Humans

Complex and dynamic interactions involving domestic animals, wildlife, and humans create environments favorable to the emergence of new diseases, or reemergence of diseases in new host species. Today, reservoirs of Mycobacterium bovis, the causative agent of tuberculosis in animals, and sometimes humans, exist in a range of countries and wild animal populations. Free-ranging populations of white-tailed deer in the US, brushtail possum in New Zealand, badger in the Republic of Ireland and the United Kingdom, and wild boar in Spain exemplify established reservoirs of M. bovis. Establishment of these reservoirs is the result of factors such as spillover from livestock, translocation of wildlife, supplemental feeding of wildlife, and wildlife population densities beyond normal habitat carrying capacities. As many countries attempt to eradicate M. bovis from livestock, efforts are impeded by spillback from wildlife reservoirs. It will not be possible to eradicate this important zoonosis from livestock unless transmission between wildlife and domestic animals is halted. Such an endeavor will require a collaborative effort between agricultural, wildlife, environmental, and political interests.

Adverse reactions to Mycobacterium bovis bacille Calmette–Guérin (BCG) vaccination against tuberculosis in humans, veterinary animals and wildlife species

The Mycobacterium bovis strain, bacille Calmette-Guérin (BCG) is one of the most widely used human vaccines and remains one of the safest vaccines available. It has been used in human populations for over 80 years and 100 million children receive the vaccine annually. It has also been employed extensively for vaccine studies in laboratory animal hosts and is currently being developed for use in a variety of livestock and wild animals. Despite the large number of doses delivered since is first usage in 1921, reports of adverse reactions arising from the use of the BCG vaccine are relatively uncommon and where serious reactions do occur they are often the result of vaccination of immuno-compromised individuals. Factors that may influence the development of adverse reactions to BCG include the potency and dose of the vaccine strain, the route of delivery, the age and immune status of the host, and the skill levels of the operator administering the vaccine. Circumstances affecting the notification of adverse reactions include the lack of clear case definitions of abnormal vaccine reactions, and a scarcity of systematic surveillance and functioning reporting systems. With continued use of the BCG and the development of a new generation of prophylactic and therapeutic vaccines against tuberculosis in different host species, the risk factors associated with adverse reactions may need to be reappraised.

Immunological responses and protective immunity in BCG vaccinated badgers following endobronchial infection with Mycobacterium bovis

European badgers (Meles meles) are a reservoir host of Mycobacterium bovis and are implicated in the transmission of tuberculosis to cattle in Ireland and Great Britain. The development of a vaccine for use in badgers is considered a key element of any campaign to eradicate the disease in livestock in both countries. In this study we have vaccinated groups of badgers with approximately 5 x 10(5)cfu of the BCG vaccine delivered via two alternative routes, subcutaneous and mucosal (intranasal/conjunctival). Following experimental endobronchial infection with approximately 10(4)cfu of M. bovis, all badgers were euthanised at 12 weeks post-infection. At post-mortem examination both vaccinated groups had significantly reduced severity of disease compared with the non-vaccinated controls. The analysis of immune responses throughout the study showed that vaccination with BCG did not generate any detectable immunological responses as measured by IFN-gamma production in antigen-stimulated peripheral blood mononuclear cells (PBMC) and IgG serological responses. However, the levels of the responses increased following M. bovis infection, and the kinetic profiles corresponded to the severity of lesions recorded post-mortem. Significant differences were observed in the timing of development of the immune responses between vaccinates and controls. The results suggest that the immunological responses are associated with the levels of protective immunity and could be used as markers to monitor control of disease in badgers following vaccination.