Progress in the development of tuberculosis vaccines for cattle and wildlife

Vaccination against bovine tuberculosis is likely to become an important disease control strategy in developing countries, which cannot afford a test and slaughter control programme, or in countries which have a wildlife reservoir of Mycobacterium bovis infection. In the past decade, considerable progress has been made in the development and evaluation of tuberculosis vaccines for cattle and for a range of wildlife maintenance hosts including possums, badgers, deer and African buffaloes. Experimental challenge systems have been established for the different target species and the resulting disease process has mimicked that seen in the field. In cattle, neonatal vaccination with BCG appeared to be more effective than vaccination of 6-month-old calves and in most situations no other vaccine has been shown to be better than BCG. However, prime-boost strategies involving combinations of BCG with a protein or DNA vaccine, to improve on BCG vaccination alone, have produced very encouraging results. Differential diagnostic tests have been developed using mycobacterial antigens that are only present in virulent M. bovis to differentiate between BCG-vaccinated and M. bovis-infected cattle. BCG vaccine has been shown to reduce the spread of tuberculous lesions in a range of wildlife species and a prototype oral bait delivery system has been developed. Prospects for the development of improved vaccines against bovine tuberculosis are promising and vaccination approaches could become very valuable in the control and eradication of bovine tuberculosis.     

Isolation of Mycobacterium bovis and other mycobacterial species from ferrets and stoats

As part of wildlife surveillance for bovine tuberculosis, pooled lymph nodes from 21,481 ferrets, 1056 stoats and 83 weasels were cultured for mycobacteria. A total of 268 isolates of Mycobacterium bovis were obtained from ferrets, 2 from stoats and none from weasels, demonstrating the presence of a wildlife reservoir of infection in ferrets. DNA typing by restriction endonuclease analysis (REA) of 48 selected isolates of M. bovis revealed 23 REA types. Twenty-one of these types had previously been isolated from cattle and farmed deer, demonstrating a complex cycle of infection involving wildlife and domestic animals. Apart from M. bovis, a further 208 mycobacterial isolates were obtained, the majority of which (178) were members of the M. avium complex. Speciation of the remaining 30 mycobacterial isolates by DNA sequencing of the 16s rRNA gene, identified half the isolates as M. triplex. Other species identified included M. fortuitum, M. florentinum, M. interjectum, M. intracellulare, M. holsaticum, and M. septicum/M. peregrinum.     

Mycobacterial diseases of deer

The most significant mycobacterial diseases of free-living, captive and farmed deer are bovine tuberculosis, caused by Mycobacterium bovis, Johne's disease (paratuberculosis), caused by Mycobacterium avium subsp paratuberculosis (basonym M. paratuberculosis), and avian tuberculosis, caused principally by M. avium subsp avium. The first case of M. bovis infection in farmed deer was identified in New Zealand in 1978. In 1983, a voluntary scheme was introduced in New Zealand to control tuberculosis in farmed deer, followed by a compulsory tuberculosis control scheme in 1990. The primary control measure is the slaughter of infected animals, detected by skin testing and blood testing, together with movement control and vector control. The number of infected deer herds peaked in the mid 1990s at over 160 herds, but by 30 June 2002 this had been reduced to 79 (1.45%), and to 67 (1.23%) by June 2003. Deer-to-deer transmission occurs, but the majority of herd breakdowns are believed to be from infected vectors. Factors likely to affect the susceptibility of deer include age, environment, population density, exposure and genetics. Avian tuberculosis occasionally causes clinical disease in wild, captive and farmed deer in New Zealand and overseas. Mycobacterium intracellulare, and subspecies of M. avium other than M. paratuberculosis, are widespread throughout New Zealand and are thought to be largely responsible for the high level of sensitisation to avian purified protein derivative (PPD), which is used for comparison purposes in tuberculosis skin testing of deer in this country. Infections with these organisms are usually subclinical in farmed deer, although M. avium subsp avium commonly causes lesions in retropharyngeal, mesenteric and ileocaecal lymph nodes. These lesions cause problems because of their gross and microscopic similarity to those due to M. bovis infection. Birds and domestic animals are most likely to become infected via environmental contamination of food, water, bedding litter or soil, while carnivores or scavengers may also become infected by ingesting infected carcasses. Johne's disease has been reported in deer in the wild and in zoos, especially in North America, the United Kingdom (UK) and Europe. Since first being confirmed in farmed deer in New Zealand in 1979, the incidence of Johne's disease has increased steadily. To date, M. paratuberculosis has been identified in >600 farmed deer on 300 properties. The majority of cases have been identified from suspected tuberculous lesions submitted from deer slaughter plants. Clinically, Johne's disease in deer is similar to the disease in sheep and cattle, with typical signs of loss of weight and condition, and diarrhoea. However, outbreaks of Johne's disease frequently occur in young red deer, 8-15 months of age, whereas the clinical disease in sheep and cattle is sporadic and usually affects adults 3-5 years of age. The disease is characterised by a chronic granulomatous enteritis and lymphadenitis, especially affecting the jejunum and ileum and the mesenteric lymph nodes. Deer affected subclinically may have lesions in these lymph nodes at slaughter, which are grossly indistinguishable from those due to bovine tuberculosis. Because of the antigenic similarity between M. intracellulare and all the subspecies of M. avium, including M. paratuberculosis, the diagnostic tests for Johne's disease lack sensitivity and specificity, making control difficult.     

A study of bovine tuberculosis in domestic animals and wildlife in the MacKenzie Basin and surrounding areas using DNA fingerprinting

The MacKenzie Basin, an area of about 5150 km2 in the South Island of New Zealand, was free of bovine tuberculosis prior to 1980. During the next 13 years, the majority of the cattle and deer herds in this area became infected with Mycobacterium bovis. The history of infection in the MacKenzie Basin has all the characteristics of a newly developed region of endemic tuberculosis with a wildlife reservoir of M. bovis. Tuberculous possums and ferrets were found in the MacKenzie Basin and both may have been a source of infection for domestic animals. DNA fingerprinting of 125 isolates of M. bovis from domestic animals and wildlife by restriction endonuclease analysis revealed two major groups of isolates. The same groups were identified using IS6110 as a DNA probe. Restriction endonuclease analysis enabled one group to be subdivided into seven restriction types and the other group into eight types. Mycobacterium bovis isolates with the most common restriction types were present in both domestic animals and wildlife, indicating that infection had spread between these two groups of animals. DNA fingerprinting also revealed that M. bovis was introduced into the MacKenzie Basin from at least two distinct sources. Furthermore, DNA finger-printing was able to identify probable sources of infection.     

New generation vaccines and delivery systems for control of bovine tuberculosis in cattle and wildlife

Advances in the understanding of protective immune responses to tuberculosis are providing opportunities for the rational development of improved vaccines for bovine tuberculosis. Protection requires activation of macrophages through stimulation of a Th 1 type immune response. Ideally, a vaccine for cattle should induce protection without causing animals to react in a tuberculin test when exposed to Mycobacterium bovis. A number of new tuberculosis vaccines including attenuated M. bovis strains, killed mycobacteria, protein and DNA vaccines have been developed and many of these are being assessed in cattle. The requirements for a tuberculosis vaccine for wildlife differ from those for cattle. The major goal of a wildlife vaccine is to prevent the transmission of M. bovis to cattle and other wildlife. Although there are a number of technical problems associated with the development of a vaccine delivery system for wildlife, attenuated M. bovis vaccines administered via oral baits or aerosol spray to possums have already been shown to reduce the severity of a subsequent M. bovis infection.     

Animal-side serologic assay for rapid detection of Mycobacterium bovis infection in multiple species of free-ranging wildlife

Numerous species of mammals are susceptible to Mycobacterium bovis, the causative agent of bovine tuberculosis (TB). Several wildlife hosts have emerged as reservoirs of M. bovis infection for domestic livestock in different countries. In the present study, blood samples were collected from Eurasian badgers (n=1532), white-tailed deer (n=463), brushtail possums (n=129), and wild boar (n=177) for evaluation of antibody responses to M. bovis infection by a lateral-flow rapid test (RT) and multiantigen print immunoassay (MAPIA). Magnitude of the antibody responses and antigen recognition patterns varied among the animals as determined by MAPIA; however, MPB83 was the most commonly recognized antigen for each host studied. Other seroreactive antigens included ESAT-6, CFP10, and MPB70. The agreement of the RT with culture results varied from 74% for possums to 81% for badgers to 90% for wild boar to 97% for white-tailed deer. Small numbers of wild boar and deer exposed to M. avium infection or paratuberculosis, respectively, did not cross-react in the RT, supporting the high specificity of the assay. In deer, whole blood samples reacted similarly to corresponding serum specimens (97% concordance), demonstrating the potential for field application. As previously demonstrated for badgers and deer, antibody responses to M. bovis infection in wild boar were positively associated with advanced disease. Together, these findings suggest that a rapid TB assay such as the RT may provide a useful screening tool for certain wildlife species that may be implicated in the maintenance and transmission of M. bovis infection to domestic livestock.     

The control of bovine tuberculosis in New Zealand

Mycobacterium bovis was probably introduced into New Zealand with cattle imported in the early 19th century. A tuberculosis control programme was introduced for cattle in 1945. However, the control of tuberculosis in cattle and deer in New Zealand over the past two decades has been hampered by the presence of an important wildlife reservoir, the Australian brushtail possum (Trichosurus vulpecula). While the importance of this source of infection has been suspected by the Ministry of Agriculture for some time, scientific proof has been lacking until recently. A new control programme is currently being finalized with the following objectives: to reduce the prevalence of herd infection in vector free areas to internationally accepted levels, to prevent the establishment of tuberculous vectors in new areas, to decrease the number and size of existing areas where tuberculous vectors exist, and to encourage land-owners to take action against tuberculosis on their properties and in their herds.     

Mycobacterium bovis infections in wild ferrets

Extract

Bovine tuberculosis is one of the more important animal health problems in New Zealand. In contrast to a number of other countries, the classical test and slaughter methods in New Zealand have not led to the eradication of bovine tuberculosis. The failure to eradicate bovine tuberculosis is due to the continual spread of Mycobacterium bovis from wildlife to cattle and farmed deer. Although the Australian brushtail possum Trichosurus vulpecula is the most important wildlife reservoir of infection in New Zealand, M. bovis has also been isolated from wild deer, feral pigs, feral goats, feral cats and feral cattle. In this letter we wish to report the finding of M. bovis-infected wild ferrets (Mustela putorius furo) in seven geographically distinct areas of New Zealand (Figure 1). While there are reports from overseas of M. bovis being isolated from domesticated ferrets, there are no reports of its isolation from wild ferrets or polecats (Mustela putorius)⁽¹⁾.     

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.     

Progress in the control of bovine tuberculosis in Spanish wildlife

Despite the compulsory test and slaughter campaigns in cattle, bovine tuberculosis (bTB) is still present in Spain, and the role of wildlife reservoirs is increasingly recognized. We provide an update on recent progress made in bTB control in Spanish wildlife, including aspects of epidemiology, surveillance, host-pathogen interaction and wildlife vaccination. At the high densities and in the particular circumstances of Mediterranean environments, wild ungulates, mainly Eurasian wild boar and red deer, are able to maintain Mycobacterium bovis circulation even in absence of domestic livestock. Infection is widespread among wild ungulates in the south of the country, local infection prevalence being as high as 52% in wild boar and 27% in red deer. Risk factors identified include host genetic susceptibility, abundance, spatial aggregation at feeders and waterholes, scavenging, and social behaviour. An increasing trend of bTB compatible lesions was reported among wild boar and red deer inspected between 1992 and 2004 in Southwestern Spain. Sporadic cases of badger TB have been detected, further complicating the picture. Gene expression profiles were characterized in European wild boar and Iberian red deer naturally infected with M. bovis. The comparative analysis of gene expression profiles in wildlife hosts in response to infection advanced our understanding of the molecular mechanisms of infection and pathogenesis, revealed common and distinctive host responses to infection and identified candidate genes associated with resistance to bTB and for the characterization of host response to infection and vaccination. Ongoing research is producing valuable knowledge on vaccine delivery, safety and efficacy issues. Baits for the oral delivery of BCG vaccine preparations to wild boar piglets were developed and evaluated. The use of selective feeders during the summer was found to be a potentially reliable bait-deployment strategy. Safety experiments yielded no isolation of M. bovis BCG from faeces, internal organs at necropsy and the environment, even after oral delivery of very high doses. Finally, preliminary vaccination and challenge experiments suggested that a single oral BCG vaccination may protect wild boar from infection by a virulent M. bovis field strain.     

Investigation of the transmission of Mycobacterium bovis from deer to cattle through indirect contact

OBJECTIVE: To investigate the infection of calves with Mycobacterium bovis through oral exposure and transmission of M. bovis from experimentally infected white-tailed deer to uninfected cattle through indirect contact. ANIMALS: 24 11-month-old, white-tailed deer and 28 6-month-old, crossbred calves. PROCEDURE: In the oral exposure experiment, doses of 4.3 x 10(6) CFUs (high dose) or 5 x 10(3) CFUs (low dose) of M. bovis were each administered orally to 4 calves; as positive controls, 2 calves received M. bovis (1.7 x 10(5) CFUs) via tonsillar instillation. Calves were euthanatized and examined 133 days after exposure. Deer-to-cattle transmission was assessed in 2 phases (involving 9 uninfected calves and 12 deer each); deer were inoculated with 4 x 10(5) CFUs (phase I) or 7 x 10(5) CFUs (phase II) of M. Bovis. Calves and deer exchanged pens (phase I; 90 days' duration) or calves received uneaten feed from deer pens (phase II; 140 days' duration) daily. At completion, animals were euthanatized and tissues were collected for bacteriologic culture and histologic examination. RESULTS: In the low- and high-dose groups, 3 of 4 calves and 1 of 4 calves developed tuberculosis, respectively. In phases I and II, 9 of 9 calves and 4 of 9 calves developed tuberculosis, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that experimentally infected deer can transmit M. bovis to cattle through sharing of feed. In areas where tuberculosis is endemic in free-ranging white-tailed deer, management practices to prevent access of wildlife to feed intended for livestock should be implemented.     

MHC class II-restricted, CD4(+) T-cell proliferative responses of peripheral blood mononuclear cells from Mycobacterium bovis-infected white-tailed deer

White-tailed deer are significant wildlife reservoirs of Mycobacterium bovis for cattle, predators, and, potentially, humans. Infection of cattle with M. bovis stimulates an antigen-specific T-cell response, with both CD4(+) and CD8(+) cells implicated in protective immunity. Few studies, however, have examined lymphocyte subset responses to experimental M. bovis infection of white-tailed deer. In this study, a flow cytometric proliferation assay was used to determine the relative contribution of individual peripheral blood mononuclear cell subsets of M. bovis-infected white-tailed deer in the recall response to M. bovis antigen. Naive deer were challenged with M. bovis by cohabitation with infected deer. These M. bovis-challenged deer developed significant in vivo (delayed-type hypersensitivity) and in vitro (proliferative) responses to M. bovis purified protein derivative (PPD). At necropsy, typical tuberculous lesions containing M. bovis were detected within lungs and lung-associated lymph nodes of infected deer. The predominant subset of lymphocytes that proliferated in response to in vitro stimulation with PPD was the CD4(+) subset. Minimal proliferative responses were detected from CD8(+), gamma delta TCR(+), and B-cells. Addition of monoclonal antibodies specific for MHC II antigens, but not MHC I or CD1 antigens, abrogated the proliferative response. Together, these findings indicate that while CD4(+) cells from infected deer proliferate in the recall response to M. bovis antigens, this response is not sufficient to clear M. bovis and immunologic intervention may require stimulation of alternate subsets of lymphocytes.     

Patterns of antimicrobial susceptibility in Michigan wildlife and bovine isolates of Mycobacterium bovis.

The state of Michigan has recognized the presence of Mycobacterium bovis in its free-ranging white-tailed deer population since 1994. This endemic infection is primarily located in a 12-county area in the northeastern lower peninsula of Michigan. A statewide surveillance and eradication program of the disease has been in effect since 1994. Worldwide, Mycobacterium tuberculosis complex organisms have a known predilection toward development of antimicrobial resistance. The objective of this study was to investigate the antimicrobial susceptibility of M. bovis isolates from white-tailed deer in Michigan and detect any changes in susceptibility over time. M. bovis isolates from 2 fall hunting seasons (1999 and 2004) were used in this study. The fall season of 2004 marked the first documented case of direct transmission of M. bovis from a wild deer to a human in Michigan. Since M. bovis is a zoonotic disease, knowledge of susceptibility can expedite treatment options in humans. M. bovis isolates were obtained from 58 deer, 4 coyotes, 3 cattle, 2 raccoons, and 1 human case from the 2 years combined. Methods of susceptibility testing included 1% proportion agar plates and Bactec radiometric broth testing. M. bovis was found to be uniformly resistant to the antibiotic pyrazinamide; this resistance is common to all M. bovis isolates. No other antimicrobial resistance was found in any of the tested M. bovis isolates, which may be, in part, attributed to the lack of any significant treatment pressure in wildlife.     

An outbreak of Mycobacterium bovis infection in fallow deer (Dama dama)

In an outbreak of Mycobacterium bovis infection in fallow deer in South Australia, 3 herds related by recent movement of deer were infected. From these 3 infected herds, 47 of 51 animals were tuberculosis at necropsy. A range of lesions was seen most of which differed from classical bovine tuberculosis in that pus was a white liquid, fibrous encapsulation was not marked and calcification was rare. Histopathology was of classical tuberculosis. M. bovis was cultured from lesions and M. avium-intracellulare was cultured from one deer with no visible lesions. The source of M. bovis infection has not been determined.     

Histological observations of bovine tuberculosis in lung and lymph node tissues from British deer

Deer are recognized as hosts of Mycobacterium bovis and assessing the role of wild cervids in perpetuating tuberculosis among cattle has motivated extensive research on several continents. In this paper, the histopathology of lymph node and lung tuberculous granulomas in M. bovis positive British deer is presented. The overall aim was to seek further insights into the potential for onward transmission from infected deer to other species, including cattle. Samples were obtained from an extensive survey of wild mammals in South-West England and from statutory tuberculosis surveillance. M. bovis culture-positive samples were characterised microscopically as to their stage of lesion advancement, number of acid-fast bacilli and granuloma encapsulation. Seventy percent of the deer developed granulomas containing far greater numbers of M. bovis bacilli than typically reported in cattle. Red and fallow deer had the largest number of poorly encapsulated granulomas often containing many hundreds of bacilli. The results are consistent with infected wild British deer being a potential source of environmental contamination and onward transmission to other species. However, further work on levels of bacillary shedding is required before this can be confirmed.     

Epidemiology of Mycobacterium bovis infections of pigs and wild boars using a molecular approach

A molecular epidemiological approach was applied to establishing a possible role for the wild boar as a natural reservoir of Mycobacterium bovis in Sierra de Villuercas, Western Spain; an area free of farmed cattle and wild deer populations. Spoligo and VNTR typing were used over a three year period to study the epidemiological relationship between the occurrence of bovine tuberculosis (TB) in extensively bred Iberian pigs and indigenous wild boar. The 37 sampled wild boar showed different degree of calcified granulomatous lesions in retropharyngeal, mediastinal and pulmonary lymph nodes. The 25 sampled Iberian pigs showed calcified lesions, mainly in the respiratory tract. Lesions located in the mesenteric lymph nodes appeared secondarily. M. bovis was isolated from all affected animals. Twenty-five and 37 isolates of M. bovis were obtained from domestic pigs and wild boar, respectively. Our findings provide evidence that supports the possibility of cross infection between wild boar and domestic pig populations. This is contrary to the generally held belief that swine represent an epidemiological dead end host and play no role in the epidemiology of M. bovis.     

Implications and challenges of tuberculosis in wildlife ungulates in Portugal: a molecular epidemiology perspective

Mycobacterium bovis and, more rarely, Mycobacterium caprae, may cause zoonotic bovine tuberculosis (bTB) in an extensive range of animal species. In Portugal, during 2009, a remarkable raise of bTB incidence was registered in cattle along with an increase of new cases in wildlife. In this work, we reassess and update the molecular epidemiology of bTB in wild ungulates by including 83 new M. bovis and M. caprae isolates from wild boar and red deer obtained during 2008-2009. Spoligotyping identified 27 patterns in wild ungulates, including 11 patterns exclusive from deer and five from wild boar. The genetic relatedness of wildlife and livestock isolates is confirmed. However, the relative prevalence of the predominant genotypes is different between the two groups. Contrasting with the disease in livestock, which is widespread in the territory, the isolation of bTB in wildlife is, apparently, geographically localized and genotypic similarities of strains are observed at the Iberian level.     

Advances in understanding disease epidemiology and implications for control and eradication of tuberculosis in livestock: the experience from New Zealand

A deteriorating tuberculosis problem in cattle and deer in New Zealand has been halted and then reversed over the last decade. Mycobacterium bovis infection in both wild and domestic animal populations has been controlled. This has been achieved by applying a multi-faceted science-based programme. Key features of this have been a comprehensive understanding of the epidemiology of tuberculosis in animals, confidence in sampling wild animal populations, effective application of diagnostic tests in cattle and deer, and the ability to map M. bovis genotypes.     

Evaluation of historical factors influencing the occurrence and distribution of Mycobacterium bovis infection among wildlife in Michigan

OBJECTIVES: To determine historical events leading to establishment of bovine tuberculosis in the white-tailed deer population in the northeastern corner of the lower peninsula (NELP) of Michigan and describe factors relevant to the present outbreak of bovine tuberculosis in Michigan. SAMPLE POPULATION: Cattle and white-tailed deer in Michigan from 1920 to 1990. PROCEDURES: A search of extant historical documents (eg, scientific journals, books, public reports, and correspondence and internal reports from governmental agencies) was conducted. Factors investigated included the number of cattle and prevalence of tuberculosis, deer population and density levels, and changes in regional environments affecting the population and management of cattle and wild deer. RESULTS: High deer numbers and severe winter feed shortages resulting from habitat destruction in the NELP in 1930 contributed to the transmission of tuberculosis from cattle to deer. Starvation increased the susceptibility of deer to infection and modified behavior such that exposure to infected cattle was increased. Relocation of deer resulted in spread of infection to other sites, including locations at which spatial clusters of tuberculosis presently exist. Ribotyping of Mycobacterium bovis from a human patient suggests that the strain of M. bovis presently infecting white-tailed deer in the region is the same strain that affected cattle farms at that time. CONCLUSIONS AND CLINICAL RELEVANCE: Feeding deer to maintain numbers above the normal carrying capacity of the NELP led to deer depending on consumption of livestock feed for survival during winter and increased contact with domestic cattle. This practice should be avoided.     

Cattle-to-cattle transmission of bovine tuberculosis

In developed countries, Mycobacterium bovis infection in cattle is now mostly confined to the respiratory system, which reflects transmission and establishment of infection mainly by this route. A single bacillus transported within a droplet nucleus is probably sufficient to establish infection within the bovine lung. Infected cattle should always be considered as potential sources of infection, since studies have demonstrated that a significant proportion of tuberculous cattle excrete M. bovis.In general, the dynamics of M. bovis transmission are poorly understood and the conditions under which a tuberculous animal becomes an effective disseminator of infection are currently not defined although environmental contamination appears to be a less effective method of disease transmission. Field studies indicate a wide spectrum of transmission rates but generally the spread of M. bovis infection is still considered to be a relatively slow process. Slaughter of diseased cattle detected by tuberculin testing and at meat plant inspection has been shown to be an effective policy for tuberculosis eradication, provided there are no other reservoirs of infection and all involved in the cattle industry are committed to a policy of eradication. Epidemiological approaches, particularly case-control studies, seem to provide the best method for quantifying the relative importance of the various sources of M. bovis transmission to cattle and modelling techniques can be used to assist in the design of cost-effective control measures that may lead to tuberculosis eradication.