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.     

The experience of new European Union Member States concerning the control of bovine tuberculosis

On 1 May 2004, 10 new States joined the European Union, including Cyprus (CY), the Czech Republic (CR), Estonia (ES), Hungary (HU), Latvia (LA), Lithuania (LI), Malta (MA), Poland (PO), Slovakia (SK), and Slovenia (SN). Using OIE and published data, this paper summarises the status of bovine and human tuberculosis in animals in these countries between 1996 and 2003. National control programmes against bovine tuberculosis in cattle have been successful: the current herd incidence of this disease in cattle is currently lower than 0.2%, so all countries meet the OIE requirements for freedom from the disease. Furthermore, two countries have already been officially declared bovine tuberculosis-free EU States: the CR on 31 March 2004 (European Commission Decision No. 2004/320/EC) and SK on 4 March 2005 (Commission Decision No. 2005/179/EC). The last outbreak of bovine tuberculosis was diagnosed in cattle in CY (1928), ES (1986), LA (1989), SK (1993), CR (1995), and MA (2001). However, several issues of concern remain including the potential existence of a wildlife reservoir, the presence of Mycobacterium bovis, M. caprae, and other members of the M. tuberculosis complex (particularly M. tuberculosis or M. microti) in imported domestic or wild animals, and the potential for delayed detection of bovine tuberculosis in those States where annual tuberculin testing is no longer performed on cattle older than 24 months.     

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.     

Tuberculosis in cattle: strategic planning for the future

In the later stages of eradication of tuberculosis in cattle there is a need to take account of the fact that Mycobacterium bovis infection in cattle presents, not as cases of clinical disease but most commonly as apparently healthy animals showing an immunological response to tuberculin. This is an entirely different scenario to that seen when national eradication programmes were first devised, at a time when the protection of public health rather than animal health was the prime motivation. In countries with active programmes to eradicate bovine tuberculosis, it is critical for the programme's success that account is taken of this redefinition of tuberculosis, side by side with changes in modern animal production systems and their impact on the transmission of M. bovis. This paper highlights factors critical to the success of a national eradication programme, including a clear identification of the goals, of the policies that guide actions, and of the sequences of actions that are required within the programme to accomplish these goals. Experience has illustrated the adverse effects of compromise on outcome when the application of fundamental principles of disease control such as sound animal management, removal of known sources of infection, early diagnosis, quarantine, movement control and environmental hygiene are less than enthusiastically promoted and applied. The reality is that where these principles are applied in a sustained manner, the outcome is more likely to be successful. Therein lies the challenge for the risk manager.     

Immunological approaches to the control of tuberculosis in wildlife reservoirs

Attempts to eradicate tuberculosis from cattle and farmed deer in some countries have been frustrated by the existence of wildlife reservoirs of Mycobacterium bovis infection. Possum control programmes in New Zealand using poisons have shown clearly that the brushtail possum is an important source of infection for cattle and farmed deer, and the sum of evidence strongly suggests that badgers serve as a source of infection for cattle in the UK. Bovine tuberculosis can only be eradicated from these countries by controlling M. bovis infection in both wildlife and domestic animals. The most promising options for control of M. bovis infection in wildlife in the longer term include the development of a tuberculosis vaccine for wildlife and a strategy for biological control of possums. The aim of this review is to address the problems and approaches involved in the control of wildlife tuberculosis from an immunological perspective.     

Pathogenesis of bovine tuberculosis: the role of experimental models of infection

In many countries, test-and-slaughter policies based on tuberculin skin testing have made a significant impact on the control of bovine tuberculosis (caused by infection with Mycobacterium bovis). However, in some countries these policies have not proved as effective and improved disease control strategies are required (including improved diagnostic tests and development of vaccines). The host pathogen interactions in bovine tuberculosis are very complex. While studies of the disease in naturally infected field cases of bovine tuberculosis have provided valuable information, detailed knowledge can also be gained through studies of disease models. A number of studies have developed M. bovis infection models employing a range of routes and challenge doses. An early objective was assessment of vaccine efficiency, and models of infection remain central to current work in this area. Development of the intra-nasal and intra-tracheal models have also advanced our understanding of the kinetics of the immune response. In many of these studies, understanding of pathogenesis has been improved by definition of the cells that respond to infection and those that are instrumental in modulation of host responses. Experimental models of infection have been adapted to study cattle to cattle transmission, modeling one of the fundamental routes of infection. This review provides a historical perspective on the types of experimental models used in over 100 years of research and outlines new opportunities to refine those methods for bovine and human tuberculosis and to contribute to improved diagnostics, advanced understanding of immunology and vaccine design.     

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.     

Effects of sustained control of brushtail possums on levels of Mycobacterium bovis infection in cattle and brushtail possum populations from Hohotaka, New Zealand

AIMS: To examine the effect of reducing the abundance of brushtail possums (Trichosurus vulpecula) on the distribution and prevalence of bovine tuberculosis (Mycobacterium bovis, Tb) in possums and the incidence of Tb in domestic cattle on a group of farms in the central North Island, New Zealand. METHODS: The cumulative yearly incidence of Tb infection from 12 cattle herds was estimated from annual tuberculin testing and abattoir inspection data over the period 1983-98. Intensive control of possum populations began for six of the herds in 1988, five herds in 1994 and the remaining herd in 1996. The prevalence and distribution of macroscopic M. bovis infection in possums and an index of possum abundance was estimated during yearly cross-sectional surveys from 1988 to 1998. This enabled formal testing of the link between the abundance of tuberculous possums and the incidence of Tb in cattle. RESULTS: Before possum control, infected possums were clustered in foci on or adjacent to the farms with the highest annual incidence of tuberculosis in cattle, and had an overall prevalence of macroscopic M. bovis infection of 2.3%. The prevalence of disease declined to zero with ongoing possum control, although infected possums continued to be found during the first 5 years of control. Maintaining the possum population at an average of 22.1% of its pre-control density significantly reduced the odds of the cumulative yearly incidence of Tb in cattle by 77% during the first 5 years of possum control and a further 65% in the second 5-year period. Nine of 11 tuberculous possums identified since the start of possum control were found within the areas where infected possums were clustered during the pre-control survey, suggesting that the persistence of infection within these clusters rather than infected immigrants was the source of ongoing disease. Annual estimates of the prevalence of tuberculous possums broadly followed the predictions of Barlow's possum-Tb model for a controlled possum population. CONCLUSION: The results support the hypothesis that tuberculous possums transmit bovine tuberculosis to domestic cattle, and therefore that reducing the abundance of tuberculous possums reduces the incidence of Tb in cattle. If the level of possum culling is sufficient, it appears that M. bovis infection may be eradicated from possum populations. Better information on population density, rate of increase and annual culling rates would have been needed for a truly independent examination of the Barlow possum-Tb model.     

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.     

Pathologic findings and association of Mycobacterium bovis infection with the bovine NRAMP1 gene in cattle from herds with naturally occurring tuberculosis

OBJECTIVE: To determine necropsy and Mycobacterium bovis culture results in cattle from herds with tuberculosis, the role of the bovine NRAMP1 gene in resistance and susceptibility to infection with M bovis, and the association between magnitude of the tuberculous lesions and various types of M bovis isolates. ANIMALS: 61 cattle from herds with tuberculosis in Texas and Mexico. PROCEDURE: 61 cattle were evaluated by necropsy; 59 had positive and 2 had negative caudal fold tuberculin intradermal test (CFT) results. Thirty-three cattle with positive CFT results were genotyped to evaluate polymorphism of the 3' untranslated region of the bovine NRAMP1 gene, using single-stranded conformational analysis, 9 were resistant to M bovis with no tuberculous lesions and negative M bovis culture results, and 24 were susceptible with tuberculous lesions and positive M bovis culture results. Isolates of M bovis were analyzed by restriction fragment length polymorphism (RFLP) on the basis of IS6110 sequences and direct-repeat fingerprinting patterns. RESULTS: 21 (35.6%; 21/59) cattle with positive CFT results had tuberculous lesions or positive culture results; in addition, 1 of 2 cattle with negative CFT results had tuberculous lesions and positive culture results. Tuberculous lesions were most common in the thorax (35/63; 55.5%) and lymphoid tissues of the head (10/63; 15.9%). Tuberculous lesions varied from 1 to 11/animal; 8 of 21 (38.1%) had solitary lesions. Associations were not found between resistance or susceptibility to infection with M bovis and polymorphism in the NRAMP1 gene or between the magnitude of the lesions and various RFLP types of M bovis isolates. CONCLUSIONS AND CLINICAL RELEVANCE: The NRAMP1 gene does not determine resistance and susceptibility to infection with M bovis in cattle.     

The Epidemiology of Mycobacterium bovis in Buffalo in Iran

Mycobacterium bovis is the cause of bovine tuberculosis (bovine Tb) in animals and is considered to be zoonotic and accordingly it infects humans, although cattle are the main host. Buffalo can also be infected and develop bovine Tb. In Iran, almost half a million buffaloes are farmed, mainly in three provinces. In West Azerbaijan, which has the largest numbers of buffaloes, cattle and buffalo are often farmed together. According to the reports of the Iranian Veterinary Organization over the last 25 years, there have been no reports of bovine Tb in buffalo, although the disease is often reported in cattle in this province. Eighteen and 140 pathology specimens from cattle and buffalo, respectively, collected from West Azerbaijani abattoirs were cultured. From one buffalo specimen out of 140, M. bovis was recovered, whereas the pathogen was isolated from 13 cattle specimens. Spoligotyping showed a relatively higher polymorphism within these isolates compared with M. bovis isolated from other Iranian provinces.     

Effect of paratuberculosis on the diagnosis of bovine tuberculosis in a cattle herd with a mixed infection using interferon-gamma detection assay

Interferon-gamma (IFN-gamma) detection assay is being applied as an ancillary test to tuberculin tests in the diagnosis of bovine tuberculosis to detect the maximum number of infected animals. Among possible factors influencing the performance of tuberculosis-diagnostic tests, paratuberculosis, a widespread disease in Spain and other European countries, has been pointed out as a cause of false positive reactions. Still, its effect on the sensitivity of these tests in cattle has yet to be fully characterized. The impact of paratuberculosis in the apparent sensitivity of IFN-gamma assay was studied in a bullfighting cattle herd with a mixed tuberculosis-paratuberculosis infection, using culture of Mycobacterium bovis and Mycobacterium avium paratuberculosis as the gold standard to determine the infection status of every animal. A total of 218 animals were slaughtered and sampled for bacteriology after blood sampling. IFN-gamma assay showed a lower apparent sensitivity in animals with a mixed infection (50%) compared to all animals suffering tuberculosis (78.3%). This finding indicates that the presence of paratuberculosis in tuberculosis-infected herds could imply a serious impairment in the sensitivity of IFN-gamma detection test.     

Immune responses in bovine tuberculosis: towards new strategies for the diagnosis and control of disease

In several countries, bovine tuberculosis (caused by infection with Mycobacterium bovis) is a major economic problem with the potential to be a significant public health risk. Where traditional test-and-slaughter policies based on skin testing with tuberculin have not been fully successful, new tools including additional diagnostic tests and improved vaccines are required urgently. This paper considers how recent developments in knowledge of immune responses and mycobacterial antigens can be used in the logical development of more efficient strategies for the identification of infected cattle.     

Mycobacterium bovis infection and tuberculosis in cattle

This review considers the possible events that can occur when cattle are exposed to Mycobacterium bovis and, where appropriate, draws on principles accepted for tuberculosis infection in humans and laboratory animal models. Consideration is given to the many complex factors which influence the outcome of challenge with tubercle bacilli. These include features inherent to the mycobacterium, the host and the environment. It is apparent that clinical disease probably occurs only in a relatively small, but undetermined, proportion of cattle that are exposed to Al. bovis. The majority of animals may clear infection or control the bacilli, possibly in a condition of latency. It is concluded that a better understanding of the dynamics of the events following M. bovis exposure and subsequent infection in cattle would be of significant benefit in developing new tools appropriate for disease control and to designing optimal approaches for their application.     

The epizootiological significance of positive bacteriological findings on Mycobacterium tuberculosis and Mycobacterium bovis in humans

The relation between the active form of tuberculosis in persons working in agriculture and incidence of tuberculosis in cattle was analyzed in 1974 to 1978, i.e. in the period after the elimination of bovine tuberculosis in Czechoslovakia (in 1968). M. tuberculosis was isolated in 15 cases and M. bovis in four cases of persons employed by the farms on which the Regional Hygienic Station, Brno, was responsible for the microbiological diagnostics of tuberculosis. Direct contact with animals was demonstrated in eight patients; M. tuberculosis was isolated from seven of these patients and M. bovis from one. Seven cattle herds were exposed to spontaneous infection by M. tuberculosis and in one of them tuberculosis was not demonstrated during complex examination. In three herds the examination revealed only a sensitivity of cattle to mammalian tuberculin. In other three herds tuberculosis was detected by allergic tests, patho-anatomic examination and bacteriological examination. M. tuberculosis in cattle was detected in two herds. The occurrence of bovine tuberculosis caused by a cattle tender with a positive finding of M. bovis in sputum was demonstrated in one herd. Virulence for the tested cattle was found in one strain (isolated from a mesenterial lymph node of cattle) of the four strains of M. tuberculosis used for the experimental infection of 17 animals. On the other hand, in three strains of M. tuberculosis, trials with experimental infection demonstrated only allergy to mammalian tuberculin and changes at the sites of subcutaneous inoculation of mycobacteria of regressive nature; these mostly disappeared within 90 days from infection.     

Value of existing serological tests for identifying badgers that shed Mycobacterium bovis

In the UK there has been a sharp rise in the incidence of bovine tuberculosis since the early 1990s and the badger has been identified as an important wildlife reservoir for this infection. Infected badgers can excrete Mycobacterium bovis, putting other badgers and cattle at risk of becoming infected. Vaccination has been proposed as an approach to reducing the excretion of M. bovis by tuberculous badgers. In order to evaluate the efficacy of a badger vaccine it will be necessary to accurately determine the number of badgers excreting M. bovis without removing them for post-mortem evaluation. The existing live tests for tuberculosis in the badger (culture, indirect ELISA, Western blot) have not been assessed for their ability to detect badgers excreting M. bovis. Over the past 18 years, badgers from 31 social groups have been trapped and sampled in a study area of the Cotswold escarpment. We have examined the serological responses of 128 badgers trapped between 1985 and 1998 from social groups where M. bovis infection was endemic. These responses were compared with culture from faeces, urine, tracheal aspirates and bite wound swabs taken from these animals while alive. ELISA was found to be more sensitive than Western blot in detecting badgers excreting M. bovis. The majority of culture-positive badgers excreted M. bovis intermittently over the period of study. As a result, there was only a 27.5% chance of sampling a badger for culture when it was excreting M. bovis. In contrast, a positive ELISA result correctly predicted 68.2% of badgers with a history of excreting M. bovis. In the absence of alternative live tests for the badger, the Brock Test indirect ELISA appears to be more valuable than culture for measuring the effect of vaccination on reducing the number of badgers at risk of transmitting tuberculosis.     

Prevalence of bovine tuberculosis in slaughter cattle in Kenya: a postmortem, microbiological and DNA molecular study

A study to determine the presence and prevalence of bovine tuberculosis in slaughter cattle in Kenya was carried out in two abattoirs from July to November 2009. Routine postmortem meat inspection was performed on a subpopulation of 929 cattle selected randomly from among 4,984. Carcases were inspected for gross tuberculous lesions which were then examined for acid-fast bacilli, (AFB), cultured for isolation of mycobacteria and the isolates characterised by DNA molecular analysis. Of the carcases examined, 176 (18.95?%, 95?% CI) had lesions suggestive of tuberculosis. AFB were observed in 63/176 of the lesioned cattle and mycobacteria were isolated from 64 of them. The isolates were identified as Mycobacterium bovis (19/64), Mycobacterium tuberculosis, (2/64) and mycobacteria other than tuberculosis (43/64). The prevalence of M. bovis by molecular analysis was 2.05?% (95?% CI). This study documents for the first time the presence of bovine tuberculosis among slaughter cattle in Kenya. There is therefore a need to formulate and implement control programmes in order to minimise transmission among animals and to humans. Isolation of M. tuberculosis from cattle underscores the risk tuberculous humans pose to animals.     

结核分枝杆菌疫源追踪的基因检测分析

目的探讨牛源性人结核疫源追踪的基因分析方法。方法采用复方PCR结核杆菌快速鉴定技术,针对特征性分枝杆菌标志基因(MTP40基因、α抗原基因和IS6110),对结核病人和患牛的标本,进行检测。结果患者标本结核分枝杆菌阳性;患牛标本牛分枝杆菌和结核分枝杆菌分别阳性。结论结核病奶牛不仅是人类牛型结核的可能来源,也可能成为人类人型结核的潜在来源。     

Evaluation of Spoligotyping and MIRU-VNTR for Mycobacterium bovis in Xinjiang, China

Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB) in cattle, is also a pathogen for human and other mammals. In this study, 406 cows were screened for bTB by both single intradermal comparative cervical tuberculin (SICCT) test and IFN-γ assay. 135 M. bovis were isolated from 31 SICCT and IFN-γ double-positive cows in Xinjiang Uygur Autonomous Region of China. Spoligotyping and MIRU-VNTR were evaluated for genotyping, and 4 and 7 genotypes were identified, respectively. A new combination of nine MIRU-VNTR loci was most discriminative for M. bovis clones from Xinjiang. Interestingly, two new spoligotypes (SB1903 and SB1904) and special repeat numbers of three loci (ETR-D, QUB 1895 and QUB 3336) were discovered in this study. These results indicated a specific epidemic conservation in Xinjiang, China. M. bovis strains with the unique genotypes were isolated from the herds maintaining parent cows imported from the bTB-free countries, suggesting a possible transmission from the local breed of Xinjiang brown cattle.     

Assessment of diagnostic tools for eradication of bovine tuberculosis in cattle co-infected with Mycobacterium bovis and M. avium subsp. paratuberculosis

The intradermal tuberculin (IDTB) test and the interferon-gamma (IFN-gamma) assay are used worldwide for detection of bovine tuberculosis in cattle, but little is known about the effect of co-infecting agents on the performance of these diagnostic tests. This report describes a field trial conducted in a cattle herd with dual infection (bovine tuberculosis and paratuberculosis) during 3.5 years. It has been based on a strategic approach encompassing serial parallel testing (comparative IDTB test, the IFN-gamma assay and serology of paratuberculosis) that was repeated 8 times over the period, and segregation of animals into two herds. The IDTB test detected 65.2% and the IFN-gamma test detected 69.6% of the Mycobacterium bovis culture-positive cattle. However, the IDTB test performed better during the first part of the trial, while the IFN-gamma test was the only method that detected infected animals during the following three samplings. The number of false positive reactors with the IDTB and/or the IFN-gamma tests was remarkably high compared to other reports, and could be caused by cross-reactivity with M. avium subsp. paratuberculosis. Also, the M. bovis isolates from cattle and wildlife from the same property were characterised using molecular techniques to disclose an epidemiological link. The IDTB test may not be appropriate to eradicate bovine tuberculosis in herds with dual mycobacterial infections. This report highlights the need to use several diagnostic techniques for the accurate detection of M. bovis infected animals in these herds.