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.     

Defining output-based standards to achieve and maintain tuberculosis freedom in farmed deer, with reference to member states of the European Union

Within the European Union (EU), detailed legislation has been developed for cattle, but not deer, to minimise disease risks associated with trade in animals and animal products. This legislation is expressed as input-based standards, providing a detailed outline of the activity required (for example, testing of animals and application of defined control measures), on the expectation that an adequate output (for example, confidence in freedom) will be achieved. Input-based standards are at odds with the increasing shift towards output-based standards, particularly in OIE rules governing international trade. In this paper, we define output-based standards to achieve and maintain freedom from tuberculosis (TB) in farmed deer, with reference to EU member states. After considering the probability of freedom achieved for cattle under existing EU legislation, we defined a ‘free farmed deer holding’ as one with a probability of freedom from infection of at least 99%. We then developed an epidemiological model of TB surveillance systems for deer holdings, incorporating different surveillance strategies, including combinations of diagnostic tests, and a variety of different scenarios relating to the potential for introduction of infection. A range of surveillance strategies were identified to achieve and maintain a free farmed deer holding, and worked examples are presented. The surveillance system sensitivity for varying combinations of screening and confirmatory tests in live animals, animals at slaughter and on-farm deaths is also presented. Using a single test at a single point in time, none of the TB tests routinely used in farmed deer is able to achieve an acceptable probability of TB freedom. If repeat testing were undertaken, an acceptable probability of TB freedom could be achieved, with differing combinations of the surveillance system sensitivity, frequency of testing and risk of introduction. The probability of introduction of infection through the importation of infected deer was influenced by the use of a pre-movement test (assumed 90% test sensitivity and negative test results), the TB prevalence in the source herd and the number of animals imported. A surveillance system sensitivity of at least 81% was achieved with different combinations of annual live animal surveillance and surveillance of animals at slaughter or on-farm deaths. This methodology has broad applicability and could also be extended to other diseases in both deer and other species with relevance to trade in animals and animal products.     

Evaluation of the Abbott LCx Mycobacterium Tuberculosis Assay for Direct Detection of Mycobacterium Bovis in Bovine Tissue Samples

The commercial LCx amplification assay, usually employed to detect the Myocobacterium tuberculosis complex in respiratory specimens, was evaluated by comparing the results it gave with those obtained using Löwenstein-Jensen solid medium and pathological findings on 55 lymph nodes from cattle with positive and 10 lymph nodes from cattle with negative skin tests for tuberculosis. Fifty-three cultures (51 and 2, respectively) were positive for M. bovis, while the results for the LCx assay and the histological method were positive in 48 (45, 3) and 24 (20, 4) samples, respectively. None of the samples from cattle from certified tuberculosis-free herds were positive by any of the procedures. The results obtained with the LCx assay, compared with the culture procedure, regarded as the gold standard among the diagnostic techniques, gave a specificity of 91.6% and sensitivity of 90.5%. Although the sensitivity of LCx was suboptimal, DNA of M. bovis was detected in 81.8% of the skin test-positive animals. Amplification techniques could provide a rapid and reasonably reliable tool for detecting bovine tuberculosis.     

Molecular fingerprinting confirms extensive cow-to-cow intra-herd transmission of a single Mycobacterium bovis strain

In this study we have characterized M. bovis isolates from a herd of cattle in Uvalde, Texas in which 52 of the 193 animals selected at random in 1994 from a herd of 331 were caudal fold skin-test positive. Thirty-two of 52 skin-test positive cattle had gross lesions at slaughter, and isolations of M. bovis were made from 29 animals. The herd was comprised of Red Devon cattle purchased between 1978 and 1980 (n = 26) and breeding bulls (n = 3) introduced at later times, and all were tuberculosis test negative at the time of purchase. Other animals were natural additions (offspring) of these cattle. One additional animal, a Holstein present on the ranch at the time of purchase in 1976, was retained to nurse orphaned and weak calves. Using several molecular fingerprinting techniques we have verified a clonal relationship among the M. bovis isolates consistent with infection originating with a single strain. The molecular fingerprint patterns demonstrate the stability of the profiles despite persistence and spread of the organism within the herd for two decades and confirms their use in epidemiological tracing.     

Spoligotype diversity of Mycobacterium bovis and Mycobacterium caprae animal isolates

The genetic diversity of 283 Mycobacterium bovis (M. bovis) and 10 Mycobacterium caprae (M. caprae) strains, isolated between 2002 and 2007 from cattle, goat, red deer and wild boar from six different geographical regions of Portugal was investigated by spoligotyping. The technique showed a good discriminatory power (Hunter-Gaston Index, h=0.9) for the strains, revealing 29 different patterns. One pattern (SB0121) was clearly predominant, accounting for 26.3% of the isolates; ten patterns, representing 20.7% of the isolates, had never been reported previously. Multiple spoligotypes were detected in thirteen cattle and one goat herd, most of which were found in beef cattle and extensive management regions, suggesting different infection sources. With the exception of two spoligotypes, those in wildlife species were also found in domestic species.     

Investigations on the Prevalence of Bovine Tuberculosis and Brucellosis in Dairy Cattle in Dar es Salaam Region and in Zebu Cattle in Lugoba Area,Tanzania

A study between August 1995 and December 1997 included 343 dairy cattle on 20 farms in the Dar es Salaam region and 2289 zebu cattle on 39 bomas in the Lugoba area (coast region). The aim was to establish the prevalence of bovine tuberculosis (Mycobacterium bovis) and bovine brucellosis (Brucella abortus). In the single intradermal tuberculin test (SIT), 0.9% (3/343) of the animals in Dar es Salaam tested positive and 1.2% (4/343) were doubtful. Positive reactors were found in 10% (2/20) of the farms. In the Lugoba area, 0.6% (14/2206) were positive and 6.8% (149/2206) doubtful, positive cases being found in 21% (8/39) of all bomas. In the slow agglutination test (SAT) for B. abortus, 14.1% (48/341) of the serum samples reacted positively in Dar es Salaam and 2.3% (8/341) were doubtful. Positive SAT reactors were identified on 25% (5/20) of the dairy cattle farms. In the Lugoba area, 12.3% (273/2221) proved to be positive SAT reactors and doubtful reactions were observed in 2.9% (64/2221). SAT-positive animals were detected on 87% (34/39) of all bomas. The prevalence in single herds in Dar es Salaam varied from 4.3% to 5.3% for the SIT and from 2.2% to 50% for the SAT. The prevalence in single herds in Lugoba area was between 1.1% and 2.9% for SIT and from 1.4% up to 62.1% for SAT. The two cattle populations differed significantly (p<0.001) in the prevalence of both bovine tuberculosis and bovine brucellosis. Two cows that were positive reactors were slaughtered and subjected to post-mortem examination, and organ samples were bacteriologically cultured. The occurrence of Mycobacterium tuberculosis was confirmed by polymerase chain reaction (PCR) in both cows.     

Pathology of naturally occurring bovine tuberculosis in England and Wales

The aim of this study was to obtain a contemporary data set of pathology in tuberculin reactor and in-contact cattle in England and Wales. Four hundred animals (200 reactors and 200 in-contacts) from 242 farms located in 14 counties in Western England and Wales were examined. The mean number of lymph nodes (LNs) with tuberculosis (TB)-like lesions per TB-confirmed animal was 1.7 in reactors and 1.5 in in-contact animals. Tuberculous lesions in both reactor and in-contact animals were most commonly observed in the LNs of the thorax, followed by the head and abdomen, particularly the mediastinal, retropharyngeal and tracheobronchial LNs. Twenty-five reactors had macroscopic lesions in the palatine tonsils. Among TB-confirmed cattle, 27% of reactors and 9% of in-contact animals had gross TB-like lesions in the lungs, particularly in the caudal lobes. Gross lesions that were not TB-confirmed were parasitic granulomas (45%), bacterial or mycotic club-forming pyogranulomas (27%) and bacterial abscesses (23%). Diagnostic sensitivity was maximised when bacteriology and histopathology were used concurrently. Stage IV granulomas, alone or in combination with other stages, constituted 63% of lesions, while 16% of lesions were stage I/II granulomas. Caseous necrosis and calcification were common features of the granulomas encountered in natural Mycobacterium bovis infections, even with pathology limited to a small number of sites. Granulomas often covered large areas of histological sections and typically contained only small numbers of acid fast bacilli.     

Risk factors of bovine tuberculosis in cattle in rural livestock production systems of Ethiopia

This study shows a representative stratified cluster sample survey of the prevalence of comparative intradermal tuberculin test in cattle from four regions in Ethiopia. Using a cut-off for positivity of 2 mm, it assesses possible risk factors for tuberculin-positive reaction in cattle. Seventy-three villages in 24 kebeles (administrative units) were randomly selected, from which 2216 cattle from 780 owners were tested. In addition, 450 of these cattle owners were interviewed for risk factor assessment. Ninety-nine percent of the tested cattle in this rural livestock production system were traditional zebus. The individual overall prevalence of cattle bovine tuberculosis (BTB)e was 3%, with the highest found in Meskan Mareko, in Central Ethiopia (7.9%) and the lowest in Woldia, in the North East edge of the Rift Valley (1.2%). Generalised Linear Mixed Models (GLMM) with random effect on kebeles was used to analyse risk factors of cattle reactors and human tuberculosis (TB) infection. Purchase of cattle and presence of other livestock in the herd were statistically significant, with OR: 1.7, p-values of 0.03 and OR: 2, p = 0.05, respectively. Family members diagnosed with TB or showing clinical signs of extra-pulmonary TB (EPTB) were reported in 86 households (19%). None of the assessed potential risk factors of disease transmission between cattle and human (food consumption, livestock husbandry and presence of BTB-positive cattle) were statistically significant.     

Monitoring of the intra-dermal tuberculosis skin test performed by Belgian field practitioners

The present study aimed to monitor skin test practices as performed by veterinarian field practitioners in Belgium. For this purpose, an anonymous postal questionnaire was elaborated and dispatched to veterinarians involved in bovine tuberculosis detection. The questionnaire included items focusing on the skin test performance. International experts in the field of bovine tuberculosis were asked to fill the questionnaire and a scoring scale was built as follows: 0 = ‘ideal’ answer, 1 = acceptable answer, whereas 2 = unacceptable answer. Furthermore, experts were asked to rank the questionnaire’s items according to their possible impact on the risk of not detecting reactors. A global score was further calculated for each participant and a comparison of practices was carried out between the two regions of the country, i.e. Wallonia and Flanders. Significant differences were observed between both regions, a harmonization at the country level is thus essential. No veterinarian summed a null score, corresponding to the ideal skin test procedure, which suggests that skin-testing is far from being performed correctly. Field practitioners need to be sensitized to the importance of correctly performing the test. The authors recommend the questionnaire is suitable for application in other countries or regions.     

Survival rate of airborne Mycobacterium bovis

Despite years of study the principle transmission route of bovine tuberculosis to cattle remains unresolved. The distribution of pathological lesions, which are concentrated in the respiratory system, and the very low dose of Mycobacterium bovis needed to initiate infection from a respiratory tract challenge suggest that the disease is spread by airborne transmission. Critical to the airborne transmission of a pathogenic microorganism is its ability to survive the stresses incurred whilst airborne. This study demonstrates that M. bovis is resistant to the stresses imposed immediately after becoming airborne, 94% surviving the first 10 min after aerosolisation. Once airborne the organism is robust, its viability decreasing with a half-life of approximately 1.5 hours. These findings support the hypothesis that airborne transmission is the principle route of infection for bovine tuberculosis.     

A Cross-sectional Study of Bovine Tuberculosis in Dairy Farms in Asmara,Eritrea

Bovine tuberculosis in dairy cattle in Asmara, Eritrea, was studied using a cross-sectional study to describe its prevalence and to identify factors associated with it. A total of 72 randomly selected herds were included in the study. The comparative intradermal tuberculin test was used for the diagnosis. Of 1813 individual animals tested, 14.5% were reactors. Thirty herds (41.7%) had at least one reactor but, by defining a reactor herd as any herd with two or more reactors, only 19 (26.4%) herds were classified as reactor herds. Based upon individual animal specificity of 98.5%, the calculated herd specificity was >99%. A multiple logistic model showed that the presence of exotic breeds was associated with a high risk (odds ratio = 5.70; 95% confidence interval 1.13–28.8). An increased risk was also linked to large herds. Keeping the animals always indoors reduced the risk, but could not be fitted to the model owing to empty cells.     

Characterization of pathogen-specific expression of host immune response genes in Anaplasma and Mycobacterium species infected ruminants

Anaplasma and Mycobacterium species are among the most prevalent bacterial pathogens in European red deer (Cervus elaphus) in south-central Spain and are known to modify gene expression in ruminants. In this study, we used microarray hybridization and real-time RT-PCR analyses to characterize global gene expression profiles in red deer in response to Anaplasma ovis and A. ovis/Mycobacterium bovis/Mycobacterium avium sub. paratuberculosis (MAP) infections, compare the expression of immune response genes between red deer infected with A. ovis, M. bovis and A. ovis/M. bovis/MAP, and characterize the differential expression of immune response genes identified in red deer in cattle infected with M. bovis and Anaplasma marginale. Global gene differential expression in A. ovis- and A. ovis/M. bovis/MAP-infected deer resulted in the modification of common and pathogen-specific cellular biological processes. The differential expression of host immune response genes showed pathogen and host-specific signatures and the effect of infection with multiple pathogens on deer immune response. These results suggested that intracellular bacteria from Anaplasma and Mycobacterium genera produce similar genes expression patterns in infected ruminants. However, pathogen and host-specific differences could contribute to disease diagnosis and treatment in ruminants.     

Transdisciplinary habitat models for elk and cattle as a proxy for bovine tuberculosis transmission risk

Zoonotic diseases such as bovine tuberculosis (TB) that infect wildlife and livestock are particularly difficult to eradicate where wild animals make extensive use of agricultural landscapes. Transmission of TB between cattle (Bos taurus) and wild elk (Cervus elaphus) in southwestern Manitoba, Canada remains poorly understood but there is a risk when comingling occurs on summer pasture. Elk use of cattle summer pastures was assessed using ecological data (187 VHF and 25 GPS collared elk monitored over four years representing 8% of the elk population). Local knowledge was documented by conducting interviews and participatory mapping exercises with 86 cattle producers (98% of those within the study area). Of the 294 cattle pastures mapped by farmers, 13% were used by radio-collared elk, 38% were reported by farmers as being used by elk, and 42% were identified as used by elk when both when all datasets were combined. Cattle pastures that had been used by elk and those that had no elk were compared using binary logistic regression based on each of the three datasets (i.e. farmer observations, radio-collared elk on pasture, and combined dataset). For all three datasets, distance to protected area and proportion of forest cover on the cattle pasture were identified as the most and second most important predictor variables, respectively. There was strong agreement among the relative probabilities of elk occurrence on each pasture derived from the resource selection function (RSF) models developed using farmer interviews and elk collaring data. The farmer interview and collar datasets were then combined to generate a final integrated RSF map summarizing the probability of elk–cattle comingling and were contrasted over each of four cattle grazing seasons (spring, early summer, late summer, and autumn). These predictive maps indicate that use of cattle pastures by elk is extensive, particularly in spring and early summer. Farmer observations indicate that elk and cattle share water sources and livestock mineral supplements on pasture. Local knowledge and conventional ecological data complement and validate one another and help us better understand the temporospatial aspects of shared space use among wildlife and livestock and more generally the risks of disease transmission in agricultural landscapes.     

An epidemiological evaluation of Mycobacterium bovis infections in wild game animals of the Spanish Mediterranean ecosystem

Recreational hunting of indigenous wild artiodactyls has been one of the most lucrative and rapidly growing industries in Western Spain over the last five years. In the absence of careful ecological management, one consequence of the commercial exploitation of this natural resource has been the appearance of outbreaks of infectious disease; most notably bovine tuberculosis. From the outset of the study in 1997, we have observed a steady increase in prevalence of Mycobacterium bovis (M. bovis) in both species reaching 1.74 (+/-0.17) in deer in 2002 and 2.32 (+/-0.24) in wild boar. The latter species seems to be most severely affected with pulmonary lesions appearing more chronic than those observed in deer. In this study, we describe the epidemiology of M. bovis in European wild boar (Sus scrofa) and Iberian red deer (Cervus elaphus hispanicus) in Extremadura (W. Spain); a region where there are large areas of natural habitat for these species.     

Veterinarian injuries associated with bovine TB testing livestock in Michigan, 2001

Testing all the cattle in an entire state with a uniform procedure for each animal affords an opportunity to relate human injury data to a known number of animals handled while carrying out a standardized procedure. Our objective was to describe the type and incidence density of injuries associated with TB-testing a large number of cattle herds, and to delineate the various factors associated with the risk of injury. A survey was mailed to all veterinarians (N = 259) who had completed at least five official bovine TB (bTB) herd tests in Michigan in 2001. We collected data regarding basic demographics and health status, work experience, veterinary specialty, and practice information. Each veterinarian was also requested to complete a separate injury questionnaire for each injury received while TB testing livestock in 2001. Accurate addresses were found for 247 eligible veterinarians, 175 (71%) of whom returned the survey. Thirty-six veterinarians reported a total of 53 injuries (10 major, 12 minor and 31 self-treated). Hands (29%) and legs (21%) were the anatomic locations most frequently injured, with sprains/strains (30%) and abrasion/contusion (30%) the most common types of injuries sustained. The overall incidence density of injuries was 1.9 per 10,000 animals tested. Female gender (RR = 3.3), being employed by the government (RR = 4.5), and smoking (RR = 6.0) were significantly associated with a higher rate of injury. Significant colliniearities were found between some risk factors associated with an increased rate of injury and participants thought 81% of their injuries could have been prevented. These results are explained by the administrative structure of the bTB testing program in Michigan, and the changing demographics of the veterinary workforce.     

Adaptation of IFN-gamma ELISA and ELISPOT tests for feline tuberculosis

There are currently no reliable immunodiagnostic tests for feline tuberculosis. Infection of domestic cats in the UK is thought to occur via their contact with the relevant reservoir of infection, e.g. cattle and badgers for Mycobacterium bovis, and rodents for M. microti. In the African National Parks, where M. bovis infection of Bovidae is an increasing problem, transmission to big cats is occurring via their ingestion of infected carcasses. We have adapted feline ELISA and ELISPOT assays to potentially provide the first cell-based diagnostic test for the detection of tuberculosis in cats. We tested peripheral blood mononuclear cell antigen-specific IFN-gamma responses of 18 cats suspected of mycobacterial infection for which biopsy material was co-submitted to the Veterinary Laboratories Agency for mycobacterial culture and identification. Seventeen cats were tested by ELISA while seven cats were tested by ELISPOT (six cats were tested by both ELISA and ELISPOT). Six healthy control cats provided baseline data for these tests. Responses to bovine and avian tuberculins (PPDB and PPDA) and a protein cocktail of ESAT6 and CFP10 were measured, together with positive mitogen (PMA and calcium ionophore) and negative (medium) controls. Overall, both ELISPOT and ELISA tests were found to be suitable for generating rapid results (2 and 4 days, respectively), which provided good predictive information for M. bovis and M. microti infections, but were unable to reliably discern M. avium infection.     

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.     

An outbreak of tuberculosis affecting cattle and people on an Irish dairy farm,following the consumption of raw milk

Bovine tuberculosis is an ongoing problem in Ireland, and herd incidence has remained at approximately 5% for some years. Spillover of infection from cattle to people remains an ever-present possibility, given the ongoing pool of infection in the Irish cattle population. This paper describes an outbreak of tuberculosis affecting cattle and people on a dairy farm in southeastern Ireland following the consumption of milk from a seven-year-old cow with tuberculous mastitis. Twenty-five of 28 calves born during autumn 2004 and spring 2005 were subsequently identified as TB reactors, and five of six family members were positive on the Mantoux test. During 2005, milk from this cow had mainly been used to feed calves, and was added only occasionally to the bulk tank. Therefore, the calves each received infected milk on an almost continuous basis between birth and weaning. The family collected milk from the bulk milk tank, and consumed it without pasteurisation. This case highlights the risks associated with the consumption of raw milk. In this family, TB has had a very significant impact on the health of two young children. These risks are well recognised, and relevant information for farmers is available. It is of concern, therefore, that raw milk consumption remains prevalent on Irish farms. New strategies are needed, in partnership with industry, to address this important issue. Keywords: bovine tuberculosis, Ireland, mastitis, milk, Mycobacterium bovis, pasteurisation, TB, zoonosis.