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1.  Surveillance of Bovine Tuberculosis and Risk Estimation of a Future Reservoir Formation in Wildlife in Switzerland and Liechtenstein 
PLoS ONE  2013;8(1):e54253.
Bovine tuberculosis (bTB) caused by Mycobacterium bovis or M. caprae has recently (re-) emerged in livestock and wildlife in all countries bordering Switzerland (CH) and the Principality of Liechtenstein (FL). Comprehensive data for Swiss and Liechtenstein wildlife are not available so far, although two native species, wild boar (Sus scrofa) and red deer (Cervus elaphus elaphus), act as bTB reservoirs elsewhere in continental Europe. Our aims were (1) to assess the occurrence of bTB in these wild ungulates in CH/FL and to reinforce scanning surveillance in all wild mammals; (2) to evaluate the risk of a future bTB reservoir formation in wild boar and red deer in CH/FL. Tissue samples collected from 2009 to 2011 from 434 hunted red deer and wild boar and from eight diseased ungulates with tuberculosis-like lesions were tested by direct real-time PCR and culture to detect mycobacteria of the Mycobacterium tuberculosis complex (MTBC). Identification of suspicious colonies was attempted by real-time PCR, genotyping and spoligotyping. Information on risk factors for bTB maintenance within wildlife populations was retrieved from the literature and the situation regarding identified factors was assessed for our study areas. Mycobacteria of the MTBC were detected in six out of 165 wild boar (3.6%; 95% CI: 1.4–7.8) but none of the 269 red deer (0%; 0–1.4). M. microti was identified in two MTBC-positive wild boar, while species identification remained unsuccessful in four cases. Main risk factors for bTB maintenance worldwide, including different causes of aggregation often resulting from intensive wildlife management, are largely absent in CH and FL. In conclusion, M. bovis and M. caprae were not detected but we report for the first time MTBC mycobacteria in Swiss wild boar. Present conditions seem unfavorable for a reservoir emergence, nevertheless increasing population numbers of wild ungulates and offal consumption may represent a risk.
doi:10.1371/journal.pone.0054253
PMCID: PMC3549981  PMID: 23349839
2.  Seroprevalence and Risk Factors Associated to Mycobacterium bovis in Wild Artiodactyl Species from Southern Spain, 2006–2010 
PLoS ONE  2012;7(4):e34908.
The control of bovine tuberculosis (bTB) is at a critical point in the last stage of eradication in livestock. Wildlife species recently have emerged infected with TB in Europe, particularly ungulates in the Iberian Peninsula. Epidemiological information regarding TB in wild ungulates including affected species, prevalence, associated risk factors and appropriate diagnostic methods need to be obtained in these countries.
A cross-sectional study was carried out on wild artiodactyl species, including Eurasian wild boar (Sus scrofa) red deer (Cervus elaphus), roe deer (Capraelus capraelus), fallow deer (Dama dama), Spanish ibex (Capra pyrenaica hispanica) and mouflon (Ovis musimon), in Spain to assess the seroprevalence against Mycobacterium bovis or cross-reacting members of the Mycobcaterium tuberculosis complex (MTBC), and to provide information on associated risk factors. Previously, two in-house indirect enzyme linked immunosorbent assays (bPPD-ELISA and MPB83-ELISA) were developed using known TB status sera. Positive reference sera were selected from infected animals confirmed by culture. The M. bovis isolates belonged to spoligotypes SB0121, SB0120, SB0295, SB0265 and SB0134. Two hundred and two out of 1367 (7.5%; 95% CI: 6.1–8.9) animals presented antibodies against M. bovis by both bPPD-ELISA and MPB83-ELISA. Significantly higher TB seroprevalence was observed in wild boar compared to the other species analyzed. Interestingly, seropositivity against M. bovis was not found in any out of 460 Spanish ibex analyzed. The logistic regression model for wild boar indicated that the seropositivity to M. bovis was associated with age, location and year of sampling, while the only risk factor associated with M. bovis seroprevalence in red deer and fallow deer was the age. The seroprevalence observed indicates a widespread exposure to MTBC in several wild artiodactyl species in southern Spain, which may have important implications not only for conservation but also for animal and public health.
doi:10.1371/journal.pone.0034908
PMCID: PMC3327704  PMID: 22523563
3.  Bovine Tuberculosis in Doñana Biosphere Reserve: The Role of Wild Ungulates as Disease Reservoirs in the Last Iberian Lynx Strongholds 
PLoS ONE  2008;3(7):e2776.
Doñana National Park (DNP) in southern Spain is a UNESCO Biosphere Reserve where commercial hunting and wildlife artificial feeding do not take place and traditional cattle husbandry still exists. Herein, we hypothesized that Mycobacterium bovis infection prevalence in wild ungulates will depend on host ecology and that variation in prevalence will reflect variation in the interaction between hosts and environmental risk factors. Cattle bTB reactor rates increased in DNP despite compulsory testing and culling of infected animals. In this study, 124 European wild boar, 95 red deer, and 97 fallow deer were sampled from April 2006 to April 2007 and analyzed for M. bovis infection. Modelling and GIS were used to identify risk factors and intra and inter-species relationships. Infection with M. bovis was confirmed in 65 (52.4%) wild boar, 26 (27.4%) red deer and 18 (18.5%) fallow deer. In the absence of cattle, wild boar M. bovis prevalence reached 92.3% in the northern third of DNP. Wild boar showed more than twice prevalence than that in deer (p<0.001). Modelling revealed that M. bovis prevalence decreased from North to South in wild boar (p<0.001) and red deer (p<0.01), whereas no spatial pattern was evidenced for fallow deer. Infection risk in wild boar was dependent on wild boar M. bovis prevalence in the buffer area containing interacting individuals (p<0.01). The prevalence recorded in this study is among the highest reported in wildlife. Remarkably, this high prevalence occurs in the absence of wildlife artificial feeding, suggesting that a feeding ban alone would have a limited effect on wildlife M. bovis prevalence. In DNP, M. bovis transmission may occur predominantly at the intra-species level due to ecological, behavioural and epidemiological factors. The results of this study allow inferring conclusions on epidemiological bTB risk factors in Mediterranean habitats that are not managed for hunting purposes. Our results support the need to consider wildlife species for the control of bTB in cattle and strongly suggest that bTB may affect animal welfare and conservation.
doi:10.1371/journal.pone.0002776
PMCID: PMC2464716  PMID: 18648665
4.  Descriptive Epidemiology of Bovine Tuberculosis in Michigan (1975–2010): Lessons Learned 
Despite ongoing eradication efforts, bovine tuberculosis (BTB) remains a challenge in Michigan livestock and wildlife. The objectives of this study were to (1) review the epidemiology of BTB in Michigan cattle, privately owned cervids, and wildlife between 1975 and 2010 and (2) identify important lessons learned from the review and eradication strategies. BTB information was accessed from the Michigan BTB Eradication Project agencies. Cattle herds (49), privately owned deer herds (4), and wild white-tailed deer (668) were found infected with BTB during the review period. BTB has occurred primarily in counties located at the northern portion of the state's Lower Peninsula. Currently used BTB eradication strategies have successfully controlled BTB spread. However additional changes in BTB surveillance, prevention, and eradication strategies could improve eradication efforts.
doi:10.4061/2011/874924
PMCID: PMC3135262  PMID: 21776355
5.  Prevalence of Bovine Tuberculosis and Risk Factor Assessment in Cattle in Rural Livestock Areas of Govuro District in the Southeast of Mozambique 
PLoS ONE  2014;9(3):e91527.
Background
Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is an infectious disease of cattle that also affects other domestic animals, free-ranging and farmed wildlife, and also humans. In Mozambique, scattered surveys have reported a wide variation of bTB prevalence rates in cattle from different regions. Due to direct economic repercussions on livestock and indirect consequences for human health and wildlife, knowing the prevalence rates of the disease is essential to define an effective control strategy.
Methodology/Principal findings
A cross-sectional study was conducted in Govuro district to determine bTB prevalence in cattle and identify associated risk factors. A representative sample of the cattle population was defined, stratified by livestock areas (n = 14). A total of 1136 cattle from 289 farmers were tested using the single comparative intradermal tuberculin test. The overall apparent prevalence was estimated at 39.6% (95% CI 36.8–42.5) using a diagnostic threshold cut-off according to the World Organization for Animal Health. bTB reactors were found in 13 livestock areas, with prevalence rates ranging from 8.1 to 65.8%. Age was the main risk factor; animals older than 4 years were more likely to be positive reactors (OR = 3.2, 95% CI: 2.2–4.7). Landim local breed showed a lower prevalence than crossbred animals (Landim × Brahman) (OR = 0.6, 95% CI: 0.4–0.8).
Conclusions/Significance
The findings reveal an urgent need for intervention with effective, area-based, control measures in order to reduce bTB prevalence and prevent its spread to the human population. In addition to the high prevalence, population habits in Govuro, particularly the consumption of raw milk, clearly may potentiate the transmission to humans. Thus, further studies on human tuberculosis and the molecular characterization of the predominant strain lineages that cause bTB in cattle and humans are urgently required to evaluate the impact on human health in the region.
doi:10.1371/journal.pone.0091527
PMCID: PMC3954769  PMID: 24632593
6.  High Prevalence of Bovine Tuberculosis in Dairy Cattle in Central Ethiopia: Implications for the Dairy Industry and Public Health 
PLoS ONE  2012;7(12):e52851.
Background
Ethiopia has the largest cattle population in Africa. The vast majority of the national herd is of indigenous zebu cattle maintained in rural areas under extensive husbandry systems. However, in response to the increasing demand for milk products and the Ethiopian government's efforts to improve productivity in the livestock sector, recent years have seen increased intensive husbandry settings holding exotic and cross breeds. This drive for increased productivity is however threatened by animal diseases that thrive under intensive settings, such as bovine tuberculosis (BTB), a disease that is already endemic in Ethiopia.
Methodology/Principal Findings
An extensive study was conducted to: estimate the prevalence of BTB in intensive dairy farms in central Ethiopia; identify associated risk factors; and characterize circulating strains of the causative agent, Mycobacterium bovis. The comparative intradermal tuberculin test (CIDT), questionnaire survey, post-mortem examination, bacteriology, and molecular typing were used to get a better understanding of the BTB prevalence among dairy farms in the study area. Based on the CIDT, our findings showed that around 30% of 2956 tested dairy cattle from 88 herds were positive for BTB while the herd prevalence was over 50%. Post-mortem examination revealed gross tuberculous lesions in 34/36 CIDT positive cattle and acid-fast bacilli were recovered from 31 animals. Molecular typing identified all isolates as M. bovis and further characterization by spoligotyping and MIRU-VNTR typing indicated low strain diversity within the study area.
Conclusions/Significance
This study showed an overall BTB herd prevalence of 50% in intensive dairy farms in Addis Ababa and surroundings, signalling an urgent need for intervention to control the disease and prevent zoonotic transmission of M. bovis to human populations consuming dairy products coming from these farms. It is suggested that government and policy makers should work together with stakeholders to design methods for the control of BTB in intensive farms in Ethiopia.
doi:10.1371/journal.pone.0052851
PMCID: PMC3532161  PMID: 23285202
7.  Epidemiological Significance of the Domestic Black Pig (Sus scrofa) in Maintenance of Bovine Tuberculosis in Sicily 
Journal of Clinical Microbiology  2012;50(4):1209-1218.
Bovine tuberculosis (bTB) is an emerging disease among wild animals in many parts of the world. Wildlife reservoir hosts may thus represent a potential source of infection for livestock and humans. We investigated the role played by the Sicilian black pig, an autochthonous free- or semi-free-ranging domestic pig breed, as a potential source of bTB infection in an area where bTB prevalence in cattle is high. We initially performed a preliminary field study to assess the occurrence of bTB in such animals. We sampled 119 pigs at abattoir and found 6.7% and 3.4% of them to be affected by gross tuberculous-like lesions (TBL) and Mycobacterium bovis culture positive, respectively. We then proceeded to investigate the dissemination and characteristics of lesions in a second field study performed on 100 animals sampled from infected herds. Here, tissues collected at the abattoir were examined macroscopically, microscopically, and by culture tests. Most pigs with TBL showed generalized lesions in both gross and histological examinations (53% and 65.5%, respectively). Head lymph nodes were the most frequently affected in both localized and generalized TB cases observed macroscopically and microscopically. M. bovis was the most frequently isolated etiologic agent. The molecular characterization of isolates from both field studies by spoligotyping and analysis of 12 mycobacterial interspersed repetitive-unit–variable number tandem repeat (MIRU-VNTR) loci, followed by their comparison to isolates of cattle origin, suggested a potential transmission of mycobacteria from domestic animals to black pigs and vice versa. Our findings, along with ethological, ecological, and management considerations, suggest that the black pig might act as a bTB reservoir in the ecosystem under study. However, additional studies will be necessary to establish the true epidemiological significance of the Sicilian black pig.
doi:10.1128/JCM.06544-11
PMCID: PMC3318573  PMID: 22322347
8.  Bovine tuberculosis (Mycobacterium bovis) in British farmland wildlife: the importance to agriculture 
Bovine tuberculosis (bTB) is an important disease of cattle and an emerging infectious disease of humans. Cow- and badger-based control strategies have failed to eradicate bTB from the British cattle herd, and the incidence is rising by about 18% per year. The annual cost to taxpayers in Britain is currently £74 million. Research has focused on the badger as a potential bTB reservoir, with little attention being paid to other mammals common on farmland. We have conducted a systematic survey of wild mammals (n=4393 individuals) present on dairy farms to explore the role of species other than badgers in the epidemiology of bTB. Cultures were prepared from 10 397 samples (primarily faeces, urine and tracheal aspirates). One of the 1307 bank voles (Clethrionomys glareolus) live-sampled, and three of the 43 badgers (Meles meles), yielded positive isolates of Mycobacterium bovis. This is the first time the bacterium has been isolated from the bank vole. The strain type was the same as that found in cattle and badgers on the same farm. However, our work indicates that the mean prevalence of infectious individuals among common farmland wildlife is extremely low (the upper 95% confidence interval is ≤2.0 for all of the abundant species). Mathematical models illustrate that it is highly unlikely the disease could be maintained at such low levels. Our results suggest that these animals are relatively unimportant as reservoirs of bTB, having insufficient within-species (or within-group) transmission to sustain the infection, though occasional spill-overs from cattle or badgers may occur.
doi:10.1098/rspb.2005.3298
PMCID: PMC1560044  PMID: 16543179
bovine tuberculosis; Mycobacterium bovis; epidemiology; voles; PCR; mycobacterium microti
9.  Classification of worldwide bovine tuberculosis risk factors in cattle: a stratified approach 
Veterinary Research  2009;40(5):50.
The worldwide status of bovine tuberculosis (bTB) as a zoonosis remains of great concern. This article reviews the main risk factors for bTB in cattle based on a three-level classification: animal, herd and region/country level. A distinction is also made, whenever possible, between situations in developed and developing countries as the difference of context might have consequences in terms of risk of bTB. Recommendations are suggested to animal health professionals and scientists directly involved in the control and prevention of bTB in cattle. The determination of Millenium Development Goals for bTB is proposed to improve the control/eradication of the disease worldwide.
doi:10.1051/vetres/2009033
PMCID: PMC2710499  PMID: 19497258
zoonosis; Mycobacterium bovis; cattle; risk factor; epidemiology
10.  Eradication of bovine tuberculosis at a herd-level in Madrid, Spain: study of within-herd transmission dynamics over a 12 year period 
Background
Eradication of bovine tuberculosis (bTB) through the application of test-and-cull programs is a declared goal of developed countries in which the disease is still endemic. Here, longitudinal data from more than 1,700 cattle herds tested during a 12 year-period in the eradication program in the region of Madrid, Spain, were analyzed to quantify the within-herd transmission coefficient (β) depending on the herd-type (beef/dairy/bullfighting). In addition, the probability to recover the officially bTB free (OTF) status in infected herds depending on the type of herd and the diagnostic strategy implemented was assessed using Cox proportional hazard models.
Results
Overall, dairy herds showed higher β (median 4.7) than beef or bullfighting herds (2.3 and 2.2 respectively). Introduction of interferon-gamma (IFN-γ) as an ancillary test produced an apparent increase in the β coefficient regardless of production type, likely due to an increase in diagnostic sensitivity. Time to recover OTF status was also significantly lower in dairy herds, and length of bTB episodes was significantly reduced when the IFN-γ was implemented to manage the outbreak.
Conclusions
Our results suggest that bTB spreads more rapidly in dairy herds compared to other herd types, a likely cause being management and demographic-related factors. However, outbreaks in dairy herds can be controlled more rapidly than in typically extensive herd types. Finally, IFN-γ proved its usefulness to rapidly eradicate bTB at a herd-level.
doi:10.1186/1746-6148-8-100
PMCID: PMC3464814  PMID: 22748007
11.  Herd-Level Risk Factors for Bovine Tuberculosis: A Literature Review 
Bovine tuberculosis (TB), caused by Mycobacterium bovis, is one of the most challenging endemic diseases currently facing government, the veterinary profession, and the farming industry in the United Kingdom and Ireland and in several other countries. The disease has a notoriously complex epidemiology; the scientific evidence supports both cattle-cattle and wildlife-cattle transmission routes. To produce more effective ways of reducing such transmission, it is important to understand those risk factors which influence the presence or absence of bovine TB in cattle herds. Here we review the literature on herd-level risk factor studies. Whilst risk factors operate at different scales and may vary across regions, epidemiological studies have identified a number of risk factors associated with bovine TB herd breakdowns, including the purchase of cattle, the occurrence of bovine TB in contiguous herds, and/or the surrounding area as well as herd size. Other factors identified in some studies include farm and herd management practices, such as, the spreading of slurry, the use of certain housing types, farms having multiple premises, and the use of silage clamps. In general, the most consistently identified risk factors are biologically plausible and consistent with known transmission routes involving cattle-cattle and wildlife-cattle pathways.
doi:10.1155/2012/621210
PMCID: PMC3395266  PMID: 22966479
12.  Risk factors for bovine tuberculosis in low incidence regions related to the movements of cattle 
Background
Bovine tuberculosis (bTB) remains difficult to eradicate from low incidence regions partly due to the imperfect sensitivity and specificity of routine intradermal tuberculin testing. Herds with unconfirmed reactors that are incorrectly classified as bTB-negative may be at risk of spreading disease, while those that are incorrectly classified as bTB-positive may be subject to costly disease eradication measures. This analysis used data from Scotland in the period leading to Officially Tuberculosis Free recognition (1) to investigate the risks associated with the movements of cattle from herds with different bTB risk classifications and (2) to identify herd demographic characteristics that may aid in the interpretation of tuberculin testing results.
Results
From 2002 to 2009, for every herd with confirmed bTB positive cattle identified through routine herd testing, there was an average of 2.8 herds with at least one unconfirmed positive reactor and 18.9 herds with unconfirmed inconclusive reactors. Approximately 75% of confirmed bTB positive herds were detected through cattle with no known movements outside Scotland. At the animal level, cattle that were purchased from Scottish herds with unconfirmed positive reactors and a recent history importing cattle from endemic bTB regions were significantly more likely to react positively on routine intradermal tuberculin tests, while cattle purchased from Scottish herds with unconfirmed inconclusive reactors were significantly more likely to react inconclusively. Case-case comparisons revealed few demographic differences between herds with confirmed positive, unconfirmed positive, and unconfirmed inconclusive reactors, which highlights the difficulty in determining the true disease status of herds with unconfirmed tuberculin reactors. Overall, the risk of identifying reactors through routine surveillance decreased significantly over time, which may be partly attributable to changes in movement testing regulations and the volume of cattle imported from endemic regions.
Conclusions
Although the most likely source of bTB infections in Scotland was cattle previously imported from endemic regions, we found indirect evidence of transmission within Scottish cattle farms and cannot rule out the possibility of low level transmission between farms. Further investigation is needed to determine whether targeting herds with unconfirmed reactors and a history of importing cattle from high risk regions would benefit control efforts.
doi:10.1186/1746-6148-9-225
PMCID: PMC3826851  PMID: 24206865
Scotland; Cattle movements; Tuberculin test; Sensitivity; Specificity; Officially tuberculosis free
13.  Risk factors associated with negative in-vivo diagnostic results in bovine tuberculosis-infected cattle in Spain 
Background
Despite great effort and investment incurred over decades to control bovine tuberculosis (bTB), it is still one of the most important zoonotic diseases in many areas of the world. Test-and-slaughter strategies, the basis of most bTB eradication programs carried out worldwide, have demonstrated its usefulness in the control of the disease. However, in certain countries, eradication has not been achieved due in part to limitations of currently available diagnostic tests. In this study, results of in-vivo and post-mortem diagnostic tests performed on 3,614 animals from 152 bTB-infected cattle herds (beef, dairy, and bullfighting) detected in 2007–2010 in the region of Castilla y León, Spain, were analyzed to identify factors associated with positive bacteriological results in cattle that were non-reactors to the single intradermal tuberculin test, to the interferon-gamma (IFN-γ) assay, or to both tests applied in parallel (Test negative/Culture + animals, T-/C+). The association of individual factors (age, productive type, and number of herd-tests performed since the disclosure of the outbreak) with the bacteriology outcome (positive/negative) was analyzed using a mixed multivariate logistic regression model.
Results
The proportion of non-reactors with a positive post-mortem result ranged from 24.3% in the case of the SIT test to 12.9% (IFN-γ with 0.05 threshold) and 11.9% (95% CI 9.9-11.4%) using both tests in parallel. Older (>4.5 years) and bullfighting cattle were associated with increased odds of confirmed bTB infection by bacteriology, whereas dairy cattle showed a significantly lower risk. Ancillary use of IFN-γ assay reduced the proportion of T-/C + animals in high risk groups.
Conclusions
These results demonstrate the likelihood of positive bacteriological results in non-reactor cattle is influenced by individual epidemiological factors of tested animals. Increased surveillance on non-reactors with an increased probability of being false negative could be helpful to avoid bTB persistence, particularly in chronically infected herds. These findings may aid in the development of effective strategies for eradication of bTB in Spain.
doi:10.1186/1746-6148-10-14
PMCID: PMC3895706  PMID: 24410926
Tuberculosis; Cattle; Diagnosis; Single tuberculin test; Interferon-gamma assay; Risk factors
14.  Localized reactive badger culling increases risk of bovine tuberculosis in nearby cattle herds 
Biology Letters  2011;8(1):50-53.
Human and livestock diseases can be difficult to control where infection persists in wildlife populations. Control of bovine tuberculosis (bTB) in British cattle is complicated by the maintenance of Mycobacterium bovis (the causative agent of bTB) in badgers, acting as reservoirs of infection. Although over 20 000 badgers were culled to control bTB between 1975 and 1997, the incidence of bTB in cattle has substantially increased in parts of Great Britain in recent decades. Our case-control study, involving 1208 cattle herds, provides further evidence of the detrimental effect of localized reactive badger culling in response to the disclosure of a confirmed bTB herd breakdown in cattle. The presence of any reactive badger culling activity and increased numbers of badgers culled in the vicinity of a herd were associated with significantly increased bTB risk, even after adjusting for other important local risk factors. Such findings may partly explain why some earlier localized approaches to bTB control were ineffective.
doi:10.1098/rsbl.2011.0554
PMCID: PMC3259956  PMID: 21752812
bovine tuberculosis; case-control study; localized reactive badger culling; Randomized Badger Culling Trial
15.  Mapping bovine tuberculosis in Great Britain using environmental data 
Trends in microbiology  2002;10(10):441-444.
The incidence of bovine tuberculosis (BTB) is increasing in Great Britain, exacerbated by the temporary suspension of herd testing in 2001 for fear of spreading the much more contagious foot and mouth disease. The transmission pathways of BTB remain poorly understood. Current hypotheses suggest the disease is introduced into susceptible herds from a wildlife reservoir (principally the Eurasian Badger) and/or from cattle purchased from infected areas, while the role of climatic factors in transmission has generally been ignored. Here, we show how remotely sensed satellite data, which provide good indicators of a variety of climatic factors, can be used to describe the distribution of BTB in Great Britain in 1997, and suggest how such data could be used to produce BTB risk maps for the future.
PMCID: PMC3173847  PMID: 12377548
16.  Bovine tuberculosis and the endangered Iberian lynx. 
Emerging Infectious Diseases  2000;6(2):189-191.
We report the first case of bovine tuberculosis in a free-living Iberian lynx (Lynx pardina), an extremely endangered feline, from Doñana National Park in Spain. The isolate (Mycobacterium bovis) correlates by molecular characterization with other isolates from wild ungulates in the park, strongly suggesting an epidemiologic link. Mycobacterium bovis infects many animal species, with wild and free-ranging domestic ungulates being the main reservoirs in nature (1).
PMCID: PMC2640855  PMID: 10756155
17.  Accurate Diagnostics for Bovine tuberculosis Based on High-Throughput Sequencing 
PLoS ONE  2012;7(11):e50147.
Background
Bovine tuberculosis (bTB) is an enduring contagious disease of cattle that has caused substantial losses to the global livestock industry. Despite large-scale eradication efforts, bTB continues to persist. Current bTB tests rely on the measurement of immune responses in vivo (skin tests), and in vitro (bovine interferon-γ release assay). Recent developments are characterized by interrogating the expression of an increasing number of genes that participate in the immune response. Currently used assays have the disadvantages of limited sensitivity and specificity, which may lead to incomplete eradication of bTB. Moreover, bTB that reemerges from wild disease reservoirs requires early and reliable diagnostics to prevent further spread. In this work, we use high-throughput sequencing of the peripheral blood mononuclear cells (PBMCs) transcriptome to identify an extensive panel of genes that participate in the immune response. We also investigate the possibility of developing a reliable bTB classification framework based on RNA-Seq reads.
Methodology/Principal Findings
Pooled PBMC mRNA samples from unaffected calves as well as from those with disease progression of 1 and 2 months were sequenced using the Illumina Genome Analyzer II. More than 90 million reads were splice-aligned against the reference genome, and deposited to the database for further expression analysis and visualization. Using this database, we identified 2,312 genes that were differentially expressed in response to bTB infection (p<10−8). We achieved a bTB infected status classification accuracy of more than 99% with split-sample validation on newly designed and learned mixtures of expression profiles.
Conclusions/Significance
We demonstrated that bTB can be accurately diagnosed at the early stages of disease progression based on RNA-Seq high-throughput sequencing. The inclusion of multiple genes in the diagnostic panel, combined with the superior sensitivity and broader dynamic range of RNA-Seq, has the potential to improve the accuracy of bTB diagnostics. The computational pipeline used for the project is available from http://code.google.com/p/bovine-tb-prediction.
doi:10.1371/journal.pone.0050147
PMCID: PMC3511461  PMID: 23226242
18.  Comparing Badger (Meles meles) Management Strategies for Reducing Tuberculosis Incidence in Cattle 
PLoS ONE  2012;7(6):e39250.
Bovine tuberculosis (bTB), caused by Mycobacterium bovis, continues to be a serious economic problem for the British cattle industry. The Eurasian badger (Meles meles) is partly responsible for maintenance of the disease and its transmission to cattle. Previous attempts to manage the disease by culling badgers have been hampered by social perturbation, which in some situations is associated with increases in the cattle herd incidence of bTB. Following the licensing of an injectable vaccine, we consider the relative merits of management strategies to reduce bTB in badgers, and thereby reduce cattle herd incidence. We used an established simulation model of the badger-cattle-TB system and investigated four proposed strategies: business as usual with no badger management, large-scale proactive badger culling, badger vaccination, and culling with a ring of vaccination around it. For ease of comparison with empirical data, model treatments were applied over 150 km2 and were evaluated over the whole of a 300 km2 area, comprising the core treatment area and a ring of approximately 2 km. The effects of treatment were evaluated over a 10-year period comprising treatment for five years and the subsequent five year period without treatment. Against a background of existing disease control measures, where 144 cattle herd incidents might be expected over 10 years, badger culling prevented 26 cattle herd incidents while vaccination prevented 16. Culling in the core 150 km2 plus vaccination in a ring around it prevented about 40 cattle herd breakdowns by partly mitigating the negative effects of culling, although this approach clearly required greater effort. While model outcomes were robust to uncertainty in parameter estimates, the outcomes of culling were sensitive to low rates of land access for culling, low culling efficacy, and the early cessation of a culling strategy, all of which were likely to lead to an overall increase in cattle disease.
doi:10.1371/journal.pone.0039250
PMCID: PMC3384660  PMID: 22761746
19.  Bovine tuberculosis in cattle: reduced risk on wildlife-friendly farms 
Biology Letters  2006;2(2):271-274.
The associations between habitat and other factors that lead to the risk of bovine tuberculosis (bTB) in diary cattle were examined in an unmatched case–control study. Data from 60 herds with recent history of bTB and 60 controls were analysed using logistic regression. The predictors included farmland habitat, topography, indices of badger density and herd size. Information-theoretic approaches were used to identify those predictor variables explaining the greatest variation in cattle herd bTB breakdowns. Reduced risk of bTB was associated with the management of farmland in ways favourable to wildlife conservation, as encouraged by recent (2005) European Common Agricultural Policy reforms.
doi:10.1098/rsbl.2006.0461
PMCID: PMC1618898  PMID: 17148380
badgers; TB; Akaike information criterion; habitat; landscape
20.  Local Cattle and Badger Populations Affect the Risk of Confirmed Tuberculosis in British Cattle Herds 
PLoS ONE  2011;6(3):e18058.
Background
The control of bovine tuberculosis (bTB) remains a priority on the public health agenda in Great Britain, after launching in 1998 the Randomised Badger Culling Trial (RBCT) to evaluate the effectiveness of badger (Meles meles) culling as a control strategy. Our study complements previous analyses of the RBCT data (focusing on treatment effects) by presenting analyses of herd-level risks factors associated with the probability of a confirmed bTB breakdown in herds within each treatment: repeated widespread proactive culling, localized reactive culling and no culling (survey-only).
Methodology/Principal Findings
New cases of bTB breakdowns were monitored inside the RBCT areas from the end of the first proactive badger cull to one year after the last proactive cull. The risk of a herd bTB breakdown was modeled using logistic regression and proportional hazard models adjusting for local farm-level risk factors. Inside survey-only and reactive areas, increased numbers of active badger setts and cattle herds within 1500 m of a farm were associated with an increased bTB risk. Inside proactive areas, the number of M. bovis positive badgers initially culled within 1500 m of a farm was the strongest predictor of the risk of a confirmed bTB breakdown.
Conclusions/Significance
The use of herd-based models provide insights into how local cattle and badger populations affect the bTB breakdown risks of individual cattle herds in the absence of and in the presence of badger culling. These measures of local bTB risks could be integrated into a risk-based herd testing programme to improve the targeting of interventions aimed at reducing the risks of bTB transmission.
doi:10.1371/journal.pone.0018058
PMCID: PMC3065452  PMID: 21464920
21.  Prevalence and risk factors for infection of bovine tuberculosis in indigenous cattle in the Serengeti ecosystem, Tanzania 
Background
Bovine tuberculosis (bTB) is a chronic debilitating disease and is a cause of morbidity and mortality in livestock, wildlife and humans. This study estimated the prevalence and risk factors associated with bovine tuberculosis transmission in indigenous cattle at the human-animal interface in the Serengeti ecosystem of Tanzania.
Results
A total of 1,103 indigenous cattle from 32 herds were investigated for the presence of bTB using the Single Intradermal Comparative Tuberculin Test. Epidemiological data on herd structure, management and grazing system were also collected.
The apparent individual animal prevalence of tuberculin reactors was 2.4% (95% confidence interval (CI), 1.7 – 3.5%), whereas the true prevalence was 0.6% CI, 0.6 – 0.7% as indicated by a reaction to avian tuberculin purified protein derivatives (PPD) which is more than 4 mm greater than the reaction to avian tuberculin PPD. The results showed that 10.6% (117/1,103) showed non-specific reactions (atypical mycobacterium). The herd prevalence of 50% (16/32) was found. Tuberculin skin test results were found to be significantly associated with age, location, size of the household and animal tested. Of 108 respondents, 70 (64.8%) individuals had not heard about bovine tuberculosis at all. Thirty five percent (38/108) of respondents at least were aware of bTB. About 60% (23/38) of respondents who were aware of bTB had some knowledge on how bTB is spread. Eighty one percent (87/108) of respondents were not aware of the presence of bTB in wildlife. There is regular contact between cattle and wild animals due to sharing of grazing land and water sources, with 99% (107/108) of households grazing cattle in communal pastures.
Conclusion
The study has demonstrated a high reported interaction of livestock with wildlife and poor knowledge of most cattle owners concerning bTB and its transmission pathways among people, livestock and wildlife. Although the overall proportion of animals with bTB is relatively low, herd prevalence is 50% and prevalence within herds varied considerably. Thus there is a possibility of cross transmission of bTB at wildlife-livestock interface areas that necessitates use of genetic strain typing methods to characterize them accurately.
doi:10.1186/1746-6148-9-267
PMCID: PMC3881215  PMID: 24377705
Risk factors; Bovine tuberculosis; Mycobacterium bovis; Human-animal interface; Serengeti ecosystem; Wildlife
22.  Prevalence of Bovine Tuberculosis in Abattoirs of the Littoral and Western Highland Regions of Cameroon: A Cause for Public Health Concern 
Bovine tuberculosis (BTB) is widespread but poorly controlled in Africa and M. bovis is posing threats to human health. The risk of cattle handlers to M. bovis prevalence and public health significance of BTB in Cameroon were assessed. Slaughter inspection records from major cities revealed that BTB detection rates in cattle from 0.18% to 4.25% and BTB lesions were most common. Analyses of tissues and sera confirmed BTB in 31% (Ziehl-Neelsen), 51% (culture), and 60% (antibody detection) of test cattle. Among cattle handlers, 81.9% were aware of BTB, 67.9% knew that BTB is zoonotic, and 53.8% knew one mode of transmission but over 27% consumed raw meat and/or drank unpasteurized milk. Respondents who had encountered tuberculosis cases were more informed about zoonotic BTB (P < .05). Tuberculosis is prevalent in cattle destined for human consumption in Cameroon with serious public health implications. Targeted monitoring of infected animal populations and concerted veterinary/medical efforts are essential for control.
doi:10.4061/2010/495015
PMCID: PMC2896641  PMID: 20613999
23.  A restatement of the natural science evidence base relevant to the control of bovine tuberculosis in Great Britain† 
Bovine tuberculosis (bTB) is a very important disease of cattle in Great Britain, where it has been increasing in incidence and geographical distribution. In addition to cattle, it infects other species of domestic and wild animals, in particular the European badger (Meles meles). Policy to control bTB is vigorously debated and contentious because of its implications for the livestock industry and because some policy options involve culling badgers, the most important wildlife reservoir. This paper describes a project to provide a succinct summary of the natural science evidence base relevant to the control of bTB, couched in terms that are as policy-neutral as possible. Each evidence statement is placed into one of four categories describing the nature of the underlying information. The evidence summary forms the appendix to this paper and an annotated bibliography is provided in the electronic supplementary material.
doi:10.1098/rspb.2013.1634
PMCID: PMC3757986  PMID: 23926157
bovine tuberculosis; epidemiology; cattle; badgers; vaccination
24.  Towards Risk-Based Test Protocols: Estimating the Contribution of Intensive Testing to the UK Bovine Tuberculosis Problem 
PLoS ONE  2013;8(5):e63961.
Eradicating disease from livestock populations involves the balancing act of removing sufficient numbers of diseased animals without removing too many healthy individuals in the process. As ever more tests for bovine tuberculosis (BTB) are carried out on the UK cattle herd, and each positive herd test triggers more testing, the question arises whether ‘false positive’ results contribute significantly to the measured BTB prevalence. Here, this question is explored using simple probabilistic models of test behaviour. When the screening test is applied to the average UK herd, the estimated proportion of test-associated false positive new outbreaks is highly sensitive to small fluctuations in screening test specificity. Estimations of this parameter should be updated as a priority. Once outbreaks have been confirmed in screening-test positive herds, the following rounds of intensive testing with more sensitive, albeit less specific, tests are highly likely to remove large numbers of false positive animals from herds. Despite this, it is unlikely that significantly more truly infected animals are removed. BTB test protocols should become based on quantified risk in order to prevent the needless slaughter of large numbers of healthy animals.
doi:10.1371/journal.pone.0063961
PMCID: PMC3661673  PMID: 23717517
25.  Is Mycobacterium bovis in the environment important for the persistence of bovine tuberculosis? 
Biology Letters  2006;2(3):460-462.
Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB) in cattle and wildlife. Direct aerosol contact is thought to be the primary route of infection between conspecifics, whereas indirect transmission via an environmental reservoir of M. bovis is generally perceived not to be a significant source for infection. Here, we report on the application of molecular technology (PCR) to quantify the prevalence of M. bovis in the environment and to explore its epidemiological significance. We show that the detectability of viable M. bovis at badger setts and latrines is strongly linked to the frequency of M. bovis excretion by infected badgers, and that putative M. bovis in the environment is prevalent on a large proportion of endemic cattle farms in Britain. These results raise important questions about the role of an environmental reservoir in bTB persistence.
doi:10.1098/rsbl.2006.0468
PMCID: PMC1686208  PMID: 17148430
Mycobacterium bovis; bovine tuberculosis; environment; survival; badger; Meles meles

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