Global phylogenomic diversity of Brucella abortus: spread of a dominant lineage.

Brucella abortus is a globally important zoonotic pathogen largely found in cattle hosts and is typically transmitted to humans through contaminated dairy products or contact with diseased animals. Despite the long, shared history of cattle and humans, little is known about how trade in cattle has spread this pathogen throughout the world. Whole genome sequencing provides unparalleled resolution to investigate the global evolutionary history of a bacterium such as B. abortus by providing phylogenetic resolution that has been unobtainable using other methods. We report on large-scale genome sequencing and analysis of B. abortus collected globally from cattle and 16 other hosts from 52 countries. We used single nucleotide polymorphisms (SNPs) to identify genetic variation in 1,074 B. abortus genomes and using maximum parsimony generated a phylogeny that identified four major clades. Two of these clades, clade A (median date 972 CE; 95% HPD, 781-1142 CE) and clade B (median date 150 BCE; 95% HPD, 515 BCE-164 CE), were exceptionally diverse for this species and are exclusively of African origin where provenance is known. The third clade, clade C (median date 949 CE; 95% HPD, 766-1102 CE), had most isolates coming from a broad swath of the Middle East, Europe, and Asia, also had relatively high diversity. Finally, the fourth major clade, clade D (median date 1467 CE; 95% HPD, 1367-1553 CE) comprises the large majority of genomes in a dominant but relatively monomorphic group that predominantly infects cattle in Europe and the Americas. These data are consistent with an African origin for B. abortus and a subsequent spread to the Middle East, Europe, and Asia, probably through the movement of infected cattle. We hypothesize that European arrival to the Americas starting in the 15th century introduced B. abortus from Western Europe through the introduction of a few common cattle breeds infected with strains from clade D. These data provide the foundation of a comprehensive global phylogeny of this important zoonotic pathogen that should be an important resource in human and veterinary epidemiology.

Genomic epidemiology of Mycobacterium bovis infection in sympatric badger and cattle populations in Northern Ireland.

Bovine tuberculosis (bTB) is a costly, epidemiologically complex, multi-host, endemic disease. Lack of understanding of transmission dynamics may undermine eradication efforts. Pathogen whole-genome sequencing improves epidemiological inferences, providing a means to determine the relative importance of inter- and intra-species host transmission for disease persistence. We sequenced an exceptional data set of 619 Mycobacterium bovis isolates from badgers and cattle in a 100 km2 bTB 'hotspot' in Northern Ireland. Historical molecular subtyping data permitted the targeting of an endemic pathogen lineage, whose long-term persistence provided a unique opportunity to study disease transmission dynamics in unparalleled detail. Additionally, to assess whether badger population genetic structure was associated with the spatial distribution of pathogen genetic diversity, we microsatellite genotyped hair samples from 769 badgers trapped in this area. Birth death models and TransPhylo analyses indicated that cattle were likely driving the local epidemic, with transmission from cattle to badgers being more common than badger to cattle. Furthermore, the presence of significant badger population genetic structure in the landscape was not associated with the spatial distribution of M. bovis genetic diversity, suggesting that badger-to-badger transmission is not playing a major role in transmission dynamics. Our data were consistent with badgers playing a smaller role in transmission of M. bovis infection in this study site, compared to cattle. We hypothesize, however, that this minor role may still be important for persistence. Comparison to other areas suggests that M. bovis transmission dynamics are likely to be context dependent, with the role of wildlife being difficult to generalize.

Internet-delivered cognitive behaviour therapy for post-traumatic stress disorder: a randomised controlled trial and outcomes in routine care.

BACKGROUND: Despite its potential scalability, little is known about the outcomes of internet-based cognitive behaviour therapy (iCBT) for post-traumatic stress disorder (PTSD) when it is provided with minimal guidance from a clinician. AIM: To evaluate the outcomes of minimally guided iCBT for PTSD in a randomised control trial (RCT, Study 1) and in an open trial in routine community care (Study 2). METHOD: A RCT compared the iCBT course (n=21) to a waitlist control (WLC, n=19) among participants diagnosed with PTSD. The iCBT group was followed up 3 months post-treatment. In Study 2, treatment outcomes were evaluated among 117 adults in routine community care. PTSD symptom severity was the primary outcome in both studies, with psychological distress and co-morbid anxiety and depressive symptoms providing secondary outcomes. RESULTS: iCBT participants in both studies experienced significant reductions in PTSD symptom severity from pre- to post-treatment treatment (within-group Hedges' g=.72-1.02), with RCT findings showing maintenance of gains at 3-month follow-up. The WLC group in the RCT also significantly improved, but Study 1 was under-powered and the medium between-group effect favouring iCBT did not reach significance (g=0.64; 95% CI, -0.10-1.38). CONCLUSIONS: This research provides preliminary support for the utility of iCBT for PTSD when provided with minimal clinician guidance. Future studies are needed to clarify the effect of differing levels of clinician support on PTSD iCBT outcomes, as well as exploring how best to integrate iCBT into large-scale, routine clinical care of PTSD.

Does Mycobacterium tuberculosis var. bovis Survival in the Environment Confound Bovine Tuberculosis Control and Eradication? A Literature Review.

Bovine tuberculosis (bTB) is one of the globe's most common, multihost zoonoses and results in substantial socioeconomic costs for governments, farming industries, and tax payers. Despite decades of surveillance and research, surprisingly, little is known about the exact mechanisms of transmission. In particular, as a facultative intracellular pathogen, to what extent does survival of the causative agent, Mycobacterium tuberculosis var. bovis (M. bovis), in the environment constitute an epidemiological risk for livestock and wildlife? Due largely to the classical pathology of cattle cases, the received wisdom was that bTB was spread by direct inhalation and exchange of bioaerosols containing droplets laden with bacteria. Other members of the Mycobacterium tuberculosis complex (MTBC) exhibit differing host ranges, an apparent capacity to persist in environmental fomites, and they favour a range of different transmission routes. It is possible, therefore, that infection from environmental sources of M. bovis could be a disease transmission risk. Recent evidence from GPS-collared cattle and badgers in Britain and Ireland suggests that direct transmission by infectious droplets or aerosols may not be the main mechanism for interspecies transmission, raising the possibility of indirect transmission involving a contaminated, shared environment. The possibility that classical pulmonary TB can be simulated and recapitulated in laboratory animal models by ingestion of contaminated feed is a further intriguing indication of potential environmental risk. Livestock and wildlife are known to shed M. bovis onto pasture, soil, feedstuffs, water, and other fomites; field and laboratory studies have indicated that persistence is possible, but variable, under differing environmental conditions. Given the potential infection risk, it is timely to review the available evidence, experimental approaches, and methodologies that could be deployed to address this potential blind spot and control point. Although we focus on evidence from Western Europe, the concepts are widely applicable to other multihost bTB episystems.

Transdiagnostic internet-delivered CBT and mindfulness-based treatment for depression and anxiety: A randomised controlled trial.

AIM: To examine the efficacy of transdiagnostic internet-delivered cognitive behavioural therapy (iCBT), mindfulness-enhanced iCBT, and stand-alone online mindfulness training compared with a usual care control group (TAU) for clinical anxiety and depression. METHOD: Individuals (N = 158) with a DSM-5 diagnosis of a depressive and/or anxiety disorder were randomised to one of the three clinician-guided online interventions, or TAU over a 14-week intervention period. The primary outcomes were self-reported depression (PHQ-9) and anxiety (GAD-7) severity at post-treatment. Secondary outcomes included adherence rates, functional impairment (WHODAS-II), general distress (K-10), and diagnostic status at the 3-month follow-up (intervention groups). RESULTS: All three programs achieved significant and large reductions in symptoms of depression (g = 0.89-1.53), anxiety (g = 1.04-1.40), and distress (g = 1.25-1.76); and medium to large reductions in functional impairment (g = 0.53-0.98) from baseline to post-treatment and 3-month follow-up. Intention-to-treat linear mixed models showed that all three online programs were superior to usual care at reducing symptoms of depression (g = 0.89-1.18) and anxiety (g = 1.00-1.23). CONCLUSION: Transdiagnostic iCBT, mindfulness-enhanced iCBT and online mindfulness training are more efficacious for treating depression and anxiety disorders than usual care, and represent an accessible treatment option for these disorders.

Genomic epizootiology of a Brucella abortus outbreak in Northern Ireland (1997-2012).

BACKGROUND: In the recent past (1997-2012), Northern Ireland in the United Kingdom suffered an outbreak of Brucella abortus, which at its height affected over 200 cattle herds. Initially, isolates were characterized using multi-locus variable number tandem repeats analysis (MLVA). While informative in this setting, hyper-variability in some loci limited the resolution necessary to infer fine-scale disease transmission networks. Consequently, we applied whole-genome sequencing to isolates from this outbreak to evaluate higher resolution markers for disease epizootiology. RESULTS: Phylogenetic analysis revealed that the B. abortus outbreak in Northern Ireland was caused by two distinct pathogen lineages. One contained isolates consistent with the 1997-2012 outbreak being linked to a previous endemic infection thought eradicated. The dominant second lineage exhibited little genetic diversity throughout the recrudescent outbreak, with limited population sub-structure evident. This finding was inconsistent with prior MLVA molecular characterizations that suggested the presence of seven clonal complexes. Spatio-temporal modeling revealed a significant association of pairwise SNP differences between isolates and geographic distances. However, effect sizes were very small due to reduced pathogen diversity. CONCLUSIONS: Genome sequence data suggested that hyper-variability in some MLVA loci contributed to an overestimate of pathogen diversity in the most recent outbreak. The low diversity observed in our genomic dataset made it inappropriate to apply phylodynamic methods to these data. We conclude that maintaining data repositories of genome sequence data will be invaluable for source attribution/epizootiological inference should recrudescence ever re-occur. However genomic epizootiological methods may have limited utility in some settings, such as when applied to recrudescent/re-emergent infections of slowly-evolving bacterial pathogens.

Characteristics of Northern Irish cattle herds without bovine tuberculosis infection.

BACKGROUND: Despite ongoing eradication efforts, bovine tuberculosis (bTB) is endemic in cattle herds in Northern Ireland (NI). This disease has serious implications for the economy, farming and animal welfare. Previous research identified a population of herds which have remained free from bTB infection for 10 years (2004-2014). Understanding the characteristics of these herds may have important implications for eradication efforts, such as spatially targeted interventions. METHODS: A cluster analysis and a retrospective case-control analysis was conducted to compare bTB- free herds with herds which experienced prolonged infection (ie, bTB breakdowns lasting more than ≥ 365 days). RESULTS: Only small, localised clusters of herds which have remained free from bTB were revealed, thus limiting the potential for spatially targeted interventions. The results illustrated the importance of herd size to disease status; over 27 per cent of the bTB-free herds had up to 10 animals. However, the data also showed that there were no inward movements in the year before the bTB skin test in those herds which remained free from bTB. CONCLUSIONS: Attention should therefore be given to the cattle movement network in NI to better understand the risk associated with cattle purchasing.

The population and landscape genetics of the European badger (Meles meles) in Ireland.

The population genetic structure of free-ranging species is expected to reflect landscape-level effects. Quantifying the role of these factors and their relative contribution often has important implications for wildlife management. The population genetics of the European badger (Meles meles) have received considerable attention, not least because the species acts as a potential wildlife reservoir for bovine tuberculosis (bTB) in Britain and Ireland. Herein, we detail the most comprehensive population and landscape genetic study of the badger in Ireland to date-comprised of 454 Irish badger samples, genotyped at 14 microsatellite loci. Bayesian and multivariate clustering methods demonstrated continuous clinal variation across the island, with potentially distinct differentiation observed in Northern Ireland. Landscape genetic analyses identified geographic distance and elevation as the primary drivers of genetic differentiation, in keeping with badgers exhibiting high levels of philopatry. Other factors hypothesized to affect gene flow, including earth worm habitat suitability, land cover type, and the River Shannon, had little to no detectable effect. By providing a more accurate picture of badger population structure and the factors effecting it, these data can guide current efforts to manage the species in Ireland and to better understand its role in bTB.

Internet cognitive-behavioural treatment for panic disorder: randomised controlled trial and evidence of effectiveness in primary care.

BACKGROUND: Internet cognitive-behavioural therapy (iCBT) for panic disorder of up to 10 lessons is well established. The utility of briefer programmes is unknown. AIMS: To determine the efficacy and effectiveness of a five-lesson iCBT programme for panic disorder. METHOD: Study 1 (efficacy): Randomised controlled trial comparing active iCBT (n=27) and waiting list control participants (n=36) on measures of panic severity and comorbid symptoms. Study 2 (effectiveness): 330 primary care patients completed the iCBT programme under the supervision of primary care practitioners. RESULTS: iCBT was significantly more effective than waiting list control in reducing panic (g=0.97, 95% CI 0.34 to 1.61), distress (g=0.92, 95% CI 0.28 to 1.55), disability (g=0.81, 95% CI 0.19 to 1.44) and depression (g=0.79, 95% CI 0.17 to 1.41), and gains were maintained at 3 months post-treatment (iCBT group). iCBT remained effective in primary care, but lower completion rates were found (56.1% in study 2 v. 63% in study 1). Adherence appeared to be related to therapist contact. CONCLUSIONS: The five-lesson Panic Program has utility for treating panic disorder, which translates to primary care. Adherence may be enhanced with therapist contact. DECLARATION OF INTEREST: None. COPYRIGHT AND USAGE: © The Royal College of Psychiatrists 2016. This is an open access article distributed under the terms of the Creative Commons Non-Commercial, No Derivatives (CC BY-NC-ND) licence.

Use of bacterial whole-genome sequencing to investigate local persistence and spread in bovine tuberculosis.

Mycobacterium bovis is the causal agent of bovine tuberculosis, one of the most important diseases currently facing the UK cattle industry. Here, we use high-density whole genome sequencing (WGS) in a defined sub-population of M. bovis in 145 cattle across 66 herd breakdowns to gain insights into local spread and persistence. We show that despite low divergence among isolates, WGS can in principle expose contributions of under-sampled host populations to M. bovis transmission. However, we demonstrate that in our data such a signal is due to molecular type switching, which had been previously undocumented for M. bovis. Isolates from farms with a known history of direct cattle movement between them did not show a statistical signal of higher genetic similarity. Despite an overall signal of genetic isolation by distance, genetic distances also showed no apparent relationship with spatial distance among affected farms over distances <5 km. Using simulations, we find that even over the brief evolutionary timescale covered by our data, Bayesian phylogeographic approaches are feasible. Applying such approaches showed that M. bovis dispersal in this system is heterogeneous but slow overall, averaging 2 km/year. These results confirm that widespread application of WGS to M. bovis will bring novel and important insights into the dynamics of M. bovis spread and persistence, but that the current questions most pertinent to control will be best addressed using approaches that more directly integrate WGS with additional epidemiological data.

Internet-based cognitive behavioural therapy (iCBT) for posttraumatic stress disorder versus waitlist control: study protocol for a randomised controlled trial.

BACKGROUND: This randomised controlled trial (RCT) with two parallel arms will evaluate the efficacy of an internet-delivered six-lesson 10-week cognitive behavioural therapy (iCBT) intervention for posttraumatic stress disorder (PTSD). It will also investigate the association between changes in PTSD symptoms, intolerance of uncertainty (IU) and emotion regulation. METHODS/DESIGN: Patients with PTSD will be recruited via the research arm of a not-for-profit clinical and research unit in Australia and randomised to a treatment group or waitlist control group. The minimum sample size for each group (alpha 0.05, power 0.80 for a g of 0.47) was identified as 72, but 10 % more will be recruited to hedge against expected attrition. PTSD diagnosis will be determined using the PTSD module from the Mini International Neuropsychiatric Interview version 5.0.0. The PTSD Checklist - Civilian version (PCL-C) will be used to measure PTSD symptoms (the primary outcome measure), with the Intolerance of Uncertainty Scale 12-item version (IUS-12) and the Emotion Regulation Questionnaire (ERQ) used to measure intolerance of uncertainty and emotion regulation, respectively. The PCL-C will be administered to the treatment group before each lesson of the PTSD program and at 3-month follow-up. The IUS-12 and ERQ will be administered before lessons 1 and 4, at post-treatment and at 3-month follow-up. The waitlist control group will complete these measures at week 1, week 5 and week 11 of the waitlist period. PTSD program efficacy will be determined using intent-to-treat mixed models. Maintenance of gains will be assessed at 3-month follow-up. Mediation analyses using PROCESS will be used to examine the association between change in PTSD symptoms over treatment and change in each of IU and emotion regulation ability in separate analyses. DISCUSSION: The current RCT seeks to replicate previous efficacy findings of iCBT for PTSD in a formally assessed PTSD sample from the general population. Findings may point to future lines of enquiry for the role of IU and emotion regulation in the mechanism of PTSD symptom change during CBT. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry: ACTRN12614001213639 , registered 18 November 2014. This trial protocol is written in compliance with the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines.

Herd-level bovine tuberculosis risk factors: assessing the role of low-level badger population disturbance.

Bovine TB (bTB) is endemic in Irish cattle and has eluded eradication despite considerable expenditure, amid debate over the relative roles of badgers and cattle in disease transmission. Using a comprehensive dataset from Northern Ireland (>10,000 km(2); 29,513 cattle herds), we investigated interactions between host populations in one of the first large-scale risk factor analyses for new herd breakdowns to combine data on both species. Cattle risk factors (movements, international imports, bTB history, neighbours with bTB) were more strongly associated with herd risk than area-level measures of badger social group density, habitat suitability or persecution (sett disturbance). Highest risks were in areas of high badger social group density and high rates of persecution, potentially representing both responsive persecution of badgers in high cattle risk areas and effects of persecution on cattle bTB risk through badger social group disruption. Average badger persecution was associated with reduced cattle bTB risk (compared with high persecution areas), so persecution may contribute towards sustaining bTB hotspots; findings with important implications for existing and planned disease control programmes.

Hui and Walter's latent-class model extended to estimate diagnostic test properties from surveillance data: a latent model for latent data.

Diagnostic test sensitivity and specificity are probabilistic estimates with far reaching implications for disease control, management and genetic studies. In the absence of 'gold standard' tests, traditional Bayesian latent class models may be used to assess diagnostic test accuracies through the comparison of two or more tests performed on the same groups of individuals. The aim of this study was to extend such models to estimate diagnostic test parameters and true cohort-specific prevalence, using disease surveillance data. The traditional Hui-Walter latent class methodology was extended to allow for features seen in such data, including (i) unrecorded data (i.e. data for a second test available only on a subset of the sampled population) and (ii) cohort-specific sensitivities and specificities. The model was applied with and without the modelling of conditional dependence between tests. The utility of the extended model was demonstrated through application to bovine tuberculosis surveillance data from Northern and the Republic of Ireland. Simulation coupled with re-sampling techniques, demonstrated that the extended model has good predictive power to estimate the diagnostic parameters and true herd-level prevalence from surveillance data. Our methodology can aid in the interpretation of disease surveillance data, and the results can potentially refine disease control strategies.

Genomic prediction for tuberculosis resistance in dairy cattle.

BACKGROUND: The increasing prevalence of bovine tuberculosis (bTB) in the UK and the limitations of the currently available diagnostic and control methods require the development of complementary approaches to assist in the sustainable control of the disease. One potential approach is the identification of animals that are genetically more resistant to bTB, to enable breeding of animals with enhanced resistance. This paper focuses on prediction of resistance to bTB. We explore estimation of direct genomic estimated breeding values (DGVs) for bTB resistance in UK dairy cattle, using dense SNP chip data, and test these genomic predictions for situations when disease phenotypes are not available on selection candidates. METHODOLOGY/PRINCIPAL FINDINGS: We estimated DGVs using genomic best linear unbiased prediction methodology, and assessed their predictive accuracies with a cross validation procedure and receiver operator characteristic (ROC) curves. Furthermore, these results were compared with theoretical expectations for prediction accuracy and area-under-the-ROC-curve (AUC). The dataset comprised 1151 Holstein-Friesian cows (bTB cases or controls). All individuals (592 cases and 559 controls) were genotyped for 727,252 loci (Illumina Bead Chip). The estimated observed heritability of bTB resistance was 0.23±0.06 (0.34 on the liability scale) and five-fold cross validation, replicated six times, provided a prediction accuracy of 0.33 (95% C.I.: 0.26, 0.40). ROC curves, and the resulting AUC, gave a probability of 0.58, averaged across six replicates, of correctly classifying cows as diseased or as healthy based on SNP chip genotype alone using these data. CONCLUSIONS/SIGNIFICANCE: These results provide a first step in the investigation of the potential feasibility of genomic selection for bTB resistance using SNP data. Specifically, they demonstrate that genomic selection is possible, even in populations with no pedigree data and on animals lacking bTB phenotypes. However, a larger training population will be required to improve prediction accuracies.

Field-isolated genotypes of Mycobacterium bovis vary in virulence and influence case pathology but do not affect outbreak size.

Strains of many infectious agents differ in fundamental epidemiological parameters including transmissibility, virulence and pathology. We investigated whether genotypes of Mycobacterium bovis (the causative agent of bovine tuberculosis, bTB) differ significantly in transmissibility and virulence, combining data from a nine-year survey of the genetic structure of the M. bovis population in Northern Ireland with detailed records of the cattle population during the same period. We used the size of herd breakdowns as a proxy measure of transmissibility and the proportion of skin test positive animals (reactors) that were visibly lesioned as a measure of virulence. Average breakdown size increased with herd size and varied depending on the manner of detection (routine herd testing or tracing of infectious contacts) but we found no significant variation among M. bovis genotypes in breakdown size once these factors had been accounted for. However breakdowns due to some genotypes had a greater proportion of lesioned reactors than others, indicating that there may be variation in virulence among genotypes. These findings indicate that the current bTB control programme may be detecting infected herds sufficiently quickly so that differences in virulence are not manifested in terms of outbreak sizes. We also investigated whether pathology of infected cattle varied according to M. bovis genotype, analysing the distribution of lesions recorded at post mortem inspection. We concentrated on the proportion of cases lesioned in the lower respiratory tract, which can indicate the relative importance of the respiratory and alimentary routes of infection. The distribution of lesions varied among genotypes and with cattle age and there were also subtle differences among breeds. Age and breed differences may be related to differences in susceptibility and husbandry, but reasons for variation in lesion distribution among genotypes require further investigation.

Detectability of bovine TB using the tuberculin skin test does not vary significantly according to pathogen genotype within Northern Ireland.

Strains of many infectious diseases differ in parameters that influence epidemic spread, for example virulence, transmissibility, detectability and host specificity. Knowledge of inter-strain variation can be exploited to improve management and decrease disease incidence. Bovine tuberculosis (bTB) is increasingly prevalent among farmed cattle in the UK, exerting a heavy economic burden on the farming industry and government. We aimed to determine whether strains of Mycobacterium bovis (the causative agent of bTB) identified and classified using genetic markers (spoligotyping and multi-locus VNTR analysis) varied in response to the tuberculin skin test; this being the primary method of bTB detection used in the UK. Inter-strain variation in detectability of M. bovis could have important implications for disease control. The skin test is based on a differential delayed type hypersensitivity (DTH) response to intradermal injections of purified protein derivative (PPD) from M. bovis (PPD-B) and Mycobacterium avium (PPD-A). We searched for an association between skin test response (PPD-B skin rise minus PPD-A skin rise) and M. bovis genotype at the disclosing test in culture-confirmed cases using a field dataset consisting of 21,000 isolates belonging to 63 genotypes of M. bovis from cattle in Northern Ireland. We found no substantial variation among genotypes (estimated responses clustered tightly around the mean) controlling for animal sex, breed and test effects. We also estimated the ratio of skin test detected to undetected cases (i.e. cases only detected at abattoir). The skin test detection ratio varied among abattoirs with some detecting a greater proportion of cases than others but this variation was unrelated to the community composition of genotypes within each abattoir catchment. These two lines of evidence indicate that M. bovis genotypes in Northern Ireland have similar detectability using the skin test.

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.