Tuberculosis in found dead badgers at the edge of the expanding bovine tuberculosis epidemic.

Tuberculosis in found dead badgers at the edge of the expanding bovine tuberculosis epidemic. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Tuberculosis in found dead badgers at the edge of the expanding bovine tuberculosis epidemic. Sian Powell, Nicola Dessi, Malcolm Bennett, Belinda Wang, Andrew Robertson, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5193016/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bovine tuberculosis (bTB) is a major disease of cattle in the UK, placing a significant economic burden on the taxpayer. The causative agent, Mycobacterium bovis , has a wide host range, including the European badger ( Meles meles) . While badgers have been implicated in the transmission and maintenance of infection in cattle in areas of endemic disease, their role at the edge of the endemic area is poorly understood. Here we present data on the prevalence of infection in badgers collected along the southern edge of England’s bTB epidemic. Stakeholders across five counties (Oxfordshire, Berkshire, Buckinghamshire, Hampshire, and East Sussex) submitted found-dead badgers for post-mortem examination and testing by bacterial culture. The overall prevalence, as confirmed by whole genome sequencing, was 6.5% (28/428), ranging between 1.1% (1/88) in Hampshire and 13.0% (14/108) in Oxfordshire. The commonest M. bovis clade in badgers was B6-62, which was predominant in 4/5 counties. B6-62 was also the commonest clade found in cattle and was detected in all counties except East Sussex where, although absent from the cattle population, it was detected in local badgers. This study highlights the co-incidence of infection in badgers and cattle in parts of the southern edge area consistent with localised clustering of infection in both species. Infectious Diseases Zoonoses Wildlife Biology Mycobacterium bovis bovis badgers southern edge RTA disease surveillance bTB Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Mycobacterium bovis ( M. bovis ) is a member of the M. tuberculosis complex (MTC), a group of host-associated, tuberculous disease-causing bacteria of high genetic similarity 1 , 2 . Despite this high level of clonality, the group varies significantly in host tropism with M. bovis having the widest known host range 3 . Indeed, in the United Kingdom (UK) alone M. bovis has been detected in nine species of farmed or companion mammals, and at least 20 species of wild mammals 4 – 9 . In cattle, M. bovis causes bovine tuberculosis (bTB), one of the most significant challenges affecting cattle health in England, which is estimated to cost the UK taxpayer over £150 million per annum, with further costs to the cattle industry 10 . In addition to the financial burden is the cost of livestock life, with over 31,000 cattle slaughtered in 2023, of which 20,200 were from England 11 . English counties are categorised by their historic prevalence and epidemiology of bTB infection in cattle (see Fig. 1 ). In the high-risk area (HRA), which encompasses the southwest of the country, the disease is endemic in cattle and extensive disease surveillance is in place, including standard six-monthly herd testing and pre-movement testing. Surrounding the HRA is the edge area (EA), where bTB prevalence has been historically lower than the HRA but into which the epidemic is expanding, resulting in highly variable disease prevalence and either six-monthly or annual cattle testing, in addition to pre-movement testing. Though not contiguous with the rest of the EA, East Sussex is included within this zone due to the prevalence of bTB in the county. Finally, in the low-risk area (LRA) the incidence of bTB in cattle has been historically low and stable, with most cattle herds routinely tested for TB every four years, supplemented by targeted enhanced testing of certain herds and mandatory post-movement testing of cattle introduced from the rest of England and Wales 12 . While cattle movements undoubtedly make a substantial contribution to the spread and persistence of bTB in herds across the three risk areas, the presence of infection in the primary wildlife reservoir, the European badger ( Meles meles ) adds further complexity to disease control 13 – 15 . Infectious badgers can excrete M. bovis in their saliva, urine, faeces or from ruptured abscesses, presenting a hazard to cattle through indirect transmission, and potentially via direct transmission in cattle housing 16 – 19 . During the Randomised Badger Culling Trial (RBCT, 1998–2005), 15.9% of culled badgers tested positive by culture or by having lesions containing acid-fast organisms 20 . There was a demonstrable association between the proportion of positive badgers and cattle TB incidents at a distance of 1–2 km 20 , 21 . In addition, spoligotyping and multi-locus variable number tandem repeat analyses (MLVA) have demonstrated the co-localisation of related M. bovis isolates from badgers and cattle 21 – 24 . Whole genome sequencing (WGS) has further evidenced the link between infections in cattle and badgers, with highly related isolates being cultured from sympatric populations indicating recent and bi-directional transmission 25 – 27 . In English areas of high cattle incidence, efforts to reduce transmission risks from wildlife have included licenced culls of badgers, with over 53,000 animals culled during the years 2022 to 2023 20,22,28–32 . For the purposes of surveillance, found-dead surveys are the most viable approach for collecting a sufficiently large sample of badgers (typically the results of road traffic collisions) for investigating infection prevalence at a relatively broad geographic scale (e.g., county-level). This approach was extensively validated alongside the RBCT and found to be a reliable proxy for M. bovis infection prevalence within the sampled population 15 . Since then, it has been employed across Wales 23 , 33 , large areas of the Republic of Ireland and Northern Ireland 24 , 34 and in the county of Cheshire to investigate an emerging cluster of TB in cattle 35 . Also, of particular relevance to the present study, was an investigation of bTB in found dead badgers in the northern EA 36 . This study took place in 2016–2017 and identified a headline MTC infection rate in badgers of 8.3% (range: 4–15%), and common areas of high-prevalence in cattle and badgers as also reported in other studies 23 , 24 , 33 , 34 . Effective control of bTB in cattle at the edge of the endemic area requires a better understanding of the potential role of badgers in the spread and perpetuation of infection in this transitional zone. Here we present the results of an investigation into the prevalence of M. bovis in found-dead badgers in the southern EA, undertaken between 1st of April 2021 and the 30th of April 2023. Our results are interpreted alongside contemporaneous data on infection in cattle, gathered during routine surveillance and monitoring, and the results of the earlier study undertaken in the northern EA 36 . Results Badger carcasses In total, 525 badger carcasses were collected from across the five counties of the southern EA, with 428 (81.5%) deemed suitable for examination and tissue sampling. RTAs accounted for 84.3% of submissions (n = 443), sixteen (3.0%) were found dead elsewhere and were typically emaciated and in poor condition, whilst the causes of death could not be determined in the remainder, usually owing to the degree of carcass decomposition. The number of carcasses submitted varied by county (χ² = 46.0, df = 4, P < 0.0001), with Oxfordshire yielding the highest number (n = 138) and Berkshire providing the fewest (n = 49) (Table 1 ). The majority of carcasses were collected by farmers and farming groups (n = 231, 44.0%), followed by conservation groups (n = 132, 25.1%), veterinarians and their affiliates (n = 85, 16.2%), government agencies or local authorities (n = 18, 3.4%), and miscellaneous other groups or individuals (n = 59, 11.2%). Of the carcasses submitted for which the sex was determined (n = 443), 226 (51.0%) were males and 217 (49.0%) were females. No temporal variation was observed in the number of carcasses submitted by month when the average number of submissions were examined. Comparisons between the submissions of male and female carcasses with reference to month had to account for variable sample sizes, the low number of repeat data and the unequal number of repeats. On average, there were fewer submissions of males across the calendar year, with the exception of December – March (Fig. 2 ). However, only in April were significantly more females submitted than males (t = 9.5, df = 2, P ≤ 0.01). The majority of carcasses were of adult badgers (estimated to be over 1 year old), with only 52/525 (9.9%) of those collected and 43/444 (9.7%) of those examined recorded as cubs. M. bovis infection in surveyed badgers Across the southern EA, 31/428 (7.2%, 95% CI: 5.0-9.7%) badger carcasses tested positive for MTC bacteria by culture, IS 6110 PCR and hsp65 sequencing. Of these, 28 were confirmed as M. bovis by WGS giving an apparent prevalence of 6.5% (95% CI: 4.4–8.9%). Despite the low number of positive badgers, and the aforementioned study design, efforts were made to examine factors that increased the likelihood of positive carcasses being detected. The prevalence of infected badgers varied significantly by county (χ² = 12.7, df = 4, P ≤ 0.01), ranging from 1.1% in Hampshire to 13.0% in Oxfordshire (Table 1 ). Additionally, the detection of positive carcasses varied with the time of year, being most likely in March (OR (95% CI) = 10.58 (1.794–20.14), P < 0.05), and least likely in October (not significant) (Fig. 2 ). All of the confirmed positives were adults (28/384 confirmed adults, 7.3%, 95% CI: 4.9–10.4%) and the prevalence of infection was more than 3 times higher in male badgers than in females (χ² = 8.3, df = 1, P < 0.01), with 22/215 male badgers testing positive (10.2%, 95% CI: 6.5–15.1%) and 6/210 females (2.9%, 95% CI: 1.1–6.1%). The distances between any given positive badger and its nearest positive neighbour were significantly smaller than those between any negative badger and their nearest positive neighbour (W = 4193, P < 0.05). The median distance from a positive badger to the nearest other positive badger was 6.1 km (interquartile range, IQR: 2.4–11.6 km), whilst negative badgers were on average 8.4 km (IQR: 5.1–16.5 km) from their nearest positive neighbour. Six M. bovis clades were identified amongst the isolates obtained from confirmed positive badger carcasses. Of these, B6-62 was the most common, representing 21/28 (75.0%) of M. bovis confirmed isolates. The remaining clades (B6-61, B6-71, B6-85, B6-91, and B1-11) were each detected only once while two isolates (7.1%) were not identified to clade level (Fig. 3 ). The distance to the nearest badger with the same strain (where available) had a mean of 7.9 km (95% CI: 5.4–10.7 km) while the distance to the nearest badger with a different strain was 17.4 km (95% CI: 12.7–22.0 km). Statistical analysis identified a significant difference between the two distributions (t = -4.57, df = 20, P < 0.001). M. bovis in cattle During the study period, there was an average of 2639 (range: 2606–2711) active herds in the southern EA per annum. Of these, 277 herds (10.5%) experienced at least one TB breakdown (i.e. detection of at least one TB test positive animal and/or laboratory-positive case at routine slaughter). During the study period, a total of 308 breakdowns occurred in the study area (including 31 repeat breakdowns) of which 132 (56.5%) were OTF-W, 174 (42.9%) were OTF-S and the remainder were unclassified. Of the breakdowns involving at least one animal with visible tuberculous lesions or positive culture (OTF-W), WGS clades were identified in 84.1% (n = 111) of incidents, meaning that amongst all incidents of OTF status being suspended or withdrawn a clade was identified in 36.0% of cases. The proportion of herds with reactor cattle during the period of study varied by county (χ² = 13.7, df = 4, P < 0.01); Oxfordshire herds were the most likely to have a TB test reactor detected with 20.9% of herds experiencing a breakdown during the study, compared to only 5.0% of herds in Hampshire (Table 1 ). Of those herds where a clade associated reactor was identified (n = 111), the most common M. bovis clade was B6-62 which was found in 83 incidents (74.8%), followed by B6-11 in twelve incidents (10.8%). Table 1 County level demography and TB status of badger carcasses submitted during the study period (1st April 2021–30th April 2023), and data on the number of active herds per county and their breakdown status (1st April 2021-30th April 2023). County Oxfordshire East Sussex Buckinghamshire Hampshire Berkshire Badgers 1 Carcasses submitted 138 128 99 107 49 Carcasses sampled 2 108 (78.2%) 103 (80.4%) 85 (85.9%) 88 (82.2%) 44 (89.8%) Carcass density (per km 2 ) 0.0415 0.0575 0.0454 0.0239 0.0349 Male (%) 46.4 51.4 48.9 55.1 57.8 Female (%) 54.6 48.6 51.1 44.9 42.2 Adults (%) 88.0 96.1 97.6 94.3 95.5 Juveniles (%) 12.0 3.9 2.4 5.7 4.5 M. bovis positive 14 (13.0%) 5 (4.9%) 4 (4.7%) 1 (1.1%) 4 (9.1%) M. bovis positive (per km 2 ) 0.00537 0.00279 0.00213 0.00027 0.00317 Mean active herds (range) 526 (509–545) 590 (584–603) 475 (467–494) 837 (820–857) 211 (208–215) Cattle 3 Mean active herd density (per km 2 ) 0.202 0.329 0.253 0.228 0.167 Number of herds with breakdown 4 110 41 53 42 31 Percentage of herds with breakdown 20.9 6.9 11.2 5.0 14.7 Density of breakdowns (per km 2 ) 0.0422 0.0229 0.0283 0.0114 0.0246 1 Note that unsampled carcasses were excluded from the analyses. 2 One badger was excluded from these analyses as the spatial data indicated it was located significantly beyond the study boundary. 3 Data is given at the herd level and analyses conducted using the mean number of active herds during the study period. 4 Estimated as the number of herds that were non-OTF during the study period; repeat breakdowns are not included. Comparison between badgers and cattle A statistically significant, positive relationship was identified between the percentage of positive badger carcasses and the herd level prevalence (defined as the percentage of herds that experienced at least one breakdown during the study period) at the county level (Fig. 4 ; Pearson’s r(3) = 0.99 (95% CI: 0.82-1.0), t(3) = 10.9, P < 0.01). The distances between badger carcasses and cattle infected with the same and different clades were analysed. The two East Sussex B6-62 infected badgers were removed from the analyses as outliers, due to the non-detection of the clade in local herds and because this county was not contiguous with any other EA county. Overall, the distance from a positive badger to a herd with the same clade was 3.6 km (IQR: 2.9–5.6 km) while the mean distance to the nearest herd infected with a different clade was 9.9 km (95% CI: 7.6–12.2 km). Badgers were significantly more likely to be found near herds infected with the same clade, than to be found near herds infected with different clades (V = 41, P < 0.01). Non-tuberculous mycobacteria (NTM) Fifteen putative species of non-tuberculous mycobacteria (NTM) were detected in 30 badgers (apparent prevalence 7.0%, 95% CI: 5.1–9.4%), including one unsexed badger. M. avium was the most prevalent NTM with an apparent prevalence of 2.6% (95% CI: 1.3–4.5%, 11/429), followed by M. vaccae with an apparent prevalence of 1.2% (95% CI: 0.4–2.7%, 5/429); some isolates were not fully identified by this method. More males tested positive for M. bovis (22/215, 10.2%) than for NTM (16/215, 7.4%) and more females tested positive for NTM (13/210, 6.2%) than for M. bovis (6/210, 2.9%), although neither difference was significant. Discussion M. bovis infection in badgers collected from the southern EA was spatially heterogeneous, with the county level apparent prevalence ranging from 1.1% in Hampshire to 13.0% in Oxfordshire. This concurs with findings from a similar study of found dead badgers in the northern EA where county-level prevalence ranged from 4–5–15% 36 . Our study wide prevalence estimate of 6.5% (95% CI: 4.4–8.9%) is also similar to that of studies in the northern EA (8.3%, 95% CI: 6.4–11.0%) and Wales (7.3%, 95% CI: 5.6–9.5%) 23 , 36 . However, direct comparisons are problematic owing to minor methodological differences, including that the current study used WGS to confirm M. bovis isolation whilst Swift et al. (2021) used IS 6110 confirmation to the MTC level, and Schroeder et al . (2020) used spoligotyping. The use of WGS in the present study excluded three samples that were identified as MTC members by hsp65 sequencing (putative M. bovis due to the host species), reducing the overall apparent prevalence from 7.2%, detected using IS6110 and hsp65 , to the reported 6.5%. The prevalence of M. bovis in badgers was significantly and positively correlated with the number of incident cases observed in cattle at the county level. Spatial correlation between infection in badgers and cattle has previously been observed in the northern Edge Area, as it has also been in the wider endemic areas of England and Wales 22 , 23 , 33 , 36 . The ratio of male to female badgers in the present study was circa 1:1, similar to that observed in other found-dead studies 33 , 36 , 37 and as identified in capture-mark-recapture studies of badger populations 38 – 40 . The prevalence of confirmed M. bovis infection in male badgers in the present study was higher than in females, consistent with previous work showing that males are more likely to become infected and experience more rapid disease progression, thus increasing the likelihood of yielding a positive culture at PME 41 , 42 . It is also hypothesised that immunological differences may enhance male susceptibility to disease 42 . The present study involved an atypically low percentage of sampled juvenile badgers (9.9%) compared to other found dead surveys (c. 30% 35,36 and population studies (c. 20–30%) 40 , 43 , 44 . As TB infection is more often observed in adult badgers than in juveniles, the high proportion of adults in the present study is likely to have inflated overall population-level prevalence 20 , 45 , 46 . The reasons for the low percentage of juveniles in our sample of carcasses is not clear, but could potentially reflect poor survival related to unfavourable conditions 47 . For example, April 2021 and 2022 and the summer of 2021 were unusually dry 48 , 49 which is likely to have reduced food availability 50 , 51 and impacted on cub survival in particular 47 . Routine WGS of M. bovis requires the culture of bacterial isolates from infected individuals. However, the success of culture can be limited by early-stage infection 52 , 53 , the metabolic state of the bacteria 54 , 55 , the slow growth rate of M. bovis 56 and the potential for overgrowth by competing microorganisms 57 ; it is therefore highly probable that the apparent prevalence given here is an underestimation of the true prevalence. Furthermore, the current routine approach of sequencing a single isolate per badger or entire cattle herd means that if multiple infections with different strains or clades were present then they would not be detected. Despite these limitations, six clades were cultured from badger carcasses during the present study, of which B6-62 was the most common, being identified in 4/5 counties. Two badgers collected from East Sussex were infected with B6-62, outside the putative home range of the clade, and this clade was undetected in the county’s cattle 58 . Due to our limited understanding of bTB in East Sussex, with a low level of isolate recovery from SICCT positive cattle, it is difficult to interpret these results. The probability that these two samples were similarly mislabelled seems low given that both the examination of carcasses and subsequent sequencing of isolates were carried out several weeks apart, and hence these results merit further investigation. Two further clades which were identified during the study (B1-11, B6-85) were also isolated from badgers in areas which were well beyond their typical home range, and one nationally rare isolate with no designated home range was detected in Oxfordshire (B6-61) 58 . While B6-85 was identified in a badger found in close proximity to cattle herd breakdowns associated with that clade, the same was not true for B1-11 and B6-61. The identification of M. bovis clades in badgers that are distinct from those detected over the same period in cattle could arise as a result of historic spillover from cattle into badgers, or the limited sensitivity of M. bovis surveillance in cattle, as only 36% of breakdowns yielded sequenceable isolates across the study area. Previous evidence has demonstrated the contemporaneous circulation of different strains within badgers and cattle, showing the value in surveying both species to entirely evaluate the disease landscape 25 . The apparent prevalence of NTM in this study was 7.0%, far greater than a previously reported value of 0.9% (424/45,705) from archived UK cull data (recorded as M. avium and other mycobacteria), but similar to that reported in Spain from a small opportunistic study 59 . Unlike M. bovis , NTMs were distributed relatively evenly between the sexes, likely related to the environmental nature of these bacteria contributing to even exposure risks. Also, in contrast to M. bovis , the likelihood of detectable NTM infection did not increase with age, though the small number of juveniles in this study likely limited our ability to detect any such effects. Estimates of prevalence from studies such as ours should be treated with caution as biases can arise as a result of the relatively small sample sizes, the unknown absolute population size, local clustering of infection, potential behavioural correlates of susceptibility to becoming a RTA, and the limitations of the diagnostic methods employed. However, the sampling approach and culture-based methodology in the present study are broadly consistent with those shared by several other badger RTA surveys, allowing for some level of comparison whilst recognising the known limitations 23 , 33 , 35 , 36 . Furthermore, the collection of found dead badgers provides a geographically broad estimate of prevalence, which would otherwise be unachievable, and the collection of epidemiologically valuable WGS data to assist in disease tracking. To conclude, the present study has provided the first estimates of M. bovis prevalence in badger populations in the southern EA. In addition to identifying county-level heterogeneity, the use of WGS has added significant value to the surveillance data available for cattle by uncovering additional clades in badgers that were not detected in local herds and has confirmed infection in wildlife where it was previously unstudied. This study has provided further evidence for the link between infection in badgers and cattle at the edge of the area of TB endemism in England. Further investigation of WGS data from this area may shed light on the relative importance of cattle or badgers in driving herd breakdowns and whether (as seems likely) cattle movements may have seeded infection in local badgers which in places may have subsequently created a self-sustaining reservoir of infection and facilitated spill-back to cattle. Understanding the proximal drivers of infection in cattle across the EA may inform management options which could include enhanced cattle measures, badger vaccination and targeted badger culling. In some parts of the EA it may not be too late to prevent infection spilling over into this wildlife host before it becomes endemic in both populations. Materials and methods Carcass collection and storage A network of collectors was established across the five counties of the southern Edge Area with the aim of collecting 100 carcasses from each county. A dedicated phoneline and e-mail address were used for communications, and the study was promoted at events, through veterinarians, and on the TB Hub website. Interested parties were provided with instructions and kits for the safe collection of found-dead badgers as well as a small financial reward. Collectors placed carcasses in three double thickness PVC bags and sealed them with pre-labelled tags. Useful metadata was recorded on the submission form by the collector, including the date and time, tag identification code, location, type of submission (e.g., road traffic accident) and any overnight storage conditions if the courier was unable to collect the same day. Carcasses were not frozen, but when storage was required, collectors were asked to place in a chiller if available, or else in a cool and dry area. If the courier was unable to transport the carcass to the post-mortem examination (PME) lab (University of Nottingham, Sutton Bonington Campus) the same day, then it would be stored in a chiller overnight or over the weekend until it could be safely received. The median time between carcass collection and PME was 3 days (range: 0–12). Carcass collection took place from April 2021 to April 2023 inclusive, with collections ceasing at the county level once 100 carcasses had been sampled. Post-mortem examination Carcasses were deemed suitable for PME if they had not suffered significant damage, were not autolysed, and arrived with the form linking them to a location. The sex, weight, length, age, (adult or juvenile), estimated from dentition, size, and pelage, condition as inferred from weight and body fat, and probable cause of death were recorded. In some cases, these data are not recorded due to the condition of the carcass, but sampling was possible, thus test data exists that cannot be related to certain population demographics. The tissue sampling protocol for isolation of mycobacteria followed that of the Northern Edge Study 36 , based on the protocols used by 33 and 34 , modified by 35 . Carcasses were examined externally and internally for TB-like lesions, and if present these were harvested for tissue processing and bacterial culture. Selected lymph nodes and lung tissue were harvested and pooled (see Table 2 ). Harvested tissues were stored at 4°C for up to 48 h before processing for bacterial culture. Table 2 Tissue pools harvested from badger carcasses for microbiological culture. Tissue pool Tissues Lesions Individual samples from each gross lesion Head and neck Right and left mandibular, parotid, and retropharyngeal lymph nodes Thorax Anterior and posterior mesenteric and left and right bronchial lymph nodes, and apical lung Abdomen Hepatic and mesenteric lymph nodes Carcass pool Right and left prescapular, axillary, inguinal, and popliteal lymph nodes Thoracic blood/fluid Frozen for serology Tissue Processing Tissue processing and microbiological examination was performed in the Containment Level 3 (CL3) facility at the University of Nottingham, according to the methods used in previous studies 35 , 36 . Briefly, lesioned tissue or pools of target tissues were gently ground with sterile sand in phosphate buffered saline (PBS). Samples were mixed 1:1 with 5% oxalic acid and incubated at room temperature for 10 min, before 200 µL of each pool was inoculated onto Stonebrink Selective agar + PACT (BD Diagnostic) and onto Middlebrook 7H11 slopes supplemented by PANTA (BD Diagnostics). Media were incubated at 37°C for a minimum of 12 weeks with checking at regular intervals for putative mycobacterial colonies. Characterisation of mycobacteria After a minimum of 12 weeks incubation, putative mycobacterial colonies were heat killed at 80°C for 30 min. DNA was extracted by crude lysis; heat killed colonies were frozen and subsequently heated to 95°C for 5 min in sterile distilled water (SDW), centrifuged at 13,000 x g for 3 min to remove cellular debris and the resultant supernatant aliquoted for molecular analyses. Isolates were screened by PCR for IS 6110 , an insertion sequence unique to the MTC, and hsp65 sequencing which also identified non-tuberculous mycobacteria (NTM). Confirmed members of the MTC were subjected to whole genome sequencing (WGS; performed at APHA laboratories, Weybridge). Sequences were compared to M. bovis reference sequence AF2122 for final confirmation and assigned to one of 30 lineages known to be in circulation in Great Britain (pipeline: https://github.com/APHA-CSU/btb-seq ). Cattle data APHA conducts routine surveillance and testing of cattle herds for bTB. In the HRA and EA cattle are tested annually or six-monthly, with increased frequency of testing following detection of infection or after high-risk cattle movements. Testing is routinely conducted in the UK using the single intradermal comparative cervical tuberculin test (SICCT) with subsequent removal of reactors (animals exhibiting a positive result). Herds that are on schedule with their testing routine and have no reactors are classed as ‘Officially bTB Free’ (OTF). If at least one reactor (a bovine with one positive or two inconclusive results) was detected, then a TB incident (herd breakdown) is declared and the OTF status is suspended (OTF-S). At PME, if lesions that are typical of TB are identified in a reactor, or a positive bacterial culture or (as of March 2022) positive PCR test is obtained from a TB test reactor, or a non-reactor animal presenting with suspected tuberculous lesions at routine slaughter, then the OTF status of the affected herd is withdrawn (OTF-W). The APHA held dataset includes the breakdown identifier, herd identifier, case reference, herd location, breakdown status, the test result and WGS clade if determined. For the present study, the data for each herd was combined across the study period and any herd that had been OTF-S or OTF-W was classed as positive. Statistical analyses Analyses were conducted in R version 4.2.2, with graphs generated using the ‘ggplot2’ and ‘patchwork’ packages. The ‘sf’ package was used to analyse spatial data and QGIS 3.8.1 to visualise it. Statistical comparisons of count, percentage and distance data were performed within this study; data were tested for normality using the Shapiro-Wilk test. Significance testing of count and distance data was performed using the following: students T-test for normal and non-percentage data otherwise a Chi-squared when sufficient data were available, else the Fisher’s Exact Test. Paired data was tested using a T-test for normal data or Wilcoxon Signed Rank Test when the data were not normal. Multiple comparisons were controlled using the Benjamini-Hochberg method to reduce the false discovery rate. Declarations Competing interests The authors S.M.P., N.D., A.R., M.B., B.W., E.W., G.C.S., R.J.D declare no competing interests. Author contributions S.M.P. was the project manager during the final year of the project, collated and analysed the data, produced the map and figures, and wrote the main manuscript. N.D. was the project manager prior to S.M.P. and collated data, conducted preliminary analyses, and reviewed the manuscript. M.B. conducted PMEs. B.W. conducted tissue processing, bacterial culture, and isolate screening. E.W. conducted clade assignment. A.R. assisted with the preliminary analyses and reviewed the manuscript. G.C.S. was involved in project conception and reviewed the manuscript. R.J.D. led on the project conception and contributed to preparation of the manuscript. Acknowledgments This project was funded by DEFRA (project APHATBOR1093). We are extremely grateful to the collectors who both promoted the study and actively participated in badger carcass collection, without whom this project would not have been possible. We also thank the pathology and microbiology teams at the University of Nottingham, Sutton Bonington Campus, and the sequencing team at APHA Weybridge. Data availability Following publication badger test results and an approximate location for each carcass will be made available online. Cattle herd data including test outcomes and locations are widely available through other platforms. References Brosch R et al (2002) A new evolutionary scenario for the Mycobacterium tuberculosis complex. Proc Natl Acad Sci U S A 99:3684–3689. 10.1073/pnas.052548299 Bespiatykh D, Bespyatykh J, Mokrousov I, Shitikov EA (2021) Comprehensive Map of Mycobacterium tuberculosis Complex Regions of Difference. mSphere 6, e0053521, 10.1128/mSphere.00535-21 O'Reilly LM, Daborn CJ (1995) The epidemiology of Mycobacterium bovis infections in animals and man: a review. Tuber Lung Dis 76(Suppl 1):1–46. 10.1016/0962-8479(95)90591-x Murhead RH, Burns KJ (1974) Tuberculosis in wild badgers in Gloucestershire: epidemiology. Vet Rec 95:552–555. 10.1136/vr.95.24.552 Delahay RJ et al (2007) Bovine tuberculosis infection in wild mammals in the South-West region of England: A survey of prevalence and a semi-quantitative assessment of the relative risks to cattle. Vet J 173:287–301. https://doi.org/10.1016/j.tvjl.2005.11.011 Delahay RJ, De Leeuw ANS, Barlow AM, Clifton-Hadley RS, Cheeseman CL (2002) The Status of Mycobacterium bovis Infection in UK Wild Mammals: A Review. Vet J 164:90–105. https://doi.org/10.1053/tvjl.2001.0667 Foyle KL, Delahay RJ, Massei G (2010) Isolation of Mycobacterium bovis from a feral wild boar (Sus scrofa) in the UK. Vet Rec 166:663–664. https://doi.org/10.1136/vr.c2681 Lee J, Hanna R, Hill R, McCormick CM, Skuce RA (2009) Bovine tuberculosis in an Eurasian otter. Vet Rec 164:727–728. https://doi.org/10.1136/vr.164.23.727-a Broughan JM et al (2013) Mycobacterium bovis infections in domesticated non-bovine mammalian species. Part 1: Review of epidemiology and laboratory submissions in Great Britain 2004–2010. Vet J 198:339–345. https://doi.org/10.1016/j.tvjl.2013.09.006 Defra (2018) Next steps for the strategy for achieving bovine tuberculosis free status for England. The government’s response to the strategy review, ( https://assets.publishing.service.gov.uk/media/5e60ad0de90e077e3d2678d2/bovine-tb-strategy-review-government-response.pdf , 2020) Defra (2024) Tuberculosis (TB) in cattle in Great Britain. (Department for Environment, Food and Rural Affairs, https://www.gov.uk/government/statistical-data-sets/tuberculosis-tb-in-cattle-in-great-britain APHA (2020) Bovine TB testing intervals , https://www.gov.uk/guidance/bovine-tb-testing-intervals Gilbert M et al (2005) Cattle movements and bovine tuberculosis in Great Britain. Nature 435:491–496. 10.1038/nature03548 Brooks-Pollock E, Roberts GO, Keeling MJ (2014) A dynamic model of bovine tuberculosis spread and control in Great Britain. Nature 511:228–231. 10.1038/nature13529 ISG. (ed (2007) Food and Rural Affairs Department for Environment). Defra Phillips CJ, Foster CR, Morris PA, Teverson R (2003) The transmission of Mycobacterium bovis infection to cattle. Res Vet Sci 74:1–15. 10.1016/s0034-5288(02)00145-5 Garnett BT, Delahay RJ, Roper TJ (2002) Use of cattle farm resources by badgers (Meles meles) and risk of bovine tuberculosis (Mycobacterium bovis) transmission to cattle. Proc Biol Sci 269:1487–1491. 10.1098/rspb.2002.2072 Benham PFJ, Broom DM (1989) Interactions between cattle and badgers at pasture with reference to bovine tuberculosis transmission. Br Vet J 145:226–241. https://doi.org/10.1016/0007-1935(89)90075-4 Woodroffe R et al (2016) Badgers prefer cattle pasture but avoid cattle: implications for bovine tuberculosis control. Ecol Lett 19:1201–1208. 10.1111/ele.12654 Jenkins HE et al (2008) The prevalence, distribution and severity of detectable pathological lesions in badgers naturally infected with Mycobacterium bovis. Epidemiol Infect 136:1350–1361. 10.1017/S0950268807009909 Woodroffe R et al (2005) Spatial association of Mycobacterium bovis infection in cattle and badgers Meles meles. J Appl Ecol 42:852–862. https://doi.org/10.1111/j.1365-2664.2005.01081.x Krebs J et al (eds) (1997) Fisheries and Food Ministry of Agriculture) (MAFF, bovinetb.info/docs/krebs.pdf Schroeder P et al (2020) Temporal and spatial Mycobacterium bovis prevalence patterns as evidenced in the All Wales Badgers Found Dead (AWBFD) survey of infection 2014–2016. Sci Rep 10:15214. 10.1038/s41598-020-72297-9 Milne G et al (2020) Mycobacterium bovis Population Structure in Cattle and Local Badgers: Co-Localisation and Variation by Farm Type. Pathogens 9. 10.3390/pathogens9070592 Crispell J et al (2019) Combining genomics and epidemiology to analyse bi-directional transmission of Mycobacterium bovis in a multi-host system. Elife 8. 10.7554/eLife.45833 Rossi G et al (2020) Identifying likely transmissions in Mycobacterium bovis infected populations of cattle and badgers using the Kolmogorov Forward Equations. Sci Rep 10:21980. 10.1038/s41598-020-78900-3 Akhmetova A et al (2023) Genomic epidemiology of Mycobacterium bovis infection in sympatric badger and cattle populations in Northern Ireland. Microb Genom 9. 10.1099/mgen.0.001023 APHA (2024) (ed Food and Rural Affairs Department for Environment) (Defra, https://www.gov.uk/government/statistics/incidence-of-tuberculosis-tb-in-cattle-in-great-britain/quarterly-tb-in-cattle-in-great-britain-statistics-notice-december-2023 N.E (2023) (ed Natural England) ( https://www.gov.uk/government/publications/bovine-tb-summary-of-badger-control-monitoring-during-2022/summary-of-2022-badger-control-operations#natural-england-chief-scientists-advice-on-the-outcome-of-badger-control-operations-2022 N.E (2023) (ed Natural England) ( https://www.gov.uk/government/publications/bovine-tb-summary-of-supplementary-badger-control-monitoring-during-2022/summary-of-2022-supplementary-badger-control-operations N.E (2024) (ed Natural England) ( https://www.gov.uk/government/publications/bovine-tb-summary-of-badger-control-monitoring-during-2023/summary-of-2023-badger-control-operations#:~:text=The%20results%20from%202023%20indicate,high%20standard%20of%20public%20safety N.E (2024) (ed Natural England) ( https://www.gov.uk/government/publications/bovine-tb-summary-of-supplementary-badger-control-monitoring-during-2023/summary-of-2023-supplementary-badger-control-operations Goodchild AV, Watkins GH, Sayers AR, Jones JR, Clifton-Hadley RS (2012) Geographical association between the genotype of bovine tuberculosis in found dead badgers and in cattle herds. Vet Rec 170:259. 10.1136/vr.100193 Byrne AW et al (2015) Spatial and temporal analyses of metrics of tuberculosis infection in badgers (Meles meles) from the Republic of Ireland: Trends in apparent prevalence. Prev Vet Med 122:345–354. 10.1016/j.prevetmed.2015.10.013 Barron S, Swift et al (2018) A study of tuberculosis in road traffic-killed badgers on the edge of the British bovine TB epidemic area. Sci Rep 8 Swift BMC et al (2021) Tuberculosis in badgers where the bovine tuberculosis epidemic is expanding in cattle in England. Sci Rep 11:20995. 10.1038/s41598-021-00473-6 Davies M, Roper J, T. J., Sheperdson DJ (1987) Seasonal distribution of road kills in the European badger (M eles meles) . J Zool 211:525–529 Anderson RM, Trewhella W (1985) Population dynamics of the badger (Meles meles) and the epidemiology of bovine tuberculosis (Mycobacterium bovis). Philos Trans R Soc Lond B Biol Sci 310:327–381. 10.1098/rstb.1985.0123 Macdonald DW, Newman C (2002) Population dynamics of badgers (Meles meles) in Oxfordshire, U.K.: numbers, density and cohort life histories, and a possible role of climate change in population growth. Journal of Zoology 256, 121–138, 10.1017/S0952836902000158 Rogers LM, Cheeseman CL, Mallinson PJ, Clifton-Hadley R (1997) The demography of a high-density badger (Meles meles) population in the west of England. J Zool 242:705–728. https://doi.org/10.1111/j.1469-7998.1997.tb05821.x Graham J et al (2013) Multi-state modelling reveals sex-dependent transmission, progression and severity of tuberculosis in wild badgers. Epidemiol Infect 141:1429–1436. 10.1017/S0950268812003019 McDonald JL, Smith GC, McDonald RA, Delahay RJ, Hodgson D (2014) Mortality trajectory analysis reveals the drivers of sex-specific epidemiology in natural wildlife-disease interactions. Proc Biol Sci 281. 10.1098/rspb.2014.0526 Harris S, Cresswell WJ, Cheeseman CL (1992) Age determination of badgers (Meles meles) from tooth wear: the need for a pragmatic approach. J Zool 228:679–684. https://doi.org/10.1111/j.1469-7998.1992.tb04467.x Macdonald DW, Newman C, Nouvellet PM, Buesching CD (2009) An Analysis of Eurasian Badger (Meles meles) Population Dynamics: Implications for Regulatory Mechanisms. J Mammal 90:1392–1403. 10.1644/08-mamm-a-356r1.1 Delahay RJ, Langton S, Smith GC, Clifton-Hadley RS, Cheeseman CL (2000) The spatio-temporal distribution of Mycobacterium bovis (bovine tuberculosis) infection in a high-density badger population. J Anim Ecol 69:428–441. https://doi.org/10.1046/j.1365-2656.2000.00406.x Gallagher J, Clifton-Hadley RS (2000) Tuberculosis in badgers; a review of the disease and its significance for other animals. Res Vet Sci 69:203–217. 10.1053/rvsc.2000.0422 Newman C, Buesching CD, Macdonald DW (2017) in Biology and Conservation of Musteloids (eds David W. Macdonald, Chris Newman, & Lauren A. Harrington) 0 Kendon M et al (2022) State of the UK Climate 2021. Int J Climatol 42:1–80. https://doi.org/10.1002/joc.7787 Kendon M et al (2023) State of the UK Climate 2022. Int J Climatol 43:1–83. https://doi.org/10.1002/joc.8167 Kruuk H, Parish T (1981) Feeding Specialization of the European Badger Meles meles in Scotland. J Anim Ecol 50:773–788. 10.2307/4136 Kruuk H (1978) Foraging and Spatial Organisation of the European Badger. Meles meles Behav Ecol Sociobiol 4:75–89 Nguyen M-VH et al (2019) Factors Associated With Sputum Culture-Negative vs Culture-Positive Diagnosis of Pulmonary Tuberculosis. JAMA Netw Open 2:e187617–e187617. 10.1001/jamanetworkopen.2018.7617 Achkar JM, Jenny-Avital ER (2011) Incipient and subclinical tuberculosis: defining early disease states in the context of host immune response. J Infect Dis 204(Suppl 4):1179–1186. 10.1093/infdis/jir451 Salina EG et al (2019) Resuscitation of Dormant Non-culturable Mycobacterium tuberculosis Is Characterized by Immediate Transcriptional Burst. Front Cell Infect Microbiol 9:272. 10.3389/fcimb.2019.00272 Mukamolova GV, Turapov O, Malkin J, Woltmann G, Barer MR (2010) Resuscitation-promoting factors reveal an occult population of tubercle Bacilli in Sputum. Am J Respir Crit Care Med 181:174–180. 10.1164/rccm.200905-0661OC Bachmann NL et al (2020) Key Transitions in the Evolution of Rapid and Slow Growing Mycobacteria Identified by Comparative Genomics. Front Microbiol 10. 10.3389/fmicb.2019.03019 Mohammed Adam MA, Ebraheem RSM, Bedri SA (2022) Statistical Investigation of High Culture Contamination Rates in Mycobacteriology Laboratory. Front Microbiol 13:789725. 10.3389/fmicb.2022.789725 APHA (2023) (ed Animal and Plant Health Agency) ( https://www.gov.uk/government/publications/bovine-tb-epidemiology-and-surveillance-in-great-britain-2022 Balseiro A et al (2011) Infection of Eurasian badgers (Meles meles) with Mycobacterium avium complex (MAC) bacteria. Vet J 188:231–233. 10.1016/j.tvjl.2010.05.003 Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5193016","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":361512459,"identity":"8f05a65d-21a4-4b96-9783-ff685fa87021","order_by":0,"name":"Sian Powell","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYDACZhiDnbnxAQPDARg3gQgtzIzNBsRpQehlbJMgSot8O/OzB4w7bBL7gVoqfu64I8/Av/iYBGNbGk4tBofZzA0Yz6QlzmxmbLvZe+aZYYPEszSglhzcWpgZzIAKDuduOMzYdoO37TBjg8QZYwOgjbgd1sz+DaxlP1BL4d+2w/YEtTAc5oHaAvQLM9CWxAb+HsMHeB12mKdMIvFMWv2Mw4zN0rJnDie3SbAlPkg4h9v78v3Ht0l83GFjzN/efPDj2x2Hbfv5Dx848KEsGbfDQCCxAcpgBDLYJBKIiEhGZC0M/AcIqR8Fo2AUjIIRBgCxs1ZhPX5KQQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-2940-5007","institution":"Animal and Plant Health Agency","correspondingAuthor":true,"prefix":"","firstName":"Sian","middleName":"","lastName":"Powell","suffix":""},{"id":361516428,"identity":"3996f3d4-b9a0-49de-b276-a6b03c4435b0","order_by":1,"name":"Nicola Dessi","email":"","orcid":"","institution":"Animal and Plant Health Agency","correspondingAuthor":false,"prefix":"","firstName":"Nicola","middleName":"","lastName":"Dessi","suffix":""},{"id":361516429,"identity":"8553ebcc-7665-4742-ac8f-2ead305df4ab","order_by":2,"name":"Malcolm Bennett","email":"","orcid":"https://orcid.org/0000-0002-5490-2910","institution":"University of Nottingham","correspondingAuthor":false,"prefix":"","firstName":"Malcolm","middleName":"","lastName":"Bennett","suffix":""},{"id":361516430,"identity":"44382c0c-d1d4-4bb4-b667-e16ee89261f0","order_by":3,"name":"Belinda Wang","email":"","orcid":"https://orcid.org/0000-0002-6963-4963","institution":"University of Nottingham","correspondingAuthor":false,"prefix":"","firstName":"Belinda","middleName":"","lastName":"Wang","suffix":""},{"id":361516431,"identity":"a3846ef7-22a3-488c-a99f-3e4f6c7a7d25","order_by":4,"name":"Andrew Robertson","email":"","orcid":"https://orcid.org/0000-0003-3720-6670","institution":"Department for Environment Food and Rural Affairs","correspondingAuthor":false,"prefix":"","firstName":"Andrew","middleName":"","lastName":"Robertson","suffix":""},{"id":361516432,"identity":"05d7fa7f-c69c-495c-9c35-da5714314d53","order_by":5,"name":"Elisabeth Waller","email":"","orcid":"https://orcid.org/0000-0003-1841-5688","institution":"Animal and Plant Health Agency","correspondingAuthor":false,"prefix":"","firstName":"Elisabeth","middleName":"","lastName":"Waller","suffix":""},{"id":361516433,"identity":"59893ebb-381a-4b80-a612-30a0ecbe11c7","order_by":6,"name":"Graham Smith","email":"","orcid":"","institution":"Animal and Plant Health Agency","correspondingAuthor":false,"prefix":"","firstName":"Graham","middleName":"","lastName":"Smith","suffix":""},{"id":361516434,"identity":"ed8d619d-a91c-4c49-baec-658e6b0a36d8","order_by":7,"name":"Richard Delahay","email":"","orcid":"https://orcid.org/0000-0001-5863-0820","institution":"Animal and Plant Health Agency","correspondingAuthor":false,"prefix":"","firstName":"Richard","middleName":"","lastName":"Delahay","suffix":""}],"badges":[],"createdAt":"2024-10-02 12:56:30","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5193016/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5193016/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":65902012,"identity":"e2bcd440-f0dc-412c-957a-daa34443d0a0","added_by":"auto","created_at":"2024-10-04 07:41:19","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":196482,"visible":true,"origin":"","legend":"\u003cp\u003eMap showing the counties of England coloured by perceived bTB risk. The insert shows the counties of the southern Edge Area which were the focus of the present study.\u003c/p\u003e","description":"","filename":"fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5193016/v1/a3cab72b523e723ba6832a5b.jpg"},{"id":65902015,"identity":"3110e25b-ebb7-471f-88c6-e944117c8995","added_by":"auto","created_at":"2024-10-04 07:41:19","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":61069,"visible":true,"origin":"","legend":"\u003cp\u003eThe mean number of badger carcasses collected per month by sex (a) and the mean percentrage of carcasses testing positive for \u003cem\u003eM. bovis\u003c/em\u003e by month and sex (b). Error bars show the standard error around the mean.\u0026nbsp;\u003c/p\u003e","description":"","filename":"fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5193016/v1/22a763b186edc8c5f959dec4.jpg"},{"id":65902156,"identity":"0753a7cf-8bdc-46b1-bb4e-69419f158720","added_by":"auto","created_at":"2024-10-04 07:49:19","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":310570,"visible":true,"origin":"","legend":"\u003cp\u003eThe spatial distribution of the sampled badger carcasses, and herd level TB test results during the study period (1st April 2021 - 30th April 2023). Stars or black crosses indicate badger carcass test results; filled circles or red crosses indicate cattle herds where Official bTB Free status was withdrawn (OTF-W) due to a TB incident (breakdown) with at least one lesion and/or \u003cem\u003eM. bovis\u003c/em\u003e culture-positive animal removed, while unfilled circles indicate suspended herd status (OTF-S).\u0026nbsp;\u003c/p\u003e","description":"","filename":"fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5193016/v1/60eae178ebc1d53f45021d15.jpg"},{"id":65902013,"identity":"897ea192-2976-409f-bb25-23937382330a","added_by":"auto","created_at":"2024-10-04 07:41:19","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":36449,"visible":true,"origin":"","legend":"\u003cp\u003eCounty-level comparison of the percentage of M. bovis infected badgers (detected by culture and WGS) and infected cattle herds (as determined by the comparative tuberculin skin test (SICCT)) in the Southern Edge Area Study (April 2021-April 2023). Repeat breakdowns are excluded from these summary data.\u003c/p\u003e","description":"","filename":"fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5193016/v1/5d2114d7ce616aee951193a9.jpg"},{"id":65902918,"identity":"7945205b-f484-40b9-be3c-da973e062bdc","added_by":"auto","created_at":"2024-10-04 07:57:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1478899,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5193016/v1/276e4100-5c73-4f47-8d3c-2df86b1ce3bd.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eTuberculosis in found dead badgers at the edge of the expanding bovine tuberculosis epidemic.\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eMycobacterium bovis\u003c/em\u003e (\u003cem\u003eM. bovis\u003c/em\u003e) is a member of the \u003cem\u003eM. tuberculosis\u003c/em\u003e complex (MTC), a group of host-associated, tuberculous disease-causing bacteria of high genetic similarity \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Despite this high level of clonality, the group varies significantly in host tropism with \u003cem\u003eM. bovis\u003c/em\u003e having the widest known host range \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Indeed, in the United Kingdom (UK) alone \u003cem\u003eM. bovis\u003c/em\u003e has been detected in nine species of farmed or companion mammals, and at least 20 species of wild mammals \u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6 CR7 CR8\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In cattle, \u003cem\u003eM. bovis\u003c/em\u003e causes bovine tuberculosis (bTB), one of the most significant challenges affecting cattle health in England, which is estimated to cost the UK taxpayer over \u0026pound;150\u0026nbsp;million per annum, with further costs to the cattle industry \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In addition to the financial burden is the cost of livestock life, with over 31,000 cattle slaughtered in 2023, of which 20,200 were from England \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eEnglish counties are categorised by their historic prevalence and epidemiology of bTB infection in cattle (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the high-risk area (HRA), which encompasses the southwest of the country, the disease is endemic in cattle and extensive disease surveillance is in place, including standard six-monthly herd testing and pre-movement testing. Surrounding the HRA is the edge area (EA), where bTB prevalence has been historically lower than the HRA but into which the epidemic is expanding, resulting in highly variable disease prevalence and either six-monthly or annual cattle testing, in addition to pre-movement testing. Though not contiguous with the rest of the EA, East Sussex is included within this zone due to the prevalence of bTB in the county. Finally, in the low-risk area (LRA) the incidence of bTB in cattle has been historically low and stable, with most cattle herds routinely tested for TB every four years, supplemented by targeted enhanced testing of certain herds and mandatory post-movement testing of cattle introduced from the rest of England and Wales \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhile cattle movements undoubtedly make a substantial contribution to the spread and persistence of bTB in herds across the three risk areas, the presence of infection in the primary wildlife reservoir, the European badger (\u003cem\u003eMeles meles\u003c/em\u003e) adds further complexity to disease control \u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Infectious badgers can excrete \u003cem\u003eM. bovis\u003c/em\u003e in their saliva, urine, faeces or from ruptured abscesses, presenting a hazard to cattle through indirect transmission, and potentially via direct transmission in cattle housing \u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. During the Randomised Badger Culling Trial (RBCT, 1998\u0026ndash;2005), 15.9% of culled badgers tested positive by culture or by having lesions containing acid-fast organisms \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. There was a demonstrable association between the proportion of positive badgers and cattle TB incidents at a distance of 1\u0026ndash;2 km \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In addition, spoligotyping and multi-locus variable number tandem repeat analyses (MLVA) have demonstrated the co-localisation of related \u003cem\u003eM. bovis\u003c/em\u003e isolates from badgers and cattle \u003csup\u003e\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Whole genome sequencing (WGS) has further evidenced the link between infections in cattle and badgers, with highly related isolates being cultured from sympatric populations indicating recent and bi-directional transmission \u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In English areas of high cattle incidence, efforts to reduce transmission risks from wildlife have included licenced culls of badgers, with over 53,000 animals culled during the years 2022 to 2023 \u003csup\u003e20,22,28\u0026ndash;32\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFor the purposes of surveillance, found-dead surveys are the most viable approach for collecting a sufficiently large sample of badgers (typically the results of road traffic collisions) for investigating infection prevalence at a relatively broad geographic scale (e.g., county-level). This approach was extensively validated alongside the RBCT and found to be a reliable proxy for \u003cem\u003eM. bovis\u003c/em\u003e infection prevalence within the sampled population \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Since then, it has been employed across Wales \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, large areas of the Republic of Ireland and Northern Ireland \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e and in the county of Cheshire to investigate an emerging cluster of TB in cattle \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Also, of particular relevance to the present study, was an investigation of bTB in found dead badgers in the northern EA \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. This study took place in 2016\u0026ndash;2017 and identified a headline MTC infection rate in badgers of 8.3% (range: 4\u0026ndash;15%), and common areas of high-prevalence in cattle and badgers as also reported in other studies \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eEffective control of bTB in cattle at the edge of the endemic area requires a better understanding of the potential role of badgers in the spread and perpetuation of infection in this transitional zone. Here we present the results of an investigation into the prevalence of \u003cem\u003eM. bovis\u003c/em\u003e in found-dead badgers in the southern EA, undertaken between 1st of April 2021 and the 30th of April 2023. Our results are interpreted alongside contemporaneous data on infection in cattle, gathered during routine surveillance and monitoring, and the results of the earlier study undertaken in the northern EA \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBadger carcasses\u003c/h2\u003e \u003cp\u003eIn total, 525 badger carcasses were collected from across the five counties of the southern EA, with 428 (81.5%) deemed suitable for examination and tissue sampling. RTAs accounted for 84.3% of submissions (n\u0026thinsp;=\u0026thinsp;443), sixteen (3.0%) were found dead elsewhere and were typically emaciated and in poor condition, whilst the causes of death could not be determined in the remainder, usually owing to the degree of carcass decomposition. The number of carcasses submitted varied by county (χ\u0026sup2; = 46.0, df\u0026thinsp;=\u0026thinsp;4, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), with Oxfordshire yielding the highest number (n\u0026thinsp;=\u0026thinsp;138) and Berkshire providing the fewest (n\u0026thinsp;=\u0026thinsp;49) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e The majority of carcasses were collected by farmers and farming groups (n\u0026thinsp;=\u0026thinsp;231, 44.0%), followed by conservation groups (n\u0026thinsp;=\u0026thinsp;132, 25.1%), veterinarians and their affiliates (n\u0026thinsp;=\u0026thinsp;85, 16.2%), government agencies or local authorities (n\u0026thinsp;=\u0026thinsp;18, 3.4%), and miscellaneous other groups or individuals (n\u0026thinsp;=\u0026thinsp;59, 11.2%). Of the carcasses submitted for which the sex was determined (n\u0026thinsp;=\u0026thinsp;443), 226 (51.0%) were males and 217 (49.0%) were females. No temporal variation was observed in the number of carcasses submitted by month when the average number of submissions were examined. Comparisons between the submissions of male and female carcasses with reference to month had to account for variable sample sizes, the low number of repeat data and the unequal number of repeats. On average, there were fewer submissions of males across the calendar year, with the exception of December \u0026ndash; March (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, only in April were significantly more females submitted than males (t\u0026thinsp;=\u0026thinsp;9.5, df\u0026thinsp;=\u0026thinsp;2, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01). The majority of carcasses were of adult badgers (estimated to be over 1 year old), with only 52/525 (9.9%) of those collected and 43/444 (9.7%) of those examined recorded as cubs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eM. bovis\u003c/b\u003e \u003cb\u003einfection in surveyed badgers\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAcross the southern EA, 31/428 (7.2%, 95% CI: 5.0-9.7%) badger carcasses tested positive for MTC bacteria by culture, IS\u003cem\u003e6110\u003c/em\u003e PCR and \u003cem\u003ehsp65\u003c/em\u003e sequencing. Of these, 28 were confirmed as \u003cem\u003eM. bovis\u003c/em\u003e by WGS giving an apparent prevalence of 6.5% (95% CI: 4.4\u0026ndash;8.9%). Despite the low number of positive badgers, and the aforementioned study design, efforts were made to examine factors that increased the likelihood of positive carcasses being detected. The prevalence of infected badgers varied significantly by county (χ\u0026sup2; = 12.7, df\u0026thinsp;=\u0026thinsp;4, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01), ranging from 1.1% in Hampshire to 13.0% in Oxfordshire (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Additionally, the detection of positive carcasses varied with the time of year, being most likely in March (OR (95% CI)\u0026thinsp;=\u0026thinsp;10.58 (1.794\u0026ndash;20.14), \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and least likely in October (not significant) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAll of the confirmed positives were adults (28/384 confirmed adults, 7.3%, 95% CI: 4.9\u0026ndash;10.4%) and the prevalence of infection was more than 3 times higher in male badgers than in females (χ\u0026sup2; = 8.3, df\u0026thinsp;=\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), with 22/215 male badgers testing positive (10.2%, 95% CI: 6.5\u0026ndash;15.1%) and 6/210 females (2.9%, 95% CI: 1.1\u0026ndash;6.1%). The distances between any given positive badger and its nearest positive neighbour were significantly smaller than those between any negative badger and their nearest positive neighbour (W\u0026thinsp;=\u0026thinsp;4193, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The median distance from a positive badger to the nearest other positive badger was 6.1 km (interquartile range, IQR: 2.4\u0026ndash;11.6 km), whilst negative badgers were on average 8.4 km (IQR: 5.1\u0026ndash;16.5 km) from their nearest positive neighbour.\u003c/p\u003e \u003cp\u003eSix \u003cem\u003eM. bovis\u003c/em\u003e clades were identified amongst the isolates obtained from confirmed positive badger carcasses. Of these, B6-62 was the most common, representing 21/28 (75.0%) of \u003cem\u003eM. bovis\u003c/em\u003e confirmed isolates. The remaining clades (B6-61, B6-71, B6-85, B6-91, and B1-11) were each detected only once while two isolates (7.1%) were not identified to clade level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The distance to the nearest badger with the same strain (where available) had a mean of 7.9 km (95% CI: 5.4\u0026ndash;10.7 km) while the distance to the nearest badger with a different strain was 17.4 km (95% CI: 12.7\u0026ndash;22.0 km). Statistical analysis identified a significant difference between the two distributions (t = -4.57, df\u0026thinsp;=\u0026thinsp;20, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eM. bovis\u003c/b\u003e \u003cb\u003ein cattle\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDuring the study period, there was an average of 2639 (range: 2606\u0026ndash;2711) active herds in the southern EA per annum. Of these, 277 herds (10.5%) experienced at least one TB breakdown (i.e. detection of at least one TB test positive animal and/or laboratory-positive case at routine slaughter). During the study period, a total of 308 breakdowns occurred in the study area (including 31 repeat breakdowns) of which 132 (56.5%) were OTF-W, 174 (42.9%) were OTF-S and the remainder were unclassified. Of the breakdowns involving at least one animal with visible tuberculous lesions or positive culture (OTF-W), WGS clades were identified in 84.1% (n\u0026thinsp;=\u0026thinsp;111) of incidents, meaning that amongst all incidents of OTF status being suspended or withdrawn a clade was identified in 36.0% of cases.\u003c/p\u003e \u003cp\u003eThe proportion of herds with reactor cattle during the period of study varied by county (χ\u0026sup2; = 13.7, df\u0026thinsp;=\u0026thinsp;4, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01); Oxfordshire herds were the most likely to have a TB test reactor detected with 20.9% of herds experiencing a breakdown during the study, compared to only 5.0% of herds in Hampshire (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Of those herds where a clade associated reactor was identified (n\u0026thinsp;=\u0026thinsp;111), the most common \u003cem\u003eM. bovis\u003c/em\u003e clade was B6-62 which was found in 83 incidents (74.8%), followed by B6-11 in twelve incidents (10.8%).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCounty level demography and TB status of badger carcasses submitted during the study period (1st April 2021\u0026ndash;30th April 2023), and data on the number of active herds per county and their breakdown status (1st April 2021-30th April 2023).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e \u003cp\u003eCounty\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOxfordshire\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEast Sussex\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBuckinghamshire\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHampshire\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBerkshire\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"8\" rowspan=\"9\"\u003e \u003cp\u003eBadgers\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCarcasses submitted\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e138\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e107\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCarcasses sampled\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e108\u003c/p\u003e \u003cp\u003e(78.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e103 (80.4%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e85\u003c/p\u003e \u003cp\u003e(85.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e88\u003c/p\u003e \u003cp\u003e(82.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e44\u003c/p\u003e \u003cp\u003e(89.8%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCarcass density (per km\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0415\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0575\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0454\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0239\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0349\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMale (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e48.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e55.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e57.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFemale (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e54.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e48.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e51.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e44.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e42.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAdults (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e88.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e97.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e94.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e95.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJuveniles (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eM. bovis\u003c/em\u003e positive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14 (13.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 (4.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4 (4.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1 (1.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4 (9.1%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eM. bovis positive\u003c/em\u003e (per km\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.00537\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.00279\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.00213\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.00027\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.00317\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean active herds (range)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e526\u003c/p\u003e \u003cp\u003e(509\u0026ndash;545)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e590\u003c/p\u003e \u003cp\u003e(584\u0026ndash;603)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e475\u003c/p\u003e \u003cp\u003e(467\u0026ndash;494)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e837\u003c/p\u003e \u003cp\u003e(820\u0026ndash;857)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e211\u003c/p\u003e \u003cp\u003e(208\u0026ndash;215)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eCattle\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean active herd density (per km\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.202\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.329\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.253\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.228\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.167\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber of herds with breakdown\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePercentage of herds with breakdown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDensity of breakdowns (per km\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0422\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0229\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0283\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0114\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.0246\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e \u003c/sup\u003eNote that unsampled carcasses were excluded from the analyses. \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003eOne badger was excluded from these analyses as the spatial data indicated it was located significantly beyond the study boundary. \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003eData is given at the herd level and analyses conducted using the mean number of active herds during the study period. \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003eEstimated as the number of herds that were non-OTF during the study period; repeat breakdowns are not included.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eComparison between badgers and cattle\u003c/h3\u003e\n\u003cp\u003eA statistically significant, positive relationship was identified between the percentage of positive badger carcasses and the herd level prevalence (defined as the percentage of herds that experienced at least one breakdown during the study period) at the county level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Pearson\u0026rsquo;s r(3)\u0026thinsp;=\u0026thinsp;0.99 (95% CI: 0.82-1.0), t(3)\u0026thinsp;=\u0026thinsp;10.9, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe distances between badger carcasses and cattle infected with the same and different clades were analysed. The two East Sussex B6-62 infected badgers were removed from the analyses as outliers, due to the non-detection of the clade in local herds and because this county was not contiguous with any other EA county. Overall, the distance from a positive badger to a herd with the same clade was 3.6 km (IQR: 2.9\u0026ndash;5.6 km) while the mean distance to the nearest herd infected with a different clade was 9.9 km (95% CI: 7.6\u0026ndash;12.2 km). Badgers were significantly more likely to be found near herds infected with the same clade, than to be found near herds infected with different clades (V\u0026thinsp;=\u0026thinsp;41, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\n\u003ch3\u003eNon-tuberculous mycobacteria (NTM)\u003c/h3\u003e\n\u003cp\u003eFifteen putative species of non-tuberculous mycobacteria (NTM) were detected in 30 badgers (apparent prevalence 7.0%, 95% CI: 5.1\u0026ndash;9.4%), including one unsexed badger. \u003cem\u003eM. avium\u003c/em\u003e was the most prevalent NTM with an apparent prevalence of 2.6% (95% CI: 1.3\u0026ndash;4.5%, 11/429), followed by \u003cem\u003eM. vaccae\u003c/em\u003e with an apparent prevalence of 1.2% (95% CI: 0.4\u0026ndash;2.7%, 5/429); some isolates were not fully identified by this method. More males tested positive for \u003cem\u003eM. bovis\u003c/em\u003e (22/215, 10.2%) than for NTM (16/215, 7.4%) and more females tested positive for NTM (13/210, 6.2%) than for \u003cem\u003eM. bovis\u003c/em\u003e (6/210, 2.9%), although neither difference was significant.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eM. bovis\u003c/em\u003e infection in badgers collected from the southern EA was spatially heterogeneous, with the county level apparent prevalence ranging from 1.1% in Hampshire to 13.0% in Oxfordshire. This concurs with findings from a similar study of found dead badgers in the northern EA where county-level prevalence ranged from 4\u0026ndash;5\u0026ndash;15% \u003csup\u003e36\u003c/sup\u003e. Our study wide prevalence estimate of 6.5% (95% CI: 4.4\u0026ndash;8.9%) is also similar to that of studies in the northern EA (8.3%, 95% CI: 6.4\u0026ndash;11.0%) and Wales (7.3%, 95% CI: 5.6\u0026ndash;9.5%) \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. However, direct comparisons are problematic owing to minor methodological differences, including that the current study used WGS to confirm \u003cem\u003eM. bovis\u003c/em\u003e isolation whilst Swift \u003cem\u003eet al.\u003c/em\u003e (2021) used IS\u003cem\u003e6110\u003c/em\u003e confirmation to the MTC level, and Schroeder \u003cem\u003eet al\u003c/em\u003e. (2020) used spoligotyping. The use of WGS in the present study excluded three samples that were identified as MTC members by \u003cem\u003ehsp65\u003c/em\u003e sequencing (putative \u003cem\u003eM. bovis\u003c/em\u003e due to the host species), reducing the overall apparent prevalence from 7.2%, detected using \u003cem\u003eIS6110\u003c/em\u003e and \u003cem\u003ehsp65\u003c/em\u003e, to the reported 6.5%.\u003c/p\u003e \u003cp\u003eThe prevalence of \u003cem\u003eM. bovis\u003c/em\u003e in badgers was significantly and positively correlated with the number of incident cases observed in cattle at the county level. Spatial correlation between infection in badgers and cattle has previously been observed in the northern Edge Area, as it has also been in the wider endemic areas of England and Wales \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe ratio of male to female badgers in the present study was circa 1:1, similar to that observed in other found-dead studies \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e and as identified in capture-mark-recapture studies of badger populations \u003csup\u003e\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. The prevalence of confirmed \u003cem\u003eM. bovis\u003c/em\u003e infection in male badgers in the present study was higher than in females, consistent with previous work showing that males are more likely to become infected and experience more rapid disease progression, thus increasing the likelihood of yielding a positive culture at PME \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. It is also hypothesised that immunological differences may enhance male susceptibility to disease \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe present study involved an atypically low percentage of sampled juvenile badgers (9.9%) compared to other found dead surveys (c. 30% \u003csup\u003e35,36\u003c/sup\u003e and population studies (c. 20\u0026ndash;30%) \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. As TB infection is more often observed in adult badgers than in juveniles, the high proportion of adults in the present study is likely to have inflated overall population-level prevalence \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. The reasons for the low percentage of juveniles in our sample of carcasses is not clear, but could potentially reflect poor survival related to unfavourable conditions \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. For example, April 2021 and 2022 and the summer of 2021 were unusually dry \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e which is likely to have reduced food availability \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e and impacted on cub survival in particular \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRoutine WGS of \u003cem\u003eM. bovis\u003c/em\u003e requires the culture of bacterial isolates from infected individuals. However, the success of culture can be limited by early-stage infection \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, the metabolic state of the bacteria \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, the slow growth rate of \u003cem\u003eM. bovis\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e and the potential for overgrowth by competing microorganisms \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e; it is therefore highly probable that the apparent prevalence given here is an underestimation of the true prevalence. Furthermore, the current routine approach of sequencing a single isolate per badger or entire cattle herd means that if multiple infections with different strains or clades were present then they would not be detected. Despite these limitations, six clades were cultured from badger carcasses during the present study, of which B6-62 was the most common, being identified in 4/5 counties. Two badgers collected from East Sussex were infected with B6-62, outside the putative home range of the clade, and this clade was undetected in the county\u0026rsquo;s cattle \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Due to our limited understanding of bTB in East Sussex, with a low level of isolate recovery from SICCT positive cattle, it is difficult to interpret these results. The probability that these two samples were similarly mislabelled seems low given that both the examination of carcasses and subsequent sequencing of isolates were carried out several weeks apart, and hence these results merit further investigation. Two further clades which were identified during the study (B1-11, B6-85) were also isolated from badgers in areas which were well beyond their typical home range, and one nationally rare isolate with no designated home range was detected in Oxfordshire (B6-61) \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. While B6-85 was identified in a badger found in close proximity to cattle herd breakdowns associated with that clade, the same was not true for B1-11 and B6-61. The identification of \u003cem\u003eM. bovis\u003c/em\u003e clades in badgers that are distinct from those detected over the same period in cattle could arise as a result of historic spillover from cattle into badgers, or the limited sensitivity of \u003cem\u003eM. bovis\u003c/em\u003e surveillance in cattle, as only 36% of breakdowns yielded sequenceable isolates across the study area. Previous evidence has demonstrated the contemporaneous circulation of different strains within badgers and cattle, showing the value in surveying both species to entirely evaluate the disease landscape \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe apparent prevalence of NTM in this study was 7.0%, far greater than a previously reported value of 0.9% (424/45,705) from archived UK cull data (recorded as \u003cem\u003eM. avium\u003c/em\u003e and other mycobacteria), but similar to that reported in Spain from a small opportunistic study \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Unlike \u003cem\u003eM. bovis\u003c/em\u003e, NTMs were distributed relatively evenly between the sexes, likely related to the environmental nature of these bacteria contributing to even exposure risks. Also, in contrast to \u003cem\u003eM. bovis\u003c/em\u003e, the likelihood of detectable NTM infection did not increase with age, though the small number of juveniles in this study likely limited our ability to detect any such effects.\u003c/p\u003e \u003cp\u003eEstimates of prevalence from studies such as ours should be treated with caution as biases can arise as a result of the relatively small sample sizes, the unknown absolute population size, local clustering of infection, potential behavioural correlates of susceptibility to becoming a RTA, and the limitations of the diagnostic methods employed. However, the sampling approach and culture-based methodology in the present study are broadly consistent with those shared by several other badger RTA surveys, allowing for some level of comparison whilst recognising the known limitations \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Furthermore, the collection of found dead badgers provides a geographically broad estimate of prevalence, which would otherwise be unachievable, and the collection of epidemiologically valuable WGS data to assist in disease tracking.\u003c/p\u003e \u003cp\u003eTo conclude, the present study has provided the first estimates of \u003cem\u003eM. bovis\u003c/em\u003e prevalence in badger populations in the southern EA. In addition to identifying county-level heterogeneity, the use of WGS has added significant value to the surveillance data available for cattle by uncovering additional clades in badgers that were not detected in local herds and has confirmed infection in wildlife where it was previously unstudied. This study has provided further evidence for the link between infection in badgers and cattle at the edge of the area of TB endemism in England. Further investigation of WGS data from this area may shed light on the relative importance of cattle or badgers in driving herd breakdowns and whether (as seems likely) cattle movements may have seeded infection in local badgers which in places may have subsequently created a self-sustaining reservoir of infection and facilitated spill-back to cattle. Understanding the proximal drivers of infection in cattle across the EA may inform management options which could include enhanced cattle measures, badger vaccination and targeted badger culling. In some parts of the EA it may not be too late to prevent infection spilling over into this wildlife host before it becomes endemic in both populations.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCarcass collection and storage\u003c/h2\u003e \u003cp\u003eA network of collectors was established across the five counties of the southern Edge Area with the aim of collecting 100 carcasses from each county. A dedicated phoneline and e-mail address were used for communications, and the study was promoted at events, through veterinarians, and on the TB Hub website. Interested parties were provided with instructions and kits for the safe collection of found-dead badgers as well as a small financial reward. Collectors placed carcasses in three double thickness PVC bags and sealed them with pre-labelled tags. Useful metadata was recorded on the submission form by the collector, including the date and time, tag identification code, location, type of submission (e.g., road traffic accident) and any overnight storage conditions if the courier was unable to collect the same day. Carcasses were not frozen, but when storage was required, collectors were asked to place in a chiller if available, or else in a cool and dry area. If the courier was unable to transport the carcass to the post-mortem examination (PME) lab (University of Nottingham, Sutton Bonington Campus) the same day, then it would be stored in a chiller overnight or over the weekend until it could be safely received. The median time between carcass collection and PME was 3 days (range: 0\u0026ndash;12). Carcass collection took place from April 2021 to April 2023 inclusive, with collections ceasing at the county level once 100 carcasses had been sampled.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePost-mortem examination\u003c/h3\u003e\n\u003cp\u003eCarcasses were deemed suitable for PME if they had not suffered significant damage, were not autolysed, and arrived with the form linking them to a location. The sex, weight, length, age, (adult or juvenile), estimated from dentition, size, and pelage, condition as inferred from weight and body fat, and probable cause of death were recorded. In some cases, these data are not recorded due to the condition of the carcass, but sampling was possible, thus test data exists that cannot be related to certain population demographics. The tissue sampling protocol for isolation of mycobacteria followed that of the Northern Edge Study \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, based on the protocols used by \u003csup\u003e33\u003c/sup\u003e and \u003csup\u003e34\u003c/sup\u003e, modified by \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Carcasses were examined externally and internally for TB-like lesions, and if present these were harvested for tissue processing and bacterial culture. Selected lymph nodes and lung tissue were harvested and pooled (see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Harvested tissues were stored at 4\u0026deg;C for up to 48 h before processing for bacterial culture.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTissue pools harvested from badger carcasses for microbiological culture.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTissue pool\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTissues\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLesions\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIndividual samples from each gross lesion\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHead and neck\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRight and left mandibular, parotid, and retropharyngeal lymph nodes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThorax\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAnterior and posterior mesenteric and left and right bronchial lymph nodes, and apical lung\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbdomen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHepatic and mesenteric lymph nodes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCarcass pool\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRight and left prescapular, axillary, inguinal, and popliteal lymph nodes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThoracic blood/fluid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFrozen for serology\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eTissue Processing\u003c/h3\u003e\n\u003cp\u003eTissue processing and microbiological examination was performed in the Containment Level 3 (CL3) facility at the University of Nottingham, according to the methods used in previous studies \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Briefly, lesioned tissue or pools of target tissues were gently ground with sterile sand in phosphate buffered saline (PBS). Samples were mixed 1:1 with 5% oxalic acid and incubated at room temperature for 10 min, before 200 \u0026micro;L of each pool was inoculated onto Stonebrink Selective agar\u0026thinsp;+\u0026thinsp;PACT (BD Diagnostic) and onto Middlebrook 7H11 slopes supplemented by PANTA (BD Diagnostics). Media were incubated at 37\u0026deg;C for a minimum of 12 weeks with checking at regular intervals for putative mycobacterial colonies.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCharacterisation of mycobacteria\u003c/h2\u003e \u003cp\u003eAfter a minimum of 12 weeks incubation, putative mycobacterial colonies were heat killed at 80\u0026deg;C for 30 min. DNA was extracted by crude lysis; heat killed colonies were frozen and subsequently heated to 95\u0026deg;C for 5 min in sterile distilled water (SDW), centrifuged at 13,000 x g for 3 min to remove cellular debris and the resultant supernatant aliquoted for molecular analyses.\u003c/p\u003e \u003cp\u003eIsolates were screened by PCR for IS\u003cem\u003e6110\u003c/em\u003e, an insertion sequence unique to the MTC, and \u003cem\u003ehsp65\u003c/em\u003e sequencing which also identified non-tuberculous mycobacteria (NTM). Confirmed members of the MTC were subjected to whole genome sequencing (WGS; performed at APHA laboratories, Weybridge). Sequences were compared to \u003cem\u003eM. bovis\u003c/em\u003e reference sequence AF2122 for final confirmation and assigned to one of 30 lineages known to be in circulation in Great Britain (pipeline: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/APHA-CSU/btb-seq\u003c/span\u003e\u003cspan address=\"https://github.com/APHA-CSU/btb-seq\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCattle data\u003c/h2\u003e \u003cp\u003eAPHA conducts routine surveillance and testing of cattle herds for bTB. In the HRA and EA cattle are tested annually or six-monthly, with increased frequency of testing following detection of infection or after high-risk cattle movements. Testing is routinely conducted in the UK using the single intradermal comparative cervical tuberculin test (SICCT) with subsequent removal of reactors (animals exhibiting a positive result). Herds that are on schedule with their testing routine and have no reactors are classed as \u0026lsquo;Officially bTB Free\u0026rsquo; (OTF). If at least one reactor (a bovine with one positive or two inconclusive results) was detected, then a TB incident (herd breakdown) is declared and the OTF status is suspended (OTF-S). At PME, if lesions that are typical of TB are identified in a reactor, or a positive bacterial culture or (as of March 2022) positive PCR test is obtained from a TB test reactor, or a non-reactor animal presenting with suspected tuberculous lesions at routine slaughter, then the OTF status of the affected herd is withdrawn (OTF-W). The APHA held dataset includes the breakdown identifier, herd identifier, case reference, herd location, breakdown status, the test result and WGS clade if determined. For the present study, the data for each herd was combined across the study period and any herd that had been OTF-S or OTF-W was classed as positive.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eAnalyses were conducted in R version 4.2.2, with graphs generated using the \u0026lsquo;ggplot2\u0026rsquo; and \u0026lsquo;patchwork\u0026rsquo; packages. The \u0026lsquo;sf\u0026rsquo; package was used to analyse spatial data and QGIS 3.8.1 to visualise it. Statistical comparisons of count, percentage and distance data were performed within this study; data were tested for normality using the Shapiro-Wilk test. Significance testing of count and distance data was performed using the following: students T-test for normal and non-percentage data otherwise a Chi-squared when sufficient data were available, else the Fisher\u0026rsquo;s Exact Test. Paired data was tested using a T-test for normal data or Wilcoxon Signed Rank Test when the data were not normal. Multiple comparisons were controlled using the Benjamini-Hochberg method to reduce the false discovery rate.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors S.M.P., N.D., A.R., M.B., B.W., E.W., G.C.S., R.J.D declare no competing interests.\u003c/p\u003e \u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eS.M.P. was the project manager during the final year of the project, collated and analysed the data, produced the map and figures, and wrote the main manuscript. N.D. was the project manager prior to S.M.P. and collated data, conducted preliminary analyses, and reviewed the manuscript. M.B. conducted PMEs. B.W. conducted tissue processing, bacterial culture, and isolate screening. E.W. conducted clade assignment. A.R. assisted with the preliminary analyses and reviewed the manuscript. G.C.S. was involved in project conception and reviewed the manuscript. R.J.D. led on the project conception and contributed to preparation of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis project was funded by DEFRA (project APHATBOR1093). We are extremely grateful to the collectors who both promoted the study and actively participated in badger carcass collection, without whom this project would not have been possible. We also thank the pathology and microbiology teams at the University of Nottingham, Sutton Bonington Campus, and the sequencing team at APHA Weybridge.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eFollowing publication badger test results and an approximate location for each carcass will be made available online. Cattle herd data including test outcomes and locations are widely available through other platforms.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBrosch R et al (2002) A new evolutionary scenario for the Mycobacterium tuberculosis complex. 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Vet J 188:231\u0026ndash;233. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.tvjl.2010.05.003\u003c/span\u003e\u003cspan address=\"10.1016/j.tvjl.2010.05.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"aa167b02-3a08-430c-9bea-e54d00e2ede9","identifier":"10.13039/501100000277","name":"Department for Environment, Food and Rural Affairs, UK Government","awardNumber":"APHATBOR1093","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Animal and Plant Health Agency","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Mycobacterium bovis, bovis, badgers, southern edge, RTA, disease surveillance, bTB","lastPublishedDoi":"10.21203/rs.3.rs-5193016/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5193016/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBovine tuberculosis (bTB) is a major disease of cattle in the UK, placing a significant economic burden on the taxpayer. The causative agent, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMycobacterium bovis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, has a wide host range, including the European badger (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMeles meles)\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. While badgers have been implicated in the transmission and maintenance of infection in cattle in areas of endemic disease, their role at the edge of the endemic area is poorly understood. Here we present data on the prevalence of infection in badgers collected along the southern edge of England’s bTB epidemic. Stakeholders across five counties (Oxfordshire, Berkshire, Buckinghamshire, Hampshire, and East Sussex) submitted found-dead badgers for post-mortem examination and testing by bacterial culture. The overall prevalence, as confirmed by whole genome sequencing, was 6.5% (28/428), ranging between 1.1% (1/88) in Hampshire and 13.0% (14/108) in Oxfordshire. The commonest \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM. bovis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e clade in badgers was B6-62, which was predominant in 4/5 counties. B6-62 was also the commonest clade found in cattle and was detected in all counties except East Sussex where, although absent from the cattle population, it was detected in local badgers. This study highlights the co-incidence of infection in badgers and cattle in parts of the southern edge area consistent with localised clustering of infection in both species.\u003c/strong\u003e\u003c/p\u003e","manuscriptTitle":"Tuberculosis in found dead badgers at the edge of the expanding bovine tuberculosis epidemic.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-04 07:41:14","doi":"10.21203/rs.3.rs-5193016/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a152617e-ae82-4865-82ca-6f84a2cfa986","owner":[],"postedDate":"October 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":38462129,"name":"Infectious Diseases"},{"id":38462130,"name":"Zoonoses"},{"id":38462131,"name":"Wildlife Biology"}],"tags":[],"updatedAt":"2024-10-04T07:41:15+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-04 07:41:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5193016","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5193016","identity":"rs-5193016","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}