Philippa Kaur

Animals and housing conditions
The present study was conducted with 24 male C57BL/6J mice, purchased from a professional breeder (Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany) at the age of five weeks. Upon arrival, mice were housed in same-sex groups of 3 individuals per cage (Makrolon cages type III, 38 × 23 × 15 cm3), since in sub-adult male mice, the occurrence of escalated aggression is very unlikely. However, with the males becoming adult, the probability of escalated agonistic encounters increases. Therefore, at the age of nine weeks, mice were transferred to single housing conditions to avoid any escalated aggressive interactions. Please note that the question whether to house male laboratory mice singly or in groups is under ongoing discussion and there is still no “gold standard” regarding its solution. For current discussions about recommendations for male mouse housing see37,38. Cages were equipped with wood chips as bedding material (TierWohl Super, J. Rettenmaier & Söhne GmbH + Co.KG, Rosenberg, Germany), a wooden stick, a semi-transparent red plastic house (11.1 × 11.1 × 5.5 cm3, Tecniplast Deutschland GmbH, Hohenpeißenberg, Germany), and a paper tissue. Housing rooms were maintained at a reversed 12 h dark/light cycle with lights off at 8 a.m., a temperature of approximately 23 °C, and a relative humidity of about 50%. The animals had ad libitum access to water and food (Altromin 1324, Altromin Spezialfutter GmbH & Co. KG, Lage, Germany) until the beginning of the touchscreen training phase. From then on they were mildly food restricted to 90–95% of their ad libitum feeding weights in order to enhance their motivation to work for food rewards. As neither distinct negative effects of such a restricted feeding protocol39, nor an interference with judgement bias assessment17,18 could be detected in previous studies, we considered this method to not affect the emotional state of the mice itself. Weights were monitored on a daily basis using a digital scale (weighing capacity: 150 g, resolution: 0.1 g; CM 150-1 N, Kern, Ballingen, Germany).
In addition to the experimental animals, 16 group-housed adult female C57BL/6J mice and 5 single-housed adult male NMRI mice, purchased from Charles River Laboratories, were used to provide the test animals with social experiences.
Ethics statement
All procedures complied with the regulations covering animal experimentation within Germany (Animal Welfare Act), the EU (European Communities Council DIRECTIVE 2010/63/EU), and the fundamental principles of the Basel Declaration, and were approved by the local (Gesundheits- und Veterinäramt Münster, Nordrhein-Westfalen) and federal authorities (Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen “LANUV NRW”, reference number 84-02.04.2015.A441).
Experimental design
In this study, the effects of different social experiences on important correlates of animal emotions, comprising cognitive (judgement bias), behavioural (anxiety-like and exploratory behaviour) as well as physiological (stress hormone levels) measures, were investigated. The experiment comprised six phases: a handling phase, a training phase, a first cognitive judgement bias (CJB) test phase, an experience phase, a second CJB test phase, and a behavioural test phase (Fig. 1).
Figure 1

Experimental design. Mice were habituated to cup handling before they underwent daily training sessions until successful acquisition of the discrimination task. Afterwards, they were tested in the cognitive judgement bias (CJB) test. During the following phase, mice repeatedly made one out of three different experiences: mildly “adverse”, “beneficial”, or “neutral”. They were then tested for their CJB again. During this second test phase, a so-called reminder was presented before each test session with the aim to re-evoke the affective state the animals experienced during the treatment phase. On the last day of each CJB test phase, faecal corticosterone metabolite concentrations (FCMs) were assessed. Subsequently, animals were tested for anxiety-like behaviour. Again, they were presented with reminders before each behavioural test.

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During the handling phase starting at PND 69, mice were first habituated to cup handling for 5 days and thereafter underwent daily training sessions to learn the discrimination task required for CJB testing, starting at PND 76. Afterwards, the animals’ initial CJB was assessed (start test phase 1: PND 223 ± 77; for details on CJB training and testing see following section).
During a subsequent experience phase starting at PND 230 ± 77, mice were exposed to one of three different experiences, each comprising three group-specific encounters, classified as either mildly “adverse”, “beneficial”, or “neutral”. Encounters took place under red light between 2:45 p.m. and 4:35 p.m. on 3 different days, always separated by a gap day. The mildly “adverse experience” group (AE group, n = 8) repeatedly encountered a dominant opponent of the aggressive NMRI strain40, with each confrontation lasting maximally 10 minutes30,41. Confrontations were terminated in cases of high aggression. The “beneficial experience” group (BE group, n = 8) was repeatedly presented with freshly collected urine of an unfamiliar C57BL/6J female for 10 minutes31. To provide all subjects with comparable experiences, we controlled for the females’ oestrus state. Since the time of oestrus in mice is relatively short42, urine from non-oestrous females was used in order to keep the total number of involved females low. The “neutral experience” group (NE group, n = 8) served as a control group and was repeatedly placed into a novel cage containing clean bedding material for 10 min.
Following the experience phase, CJB was assessed again to investigate the influence of the respective experience on the animals’ judgement bias (start test phase 2: PND 237 ± 77). In this second test phase, a so-called reminder was presented immediately before each test session. These reminders were introduced to acutely re-evoke the affective state the mice experienced during the encounters of the treatment phase. Reminders took place immediately before each test session of the second CJB test phase. For this purpose, mice were placed into a cage (Makrolon type II cage; 22 × 16 × 14 cm3) filled with bedding for 3 min. For AE mice, another 25 ml of soiled bedding from the home cage of the last NMRI male encountered were added. For BE mice, the same was done with soiled bedding from the home cage of the last female of which urine had been presented.
On the last day of each CJB test phase, faeces samples were obtained to assess corticosterone metabolite (FCM) concentrations. Finally, animals underwent a battery of standard behavioural tests for anxiety-like behaviour and exploratory locomotion (elevated plus maze test (EPM), dark-light test (DL), and open field test (OF); start: PND 245 ± 77). Before each test session, a reminder was presented again.
The allocation of mice to the treatment groups was pseudo-randomised, so that balanced numbers of mice with different learning speeds were present in each group. The testing order of mice was randomised once before the first CJB test and subsequently maintained for the following CJB and behavioural test sessions. As reminders were provided immediately before CJB testing as well as before the subsequent behavioural tests, blinding of the experimenter was not possible.
The touchscreen-based cognitive judgement bias test
Procedure
The same apparatus as described previously was used28,36 (Bussey-Saksida Mouse Touch Screen Chambers, Model 80614, Campden Instruments Ltd., Loughborough, Leics., UK). Mice underwent daily touchscreen sessions at intervals of approximately 24 h on maximally 6 consecutive days. Before each session, each mouse was taken out of its home cage and weighed. In a red semi-transparent box (21 × 21 × 15 cm3) the animal was then transported to a separate room where it was placed into the touchscreen chamber. The session was started and ended after a maximum number of trials had been performed or after a training step-specific duration. All touchscreen sessions were conducted during the dark phase between 8.15 a.m. and 1 p.m.
Paradigm
The paradigm applied here was the same as described previously with minor modifications36. Briefly, mice were trained to distinguish between a positive and a negative condition (Fig. 2). The positive condition was signalled by a bar at the bottom (5 cm below upper edge) of the cue-presentation field, the negative condition by a bar at the top (1 cm below upper edge). Mice had to touch either the left or right touch field in response to the cues. A correct touch in the positive condition led to the delivery of a large reward (12 μl of sweet condensed milk, diluted 1:4 in tap water, in the following “SCM”). An incorrect touch resulted in the delivery of a small reward (4 μl of SCM). In the negative condition, correct touches led to the delivery of a small reward (4 μl of SCM), while incorrect touches resulted in a mild “punishment” (5 s time out and houselight on). Mice had to learn to touch the high-rewarded side in the positive condition and the small-rewarded side in the negative condition. The small-rewarded touch field was the same in both conditions. The association between condition and correct touch side was the same for each individual but counterbalanced between mice. For a detailed description of the training procedure please see the supplementary material. After successful training, animals underwent CJB testing. The two cognitive bias test phases took place on five consecutive days each. During each CJB test session, three types of ambiguous cues, interspersed with the learned reference cues, were presented. These were bars at three intermediate positions: near positive (NP, 4 cm below upper edge), middle (M, 3 cm below upper edge) and near negative (NN, 2 cm below upper edge). Touches in response to these ambiguous cues resulted in a neutral outcome (neither a reward nor a “punishment”). The animals’ judgements made in response to these cues indicated whether they interpreted them according to the positive (“optimistic” response) or negative (“pessimistic” response) reference cue, serving as a measure of CJB.
Figure 2

Touchscreen-based cognitive bias paradigm. Mice were trained to distinguish between bars displayed at the top (negative condition) or bottom (positive condition) of a central field of a touchscreen. In this example, mice learned to touch right for a large reward during the positive condition and to touch left for a small reward during the negative condition (the association between positive/negative cue and the correct touch side was counterbalanced across mice). During the test, mice were presented with cues displayed at three intermediate positions: near positive, middle and near negative. The relative number of “optimistic”-like responses to these ambiguous conditions served as outcome measures of the animals’ cognitive judgement bias. Figure adopted from Krakenberg et al. (2019) with permission from Elsevier36.

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Each test session comprised 54 trials. Per session, each type of ambiguous cue was presented twice, interspersed with 48 training trials. Per test phase, each mouse was presented with each ambiguous cue ten times and each trained cue 120 times.
Behavioural measures
Responses to ambiguous cues served as a measure of the animals’ CJB. Touches according to the positive condition were defined as “optimistic” choices, touches according to the negative condition were defined as “pessimistic” choices. Out of all responses per condition, a “choice score” was calculated as previously28,36 according to the following formula:

$$ Choice,Score = frac{{N,choices ( {text{“}}optimistic{text{”}} ) – N,choices ({text{“}}pessimistic{text{”}})}}{ N,choices ({text{“}}optimistic{text{”}} + {text{“}}pessimistic{text{”}})} $$

The choice score could range between − 1 to + 1. Higher scores indicated a higher proportion of “optimistic” choices and consequently a relatively positive CJB compared to lower scores. Please note that choice scores are not an absolute, but a relative measure of CJB and that the term was chosen for the sake of intuitiveness.
Anxiety-like behaviour and exploratory locomotion
Mice were tested in three tests on anxiety-like behaviour and exploratory locomotion in the following order: the elevated plus-maze test (EPM), the dark-light test (DL) and the open field test (OF). The sequence of tests followed recommendations to schedule tests that are more sensitive to previous experience at the beginning of such a battery, and to conduct potentially more stressful tests towards the end43,44. Tests were carried out at intervals of at least 48 h and were performed in a room different from the housing room between 12:45 p.m. and 3:35 p.m. Test equipment was cleaned with 70% ethanol between subjects. Behaviour was recorded with a webcam (Logitech Webcam Pro 9000) and the animals’ movements during the EPM and OF were automatically analysed by the video tracking system ANY-maze (ANY-maze version 4.99, Stoelting Co., Wood Dale, IL, USA). Videos of the DL were analysed manually by an experienced observer (Sophie Siestrup). For apparatus descriptions and details about testing procedures see supplementary material.
Faecal corticosterone metabolites
The basal levels of adrenocortical activity of the subjects were monitored non-invasively by measuring faecal corticosterone metabolites45,46,47 (FCMs). Faeces samples of each individual were collected on the last day of the first CJB test week (= before the experience phase) and on the last day of the second CJB test week (= after the experience phase). During the dark phase, a peak of FCMs can be found in the faeces 4–6 h after the exposure to a stressor45. For this reason, faeces samples were collected 5.5–8.5 h after an individual finished CJB testing to ensure that faeces collection could be finished in the dark phase. For sample collection, mice were placed in Makrolon cages type III with a thin layer of bedding material and clean enrichment items as present in the home cage. Water was available ad libitum. After the sampling period of 3 h, mice were transferred to novel clean cages together with the enrichment items. All faeces produced during this time were collected and frozen at − 20 °C. Faecal samples were dried and homogenised and aliquots of 0.05 g were extracted with 1 ml of 80% methanol. Samples were then analysed using a 5α-pregnane-3β, 11β, 21-triol-20-one enzyme immunoassay (for details see45,46). Intra- and inter-assay coefficients of variation were More

Occurrence of the target OCs
The following individual OCs were found in fish samples with a detection frequency lower than 50%: i.e. p,p’-methoxychlor, heptachlor, heptachlor exo-epoxide, CB-66, 77, 81, 105, 114, 118, 123, 126, 128, 138, 153, 156, 157,167, 169, 170, and 180. These OCs are not discussed later. The concentrations of seven OC compounds in the muscle samples of 41 Nile tilapia and 34 Redbelly tilapia are shown in Supplementary Table S4. Since there were no significant interspecies differences between Nile tilapia and Redbelly tilapia (t-test, p = 0.16), the results of OCs analysis will be reported by tilapia genus in this study.
Median concentrations of OCPs and PCBs in tilapia samples from the main rivers system in the southern Guangxi are summarized in Table 1. PCBs and OCPs were detected in the muscle of all tilapia samples. DDTs were the predominant contaminant with a median concentration of 15.2 ng/g lw, and endosulfan was the second most common contaminant with a median concentration of 12.2 ng/g lw. PCBs, Drins, HCB, and HCHs concentrations in the fish were relatively low with median concentrations between 1.37 and 9.11 ng/g lw. The concentrations of the various OC compounds measured in the tilapia samples in this study were lower than those in tilapia collected from Guangdong province, China9, Africa10,11,12, Europe, and America13,14,15 (see Supplementary Table S5 online). This study showed that the main rivers in the southern Guangxi have low levels of OCs pollution, and the fish muscle contamination might be related to the low levels of pollution in the water and sediment. According to data from the National Bureau of statistics of China, the gross output value of industry and agriculture in Guangxi has been lower than that of other provinces or regions in China in the past few decades16. Therefore, the low levels of OCs pollution found in this study area are mainly the result of lower pollution input. In addition, most of the study area is located in the tropics, which have a relatively high perennial temperature. A warm climate is very conducive to enhance the metabolism rate of OCs by organisms17. The metabolism of organic pollutants by organisms occurs under the catalysis of a series of enzymes18,19. Factors affecting the enzymatic reaction, such as enzyme concentration and temperature, will affect the metabolism of OCs in organisms. Temperature also affects the air–water partitioning, which influences the volatilization of chemical pollutants from water20. Thus, dissolved chemical concentrations tend to be higher in cooler than in warmer waters21. In alignment with this supposition, Sobek et al. (2010) reported a largely reduced difference in bioaccumulation factor of PCBs between the Arctic and the temperate food webs, after adjustment for temperature effect22.
Table 1 Organochlorine concentration [median (range), ng/g lw] in the wild tilapia from the main rivers in Guangxi, South China.
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Distribution characteristics
Spatial distribution of OCs
The spatial distributions of seven OC compounds are presented in Fig. 1. The spatial distribution did not show a gradient in selected OCs concentrations. The spatial distribution of OCPs was under the double influence of a global distillation effect and the usage of OCPs23. Human activities can affect the distribution of OCPs in hilly areas24. However, there was no significant correlation between elevation and the residues of OCPs in this study (non-parametric test, p  > 0.05) (Fig. S2). Therefore, the distribution pattern of OCs in this study was hardly affected by global distillation. High levels of OCPs were found in TD and GG, where endosulfan or DDTs were the predominant contributors. Endosulfan is a cyclodiene pesticide extensively used throughout the world to control a wide variety of insects and mites23. Endosulfan levels were remarkably higher (10–411 times) in tilapia samples from TD than in samples from other sites. This observation was consistent with the fact that the local fruit and vegetable farming industry is the primary income source in the TD25. Therefore, we believe that the high levels of endosulfan in this study could be attributed to local pesticide practices specific to pest control needs over a short period26. Similarly, the higher levels of DDTs observed in GG also might be related to local short-term agricultural activities.
Figure 1

Spatial variations of log-transformed concentrations of OC compounds (ng/g lw) residues in wild tilapia from the main rivers in Guangxi, South China. TD: Tiandong County; LA: Longan County; CZ: Chongzuo City; FS: Fusui City; NN: Nanning City; GG: Guigang City; WX: Wuxuan County; PN: Pingnan City; TX: Tengxian County; WZ: Wuzhou City.

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PCBs are ubiquitous in tilapia samples from the study area, with a detection rate of 100%. In contrast to the OCP compounds, the overall trend of the PCBs was fairly homogenous. A relatively high median PCB concentration was detected in tilapia samples from TX, while slightly lower concentrations were detected from PN. There were no significant differences among different sampling locations (t-test, p  > 0.05). The minor differences could be explained by the migration and spread of PCBs in the environment. The limited historical use of PCBs in the present study area is another important factor contributing to this phenomenon25.
Spatial differences in pollutant metabolites
The ratio of parent compounds to their metabolites can provide useful information for the diagnosis of their sources23,24,27. The scatter plots for isomeric ratios of selected OCPs are shown in Fig. 2.
Figure 2

Scatter plots of molecular indices to identify DDTs (a) and endosulfan (b) contamination sources.

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The ratios between p,p’-DDT, p,p’-DDE and p,p’-DDD have been regarded as an indication of increasing or decreasing inputs to the environment. A ratio of (p,p’-DDE + p,p’-DDD)/p,p’-DDT  1.3) were found in two fish samples from FS (1.80) and CZ (1.71) districts, which indicates that Dicofol may be the main contributor to DDTs in these areas. In summary, the DDT residues in wild tilapia from rivers of the southern Guangxi originated mainly from the historical application of Dicofol and technical DDTs, whereas recent application of technical DDTs are indicated in TD.
Technical endosulfan includes two active ingredients: α-endosulfan (70%) and β-endosulfan (30%)23. Because α-endosulfan decomposes more easily than β-endosulfan, a α-/β-endosulfan ratio of  2.33) present in the tilapia samples from TD and FS, indicate continual use of endosulfan in these areas. In the other sites, those ratios are all below 2.33, indicating there was no recent application of technical endosulfan in that area. It is noteworthy that one sample (from TX site) contained β-endosulfan at a level below the limit of detection, but had appreciable levels of α-endosulfan, which may have been transported in from other areas. Because the Henry’s law constant for α-endosulfan is higher than the constant for β-endosulfan, there is a greater tendency for α-endosulfan to evaporate from the surface medium to air23,30.
The concentrations of ten PCB congeners in the present study area are illustrated in Fig. 3. Using degree of chlorination, these congeners can be divided into light PCBs (2–3 chlorines), medium PCBs (4–6 chlorines), and heavy PCBs (7–10 chlorines). The PCB sources of the 75 fish samples can be classified into the same categories since the PCBs in all sampling sites generally exhibited the following order: heavy PCBs (63.3–86.1% of ∑10PCBs)  > medium PCBs (9.72–18.2% of ∑10PCBs)  > light PCBs (4.66–18.3% of ∑10PCBs). The higher residual content of heavy PCBs may be related to historical production and use, or relate to their stably chemical structure31. Tri-CBs and penta-CBs were the major PCB products manufactured in China from 1965 until they were banned in 197431,32. The proportion of these compounds in PCBs was only 2.86–24.7% in this study. On the other hand, light PCBs have higher volatility and a lower octanol–water distribution coefficient than heavy PCBs33. Once absorbed into the organisms, light PCBs are usually more rapidly metabolized than the more highly chlorinated congeners34. Our results also indicated that the proportion of deca-CBs in heavy PCBs and PCBs was 78.3–98.1% and 51.8–88.6%, respectively. And the sampling sites with high deca-CBs ratio were distributed in the main agricultural farming areas (middle and upper reaches of rivers). And China banned the production of deca-CBs as early as 197435. Therefore, we believe that historical heritage was the main source of deca-CBs in the study area.
Figure 3

Composition profiles of PCB congeners in the main rivers from Guangxi, South China.

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Correlation among biological parameters and OC compounds
We studied the effects of biological parameters (including total length, body mass, age, and lipid content) on the bioaccumulation of individual contaminants in tissue samples of wild tilapia (based on dry weight). The loading and scores plot of PCA based on the concentrations of OCs in the tilapia muscle samples are displayed in Fig. 4. The PC1 explained 58.6% of the total variance and PC2 accounted for 23.5% of the variance. Table S7 lists the correlation coefficients between OC compounds and biological parameters, while the correlation coefficients between OC congeners and biological parameters are listed in Table S8. A significant relationship between growth parameters (i.e. total length, age, and body mass) was found in the tilapia samples, but only age and lipid content were significantly correlated (p  More

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Challenger Deep seawater and surface sediment samples were taken from its entire ~11,000 depth profile (Fig. 1a and Supplementary Table 1). The clines in temperature (29.8 °C in surface waters, decreasing to ~1.0 °C below 3000 m) and pressure (0.1 MPa in surface waters to ~104 MPa at the bottom of the trench) were recorded. The waters were oxic throughout the water column and the salinity ranged between 34 and 35 Practical Salinity Units (PSUs) (Supplementary Table 1). Seawater total DMSP and DMS concentrations were similar to those in previous studies21,26,27,28 and were positively correlated with Chl-a levels, being highest in the Chl-a maximum layer (10.51 × 10−3 nmol ml−1 DMSP and 4.97 × 10−3 nmol ml−1 DMS) and at lower but relatively stable levels (0.96–2.39 × 10−3 nmol ml−1 DMSP and 0.15–1.06 × 10−3 nmol ml−1 DMS) in the aphotic waters below 200 m (Fig. 1b, c and Supplementary Table 1). It should be noted that a small portion of this ‘background DMSP’ (3 μm) bacteria, which dominated the metagenomes of both these fractions, contained DMSP biosynthesis and catabolic genes (Figs. 2a and 3a, and Table 1), indicating that size fractionation is not a foolproof method of separating DMSP-producing bacteria from phytoplankton. Bacteria with dysB were shown by qPCR and metagenomics to be relatively abundant in the surface waters (dsyB total abundance of 2.61 × 105 copies L−1; 0.78–0.98% of surface water bacteria) representing ~3.49–4.38 × 103 bacteria ml−1 of surface seawater. These numbers are comparable to those predicted from the ocean microbial reference gene catalog metagenomic database (OM-RGC)37 in Williams et al.10, at ~4.8–9.6 × 103 bacteria ml−1. The abundance of these potential DMSP-producing bacteria initially decreased in 1000–2000 m deep seawater samples (3.46 × 104 copies L−1; ~0.43% bacteria at these depths), but then steadily increased with depth to reach maximal levels at 10,500 m (3.95 × 106 copies L−1; 4.03% of bacteria at 10,500 m), which were up to 15-fold higher than in the surface water (Fig. 2b, Table 1, and Supplementary Table 2). All detected dsyB sequences, including 37/162 metagenome assembled genomes (MAGs), were Alphaproteobacterial, mainly Rhodobacterales, Rhizobiales, and Rhodospirillales (Supplementary Data 1). At the genus level, Pseudooceanicola and Roseovarius were the most abundant potential DMSP producers at all depths, with much higher abundances (P  More

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Locating and collecting badgers
Members of the public, local authorities and countryside organisations in Wales reported the locations of found dead badgers to the Welsh Government or APHA Field Services who recorded the map reference details. The collection by APHA staff of badger carcases that met pre-defined criteria took place between 1st September 2014 and 31st December 2016. There were instances where the collection of reported carcases was not attempted. Reasons for not attempting collection included safety concerns arising from the specific location of the carcass or the non-availability of staff (or other necessary resource) to undertake the collection (supplementary table online ST1). In some further instances collection was attempted but was unsuccessful because either the reported carcase could not be found or the condition of the carcase made it unsuitable for further investigation (for example viscera were herniated externally through wounds, there was severe myiasis (flystrike), the carcase was distended with gas or it was flattened).
Depending on where they were found, the carcases were delivered to the APHA Veterinary Investigation Centres in Carmarthen and Shrewsbury and to the Wales Veterinary Science Centre in Aberystwyth (from 28/01/2016) where they were stored at between 2 and 8 °C for no more than four days before post-mortem examination.
Post-mortem examination and sampling
Of the 1863 dead badgers reported, 841 were collected and 681 (37% of reported carcasses) were suitable for post-mortem examination. The prevalence of bTB was calculated for suitable carcasses only. The sampling protocol was adapted from the standard and detailed protocols described in a comparison by Crawshaw and others24, so that fewer overall samples were taken than the detailed protocol but that the samples chosen were the ones most likely to yield M. bovis if present.
The initial external examination comprised the following: measuring the length from nose tip to tail base (cm), assessing and recording tooth wear, recording the sex of the animal and whether female animals were lactating, andrecording any evidence of vaccination: To temporarily identify vaccinated badgers, the guard hairs of the dorsal back (usually caudal) were trimmed and a coloured marker applied at the same site at the time of vaccination. During the external examination of the badger carcases, to attempt to detect recent vaccination, the skin and hair of the back was visually examined for guard hair trimming and coloured marker. Furthermore the examination recorded any evidence of trap injury, or of illegal interference with the animal and the presence and location of bite wounds.
A detailed examination of the lungs, pericardial sac, liver and kidneys was conducted at post-mortem examination. The lungs were examined by making multiple longitudinal incisions approximately one centimetre apart. At least four slices were made in the liver and three slices in the kidney24.
Each lymph node of all suitable badgers was incised at least once and a pool of lymph nodes (pool 1) consisting of the retropharyngeal, bronchial, mediastinal and hepatic lymph nodes (or as many as were detectable) was collected for subsequent bacteriological culture. If any gross internal lesions suggestive of tuberculosis were observed or if bite wounds were detected, the lesioned tissue and/or excised bite wounds were added to a separate container (pool 2). The pooled samples for bacteriological culture were preserved in 15 ml of 1% aqueous cetylpyridinium chloride. Samples were sent to the Animal Plant & Health Agency Laboratory inStarcross, Devon on the day of examination, for next day receipt. After taking samples, two or three incisions were made in the muscles of the anterior thigh of both hind legs to look for any potential adverse reactions to BCG vaccination24.
Culture and molecular typing
On receipt at APHA Starcross, the samples were washed in sterile 0.85% saline solution, homogenized by standard methods, inoculated onto six modified Middlebrook 7H11 agar slopes26 and incubated at 37 °C. Pool 1, and Pool 2 (if collected), were cultured separately. The slopes were examined weekly from the end of week 2 for a maximum of 12 weeks. M. bovis isolates were harvested when growth was sufficient for genotyping and sent to APHA Weybridge.
Genotyping was performed using spoligotyping27 and VNTR typing (Exact Tandem Repeat Loci A to F)9,28. Spoligotyping confirmed that the isolates were M. bovis. Genotypes of M. bovis were labelled according to the current APHA convention, using numbers to represent spoligotypes and lower case letters to represent the VNTR pattern within each spoligotype. The same genotyping methods were applied to the cultures of M. bovis from cattle slaughtered as part of the national bTB control programme. The cattle genotype home ranges were determined for 2015 (Data Systems Group, Dept of Epidemiological Sciences, Animal & Plant Health Agency Weybridge); for inclusion a genotype had to have been present for at least three years, on at least two different 5 km × 5 km grid squares, in the last 5 years (with a 10 km buffer applied).
Disease status in cattle herds
The disease status in cattle herds from the five TB regions of Wales has been calculated for all Spatial Units (for definition please refer to introduction) with at least 10 badger submissions and compared with prevalence estimates for the badger populations in these areas. The metric used to describe disease status in cattle is average annual herd prevalence during most of the AWBFD sampling period (2015–2016, number of herds under restriction at any point of year/registered live herds in region). Herd size is a known risk factor for bovine TB29; therefore, direct standardisation was used to adjust for varying herd sizes in the Spatial Units3,30,31. Briefly, annual stratum-specific prevalence was calculated for each Spatial Unit across four strata (reduced from six in the cited studies due to the small herd denominator in Wales Spatial Units) of herd size and then applied to the reference population, which comprised the sum of cattle populations across all Spatial Units. The standardised population was then used for herd-level disease measures, resulting in a standardised herd prevalence (Fig. 4).
Badger prevalence mapping
For the Spatial Unit badger prevalence map each Spatial Unit was labelled with the number of submissions, the number of positive results and the resulting prevalence estimate. When added to ArcMap (Esri ArcGIS 10.2.2) the Spatial Unit layer was symbolised using the prevalence value and six pre-defined range values or classes were applied and colour ramped. Spatial Units with less than 10 AWBFD submissions (“insufficient data”) were not colour ramped. All Spatial Units were labelled with the number of positives/number of submissions.
Statistical analysis
The prevalence of bTB in badgers was estimated among the sampled badgers as in previous studies3,8 with the underlying assumption that the carcasses collected were representative of the overall population.
Analysis was performed to test the null hypotheses (Ho) that:

There are no differences in badger bTB prevalence between the five TB Areas in Wales.

There was no change in overall badger bTB prevalence in Wales between the surveys in 2005–2006 and in 2014–2016.

There was no change in badger bTB prevalence within the five TB Areas between the surveys in 2005–2006 and in 2014–2016.

There was no change in badger bTB prevalence between the surveys in 2005–2006 and in 2014–2016 within the Intensive Action Area (IAA), site of a 2012–2015 badger vaccination trial.

There is no correlation between cattle herd prevalence and estimated bTB prevalence in dead badgers in different geographic regions.

There is no difference in cattle herd prevalence between areas with infected badgers and those with no evidence of bTB in badgers.

For statistical purposes, for the comparisons of prevalence estimates between the TB Areas and over time, both Intermediate TB Areas were combined. All data were tested for normality with the Kolmogorov–Smirnov test (SPSS Version 21 for Windows). Since the dependent variable in these analyses, prevalence, is a rate and fulfilled the criteria for normality, the z-test for comparisons of population proportions was used to test for the statistical significance of differences between prevalence estimates between the TB Areas and over time. A condition of the z-test is that each sample contains at least 10 observations in each category of the dependent variable and for comparisons between samples with less than 10 submissions in at least one category, Fishers Exact test was used32. To explore the correlation between prevalence in badgers and cattle, linear regression analysis was undertaken to calculate Pearson’s coefficient of correlation (SPPS 22 for Windows). Spatial Units with  > 10 badger submissions with one or more positives were compared with those which had none, using the two sample t test with unequal variances. In order to compare genotypes between badgers and cattle, as in the previous Wales badger survey3, the authors calculated the associations between the frequency distribution of the genotypes in badgers and the frequency distribution in cattle for each TB Area. In order to prevent ties and to account for the small number of positive badger submissions, frequencies were adjusted, by replacing them with their deviations from expected values which were calculated as (TB Area subtotal) × (Wales genotype subtotal)/(Wales grand total). A Spearman rank correlation between the ranks of genotypes in badgers and their ranks in cattle was then calculated for each TB Area. More

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