Supplementary Material s39

SUPPLEMENTARY MATERIAL

Weber et al. Denning behaviour of the European badger (Meles meles) correlates with bovine tuberculosis infection status

S1 Diagnostic Tests

We had the results from three diagnostic tests available to use in our study:

1) BrockTB Stat-Pak lateral flow serum antibody test (Chambers et al. 2008; Chambers et al.

2009);

2) an enzyme immunoassay for interferon-gamma (IFNγ) (Dalley et al. 2008; Chambers et al.

2009); and

3) culture testing.

We did not include the results from the culture test as only one animal was found to be actively excreting Mycobacterium bovis (the causative agent of bTB) at the time of the study.

Culture is a much less sensitive predictor of infection than either the StatPak or IFNγ, both of which have been used to indicate infection in free-living badgers (see Chambers et al. 2011).

Moreover, the Stat-Pak detects the presence of M. bovis antigens, the production of which has been shown to positively correlate with the extent and severity of bTB infection in both naturally and experimentally infected badgers (reviewed in Chambers 2009). In the main body of text we present the results using the outcome of either the Stat-Pak or the IFNγ diagnostic tests to infer infection as this provides the greatest values for test sensitivity

(sensitivity 85%, specificity 93% Chambers et al. 2009). Here, we additionally present the findings using the results of the Stat-Pak immune-assay only, as this test is the best available indicator of established infection in badgers and the sensitivity of the test increases with disease severity (Chambers et al. 2008). The Stat-Pak assay used to assess bTB status has a sensitivity of approximately 50% in adult and yearling badgers, increasing to between 66%-

78% with disease progression and 93% specificity (Chambers et al. 2008; Chambers et al.

2009). All animals that gave a positive test result with the Stat-Pak (barring one whose most recent blood sample was contaminated and so could not be tested) also gave a positive test result with the IFNγ test. There were an additional four animals that gave a positive IFNγ test result but a negative Stat-Pak result. Statistical results are similar whether or not these individuals are included (Table S1) and the conclusions drawn and discussed in the manuscript are the same. The only notable effect of assigning disease status solely using Stat-

Pak is that the difference in time spent at the main sett between positive and negative animals in the summer is increased (Figure S1). This suggests that the mechanism(s) driving the differences in behaviour between test-positive and negative individuals (discussed in the main text) may be particularly important in the summer. Table S1 Factors determining the proportion of time spent by badgers at the main sett vs. outlier setts. P values were obtained by step-wise deletion from maximal model, starting with the least significant term. Minimum adequate model was: Proportion of time at main sett

~ bTB test outcome season + sex age season (+ sex age + sex season + age season + bTB test outcome + season + sex+ age) + (1 | social group / badger ID).

Variables Estimate ± S.E. d.f. P Fixed Effects Sex × Age × Season 1.73 ± 0.91 9.96 3 0.01 Sex × Age 0.33 ± 1.25 0.10 1 0.80 Sex × Season 2.03 ± 0.39 52.2 3 <0.001 Age × Season 0.87 ± 0.44 8.18 3 0.04 bTB test outcome × Season 1.08 ± 0.74 15.7 3 0.001 Sex 0.23 ± 0.53 0.17 1 0.67 Age 0.21 ± 0.53 0.15 1 0.69 Season 2.13 ± 0.51 388 3 <0.001 bTB test outcome 1.90 ± 0.48 12.1 1 <0.001 Proportion of adults 3.20 ± 6.68 0.22 1 0.64 Number of outlier setts 0.52 ± 0.49 3.82 1 0.07 Bite wound score 0.23 ± 0.24 1.05 1 0.31 Territory size 0.98 ± 0.95 0.15 1 0.70 BCI 3.86 ± 3.86 0.10 1 0.32 Number of main setts 0.43 ± 0.98 0.18 1 0.67 Proportion of males 2.51 ± 2.45 1.03 1 0.31 Group size 0.29 ± 0.27 1.77 1 0.10 Random Effects Individual NA 184.5 1 < 0.001 Social Group NA 3.850 1 0.049 Figure S1 The proportion of time spent at the main sett across the four seasons in relation to bTB test outcome as inferred from the Stat-Pak immuno-assay (estimates ± 1 SE from model 1 where sex, age and social group identity are controlled for). For the badgers that tested positive; Sp n = 7, S n = 9, A n = 5, W n = 8. For those that tested negative; Sp n = 20, S n = 24, A n = 25, W n = 27. REFERENCES:

Chambers MA (2009) Review of the diagnosis and study of tuberculosis in non-bovine wildlife using immunological methods. Transboundary and Emerging Diseases. 56, 215 – 227.

Chambers MA, Crawshaw T, Waterhouse S, Delahay RJ, Hewinson RG, Lyashchenko KP (2008) Validation of the BrockTB stat-pak assay for detection of tuberculosis in Eurasian badgers (Meles meles) and influence of disease severity on diagnostic accuracy. Journal of Clinical Microbiology, 46, 1498-1500.

Chambers MA, Waterhouse S, Lyashchenko K, Delahay RJ, Sayers R, Hewinson RG (2009) Performance of TB immunodiagnostic tests in Eurasian badgers (Meles meles) of different ages and the influence of duration of infection on serological sensitivity. BMC Veterinary Research 5, 42.

Chambers MA, Rogers F, Delahay RJ, Lesellier S, Ashford R et al (2011) Bacillus Calmette- Guérin vaccination reduces the severity and progression of tuberculosis in badgers. Proceedings of the Royal Society B, 278, 1913-1920.