1
Electronic supplementary material
2
Warnecke et al., Pathophysiology of white-nose syndrome in bats: a mechanistic model
3
linking wing damage to mortality
4
Material and methods
5
This study was part of a larger project and further details on methods are published in
6
Warnecke et al. [1]. We collected bats from a WNS-negative cave near Grand Rapids,
7
Manitoba, Canada, in November 2010. We immediately transported bats in cotton bags within
8
a temperature-controlled cooler to the Animal Care Unit of the Western College of Veterinary
9
Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
10
Bats were divided into three groups of 18 individuals and inoculated with either a
11
North American isolate of Gd (designated type isolate 20631-21, see [2]), or a European
12
isolate of Gd (MmyotGER2, see [3]). We applied 20 μl of a liquid conidial suspension
13
containing 500,000 Gd spores in PBS-Tween20 directly to the dorsal side of both wings [4].
14
The control group was sham-inoculated with a PBS-Tween20 solution lacking conidia.
15
Each group was kept in a nylon mesh cage (Apogee, Dallas, Texas, USA), modified
16
with sheet plastic to an overall size of 37 × 37 × 104 cm, and contained within separate, dark,
17
water-saturated temperature-controlled environment cabinets (VWR BOD 2020; VWR
18
International LLC., Mississauga, Ontario, Canada). Bats were left undisturbed for four
19
months and observed only via an infrared, waterproof security camera (VL650IRVFS; Speco
20
Technologies, Amityville, New York, USA) connected to a motion-detecting digital video
21
recorder (SHR- 3040; Samsung Techwin, Ridgefield Park, New Jersey, USA). During this
22
time bats were not fed as they do not eat during hibernation in the wild, but water was freely
23
available. Without opening the cabinets, we refilled water dishes weekly from the outside
24
through plastic tubing.
25
At the conclusion of the study in spring 2011 the temperature of the cabinets was
26
increased to 25°C to assist bats to rewarm from torpid body temperatures to normothermia.
27
Bats were anaesthetised using 4.5-5.0% isoflurane in oxygen and body temperature was
28
measured rectally using a thermocouple and digital thermometer (800008 Sper Scientific
29
LTD, Scottsdale, Arizona, USA) immediately before blood collection. Anaesthetised bats
30
were euthanised and whole-blood samples collected using 200 μl lithium-heparin treated
31
capillary tubes and transferred into micro tubes (SSH2 StatSampler, Iris Sample Processing,
32
Westwood, Massachusetts, USA). Using a pipette we delivered ~90 µl of blood to a cartridge
33
that was inserted into a handheld blood analyser (i-STAT®1 Vet Scan, Abaxis, Union City,
34
California, USA). We used the CG8+ cartridge, which measures a range of blood variables
35
including glucose, haematocrit, partial pressure carbon dioxide, pH, potassium and sodium,
36
but not chloride.
37
To quantify severity of infection we removed all skin from one wing, rolled it on a 5
38
mm diameter rod of dental wax and then cut ~10 sections from across the length of the rolled
39
skin. These samples were processed using routine histological techniques before 5 µm
40
sections were cut, mounted on glass slides and stained with periodic acid-Schiff stain. A total
41
of 15 to 20 cm of 5 µm thick sections from each bat were examined with a light microscope to
42
derive a lesion score. Pathogenicity for both isolates of Gd was similar and lesions caused by
43
both isolates were identical based on histopathologic examination [1]. We, therefore, pooled
44
results for bats inoculated with the North American (n = 6) and European Gd isolates (n = 2)
45
for both blood physiology and tissue necrosis scoring. We modified the histopathologic
46
scoring system reported by [5]. Necrosis score was based on the percentage of skin surface
47
with fungal hyphae present that was necrotic where 0 = no necrotic tissue; 1 = minimal
48
(<1%); 2 = mild (1-10%); 3 = moderate (10-30%); 4 = severe (30-50%); 5 = very severe
49
(>50%).
50
To account for the multiple pairwise comparison design of our study we adjusted
51
alpha levels as described by Narum [6]. We used an individual-experiment alpha level of 0.1
52
because of our small sample size and then adjusted this alpha using the false discovery rate
53
method of Benjamini and Hochberg [7]. The resulting experiment-wide alpha level was 0.07
54
for t-tests and 0.04 for regression analyses. Before conducting statistical analyses we tested
55
for normality of all variables using Shapiro-Wilk test statistics. Non-normal data were log-
56
transformed and normality was confirmed. For Student’s t-tests the assumption of
57
homogeneity of variance was tested using Levene’s test of equality of variances. We
58
corrected for violation of equal variances by using the pooled estimate for the error terms and
59
making adjustments to the degrees of freedom. We used least-squares linear regression to test
60
for relationships between blood parameters and necrosis scores. All statistical tests were run
61
with StatistiXL v.1.9. (http://www.statistixl.com/).
62
63
References
64
1.
65
Warnecke L., Turner J.M., Bollinger T.K., Lorch J.M., Misra V., Cryan P.M., Wibbelt
G., Blehert D.S., Willis C.K.R. 2012 Inoculation of bats with European Geomyces
66
destructans supports the novel pathogen hypothesis for the origin of white-nose
67
syndrome. Proc. Nat. Acad. Sci. USA 109(18), 6999-7003.
68
2.
Gargas A., Trest M.T., Christensen M., Volk T.J., Blehert D.S. 2009 Geomyces
69
destructans sp. nov. associated with bat white-nose syndrome. Mycotaxon 108, 147-
70
154.
71
3.
Wibbelt G., Kurth A., Hellmann D., Weishaar M., Barlow A., Veith M., Prüger J.,
72
Görföl T., Grosche L., Bontadina F., et al. 2010 White-nose syndrome fungus
73
(Geomyces destructans) in bats, Europe. Emerg. Inf. Diseas. 16(8), 1237-1242.
74
4.
Lorch J.M., Meteyer C.U., Behr M.J., Boyles J.G., Cryan P.M., Hicks A.C., Ballmann
75
A.E., Coleman J.T.H., Redell D.N., Reeder D.M., et al. 2011 Experimental infection
76
of bats with Geomyces destructans causes white-nose syndrome. Nature 480, 376-378.
77
5.
Reeder D.M., Frank C.L., Turner G.G., Meteyer C.U., Kurta A., Britzke E.R., Vodzak
78
M.E., Darling S.R., Stihler C.W., Hicks A.C., et al. 2012 Frequent arousal from
79
hibernation linked to severity of infection and mortality in bats with white-nose
80
syndrome. PLoS ONE 7, e38920.
81
6.
82
83
84
85
86
87
88
89
Narum S.R. 2006 Beyond Bonferroni: less conservative analyses for conservation
genetics. Conserv. Genet. 7, 783-787.
7.
Benjamini Y., Hochberg Y. 1995 Controlling the false discovery rate: a practical and
powerful approach to multiple testing. J. R. Stat. Soc. B 57(1): 289-300.
90
Results
91
Raw data:
92
Treatment
pH
PCO2
HCO3
Na
K
Glu
Hct
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
I
I
I
I
I
I
I
I
7.552
7.489
7.446
7.553
7.744
7.587
7.549
7.555
7.539
7.683
7.503
7.531
7.557
7.600
7.449
7.548
7.602
7.627
7.634
7.502
7.656
7.576
7.458
7.485
7.426
7.397
33.46
45.75
49.20
44.90
25.80
33.49
39.30
34.71
43.15
30.00
44.91
35.32
37.65
27.28
50.04
42.39
35.21
29.39
26.84
33.88
24.48
36.24
29.28
34.89
26.44
17.87
29.4
34.8
33.9
39.6
35.3
31.9
34.3
30.7
36.8
35.6
35.3
29.6
33.5
26.8
34.7
36.9
34.7
30.7
28.5
26.5
27.4
33.7
20.7
26.3
17.4
11.0
147
153
155
161
136
143
150
143
145
136
153
147
151
143
165
153
138
144
133
133
134
143
129
134
126
121
4.1
4.6
4
4.9
3.9
3.6
3.8
7.4
4
5.1
4.4
4.9
4.5
6
6
3.9
5.1
5.2
3.4
4
3.7
5
7
5.5
5.8
5.8
138
155
127
160
97
123
87
36
96
61
99
108
110
87
87
86
92
116
106
66
93
45
73
98
29
57
42
48
50
51
34
43
46
44
41
41
40
51
44
40
50
49
40
38
49
48
42
48
59
46
73
63
Key:
C
Sham-inoculated control group
I
Gd-inoculated group
PCO2
Partial pressure carbon dioxide (mmHg)
HCO3
Bicarbonate (mmol/L)
Na
Sodium (mmol/L)
K
Potassium (mmol/L)
Glu
Glucose (mg/dL)
Hct
Haematocrit (% packed cell volume)
Further details see text.