1
Oaks, et al., Diclofenac Residues as the Cause of Population Decline of
Vultures in Pakistan
Supplementary Information
Histopathology. Histopathology on tissues representing all major organ systems was
performed by a panel of three pathologists: Bruce Rideout, Carol Meteyer and H.L.
Shivaprasad. All of the vultures collected in Pakistan with gross necropsy evidence of
visceral gout had severe necrosis of the renal proximal convoluted tubules with
deposition of urate material in the kidney. Deposition of urate material also occurred on
the visceral surface of many organs and was associated with tissue necrosis when urate
precipitation occurred within the parenchyma of organs such as heart, liver, spleen and
lung.
The microscopic renal lesions were similar in all cases, and a consensus histologic
description for the cases is as follows: severe renal tubular necrosis with marked urate
precipitation usually without giant cell or granulomatous response. Regions of proximal
convoluted tubules that were not necrotic had swollen epithelial cells with very large
nuclei, prominent nucleoli, and granular cytoplasm. Inflammation was not associated
with this necrotizing process. There was no evidence of tubular epithelial regeneration.
Cellular casts were present in the lumen of some tubules including collecting tubules.
Although the cells of Bowman’s capsule were prominent, the glomeruli appear to be
spared. There was also sparing of the distal convoluted tubules and collecting tubules in
most cases. Oxalate crystals in the kidneys, indicating ethylene glycol toxicity, were not
observed.
2
Various other lesions were noted, including enteritis (11/44), meningitis (3/44),
arteriolitis (4/44), tracheitis (2/44), hepatopathy (5/44), cardiomyopathy (2/44), and
osseous metaplasia (11/44). However, these were mild, probably subclinical, and were
not present consistently in either gout or non-gout vultures. All of the pathologists
regarded these as incidental findings.
Metal testing. Wet-weight metal concentrations in liver or kidney tissues were analyzed
for toxic or deficient levels. The results are summarized in Supplementary Tables 1 and
2. Lead poisoning was identified in one non-gout OWBV (#14). One non-gout OWBV
(#2) had a potentially toxic level of arsenic according to one reference1, however the
concentration was less than that reported from other apparently normal birds2 and thus
may not be diagnostic of toxicity without additional supporting evidence. One other
OWBV (#53) with gout had a potentially toxic level of manganese according to one
reference1, but again higher levels have been reported from other apparently normal
birds2. However, even if these cases were real toxicities, none are consistently associated
with renal disease. Toxic levels of arsenic, cadmium, copper, iron, manganese,
molybdenum, lead, mercury, and zinc were not detected in any other birds1,3. The levels
of iron, copper, molybdenum and zinc may be deficient in some individuals, but a clear
pattern of deficiency did not distinguish the gout from the non-gout birds. Moreover,
growth or musculoskeletal abnormalities often associated with metal deficiencies1 were
not observed in any nestling OWBV.
3
PCR assays for infectious bronchitis, avian influenza, and West Nile viruses. These
results are summarized in Supplementary Table 3. RT-PCR assays for infectious
bronchitis, avian influenza and West Nile viruses did not detect these agents in any
OWBV.
Organophosphate and carbamate analysis. Brain acetylcholinesterase (ACE) results
indicated a single non-gout case (#58) was intoxicated by an organophosphate (OP)
pesticide. Normal brain ACE values for OWBV are not available, therefore we used an
average value of 12.06 μmole/g/min derived from 5 cases (#’s 2, 14, 28, 46, 47) that were
known to be non-OP toxicity cases. This mean value was also comparable to normal
brain ACE values reported from other bird species4. Because most cases of acute OP
toxicity have >70% suppression of brain ACE4, a value of less than 3.6 μmole/g/min was
used to define OP or carbamate pesticide toxicity. The inability to thermally reactivate
ACE levels indicated that the suppression in case #58 was due to an OP rather than a
carbamate compound5. There was no significant difference (P > 0.86) between the ACE
activity levels of gout and non-gout cases, indicating that sublethal intoxications were
also not associated with renal failure. The ACE results are summarized in Supplementary
Table 4. OPs, OP metabolites, or carbamate residues were not detected in any brain or
liver samples by liquid chromatography and mass spectroscopy. The absence of
phosphates indicative of other organophosphate pesticides or metabolites was also noted
by the laboratory. These results are summarized in Supplementary Table 5. The inability
4
to detect specific OP residues in case #58 precluded identification of the specific
compound responsible.
Organochlorine and polychlorinated biphenyl analysis. Organochlorine (OC)
pesticide residues (lindane, dieldrin, DDD, DDE, and DDT) were detected in fat by liquid
chromatography and mass spectroscopy, indicating exposure to these pesticides.
However, the levels of these pesticides in brain or liver were all less than 1 g/g, well
below the ~20 g/g levels associated with acute intoxication6. Polychlorinated biphenyl
(PCB) residues were not detected in any samples. These results are summarized in
Supplementary Table 6.
Isolation of viruses and intracellular pathogens. Viruses were not isolated from any
samples. A novel mycoplasma was isolated from case #33, but subsequent investigation
did not show an association of this mycoplasma with gout7.
Identification of Mycobacterium avium. One case (#4) was diagnosed in the laboratory
of J. L. Oaks as splenic tuberculosis based on the gross necropsy findings of severe
splenomegaly, detection of acid-fast bacilli on Ziehl-Neelsen stained impression smears,
PCR amplification of the mycobacterial heat shock protein gene and restriction enzyme
polymorphisms in the PCR-amplified product as described by Mendenhall8. This vulture
was also shown to have diclofenac residues. The tuberculosis was a significant lesion in
this case. However, because this vulture was in excellent physical condition, and did not
5
demonstrate the weight loss typically associated with chronic M. avium disease, we
believe that the cause of gout was diclofenac. Had this vulture not been intoxicated with
diclofenac, it very likely would have eventually succumbed to M. avium infection.
________________________________________________________________________
1.
Puls, R. Mineral Levels in Animal Health, 2nd edn (Sherpa International,
Clearbrook, CA, 1994).
2.
Trust, K. A., Rummel, K. T., Scheuhammer, A. M., Brisbin, I. L. & Hooper, M. J.
Contaminant exposure and biomarker responses in spectacled eiders (Somateria
fischeri) from St. Lawrence Island, Alaska. Arch. Environ. Contam. Toxicol. 38,
107-113 (2000).
3.
Reviewed in Environmental Contaminants in Wildlife. Interpreting Tissue
Concentrations (eds Beyer, W., Heinz, G. & Redmon-Norwood, A.) chaps. 10, 11,
14, 17 (CRC Press, Boca Raton, 1996).
4.
Hill, E. F. & Fleming, W. J. Anticholinesterase poisoning of birds: field monitoring
and diagnosis of acute poisoning. Environ. Toxicol. Chem. 1, 27-38 (1982).
5.
Hunt, K. A. & Hooper, M. J. Development and optimization of reactivation
techniques for carbamate-inhibited brain and plasma cholinesterases in birds and
mammals. Analytical Biochem. 212, 335-343 (1993).
6.
Blus, L. J. in Environmental Contaminants in Wildlife. Interpreting Tissue
Concentrations (eds Beyer, W., Heinz, G. & Redmon-Norwood, A.) 49-71 (CRC
Press, Boca Raton, 1996).
7.
Oaks, J. L. et al. Identification of a novel Mycoplasma species from an oriental
white-backed vulture (Gyps bengalensis). Submitted.
8.
Mendenhall, M. K. et al. Detection and differentiation of Mycobacterium avium
and Mycobacterium genavense by polymerase chain reaction and restriction
enzyme digestion analysis. J. Vet. Diagn. Invest. 12, 57-60 (2000).
6
Supplementary Table 1 Metal results (wet-weight basis) in vultures with visceral gout
Case
No.
Tissue
15
16
20
21
33
34
35
36
38
39
40
41
42
44
45
53
54
55
56
57
59
60
61
62
63
64
65
66
67
68
70
71
72
73
74
75
Liver
Liver
Kidney
Kidney
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
MDL
Toxic level
Deficient level
Metal levels (g/g)
As
Cd
Cu
Fe
Mn
Mo
Pb
Zn
Hg
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
0.10
BDL
0.57
0.76
BDL
0.13
BDL
0.16
0.20
BDL
0.12
BDL
BDL
0.23
BDL
0.36
BDL
BDL
0.11
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
7.4
3.7
2.1
1.8
16.0
5.8
4.6
8.4
6.8
6.1
6.1
8.2
10.0
12
6.8
34
4.7
4.6
6.1
14.0
7.0
3.4
4.8
14.0
18.0
19.0
19.0
17.0
17.0
22.0
24.0
5.6
26.0
11.0
12.0
3.8
590
350
140
130
1900
820
450
390
302
670
1200
413
930
288
160
1605
490
838
322
590
720
670
775
240
360
230
270
210
300
250
270
570
370
360
460
490
4.5
4.5
1.6
2.2
3.4
3.4
2.9
3.8
4.0
3.7
4.0
3.1
2.7
5.8
5.4
11.0
3.8
3.8
3.9
4.5
4.3
3.0
3.6
3.0
2.4
3.2
2.5
2.6
3.1
2.8
4.7
3.2
2.6
4.3
4.1
3.0
0.19
BDL
0.42
BDL
0.27
0.30
0.24
0.28
0.17
0.19
0.26
0.25
0.32
0.40
0.30
0.89
0.28
0.15
0.30
0.33
0.25
0.26
0.13
0.35
0.30
0.30
0.18
0.33
0.23
0.17
0.20
0.15
0.16
0.15
0.24
0.15
0.91
BDL
0.69
BDL
1.70
BDL
BDL
BDL
2.10
1.20
1.10
BDL
1.10
0.90
BDL
1.00
0.68
1.00
BDL
0.80
0.77
BDL
0.45
0.60
0.50
0.90
1.00
0.63
0.91
BDL
BDL
0.63
BDL
BDL
0.60
BDL
29
26
18
21
41
43
33
36
32
35
36
33
34
43
37
100
29
29
38
42
29
27
34
40
34
38
37
38
39
36
55
25
37
47
37
28
0.032
0.005
N/D
N/D
0.023
0.027
0.011
0.018
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
1.00
>0.5(1)
N/A
0.10
>1.0(3)
N/A
0.10
>50(1)
<3.3(1)
0.50
>2000(1)
<160(1)
0.10
>9.0(1)
<2.6(1)
0.10
>6.0(1)
<0.3(1)
0.05
>8.0(3)
N/A
0.10
>200(1)
<18(1)
0.015
>20(3)
N/A
Cases 62, 63, 64, 65, 66, 67, 68, 70, 72, 73 are captive vultures that were experimentally fed diclofenactreated buffalo. As, arsenic; Cd, cadmium; Cu, copper; Fe, iron; Mn, manganese; Mo, molybdenum; Pb,
lead; Zn, zinc; Hg, mercury. BDL, below detection limit; MDL, minimum detection limit. N/D, analysis
not done; N/A, not applicable. Mercury analyses were performed on kidney samples. Abnormal values
are indicated by shaded boxes.
7
Supplementary Table 2 Metal results (wet-weight basis) in vultures without visceral gout
Case
No.
2
3
12
14
28
31
46
47
49
50
51
52
58
Tissue
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
MDL
Toxic level
Deficient level
Metal levels (g/g, wet weight)
As
Cd
Cu
Fe
Mn
Mo
Pb
Zn
Hg
2.60
N/D
BDL
BDL
BDL
N/D
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
N/D
BDL
0.16
0.15
N/D
0.17
0.16
0.11
BDL
BDL
BDL
BDL
13
N/D
6.6
9.4
5.0
N/D
12
30
23
17
26
13
10
74
N/D
880
630
520
N/D
441
77
608
212
174
1807
930
4.7
N/D
3.7
3.0
4.6
N/D
4.3
4.0
3.8
4.6
4.2
6.1
3.3
0.45
N/D
0.23
0.10
0.30
N/D
0.26
0.72
0.21
0.42
0.34
0.49
0.42
BDL
N/D
BDL
13
BDL
N/D
0.94
2.0
0.77
0.85
1.4
1.40
0.84
44
N/D
30
51
42
N/D
43
73
86
44
80
107
36
0.120
0.022
N/D
N/D
N/D
0.014
BDL
BDL
BDL
BDL
BDL
BDL
BDL
1.00
>0.5(1)
N/A
0.10
>1.0(3)
N/A
0.10
>50(1)
<3.3(1)
0.50
>2000(1)
<160(1)
0.10
>9.0(1)
<2.6(1)
0.10
>6.0(1)
<0.3(1)
0.05
>8.0(3)
N/A
0.10
>200(1)
<18(1)
0.015
>20(3)
N/A
As, arsenic; Cd, cadmium; Cu, copper; Fe, iron; Mn, manganese; Mo, molybdenum; Pb, lead; Zn, zinc;
Hg, mercury. BDL, below detection limit; MDL, minimum detection limit. N/D, analysis not done; N/A,
not applicable. Mercury analyses were performed on kidney samples. Abnormal values are indicated by
shaded boxes.
8
Supplementary Table 3 Results of RT-PCR assays for infectious bronchitis, avian
influenza, and West Nile viruses
Case
No.
Gout
Tissue
β-actin
RNA
IBV
AI
WNV
15
15
33
33
35
35
38
40
44
53
55
56
57
60
61
62
63
64
65
71
28
28
46
47
49
50
51
52
58
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
No
No
No
No
No
No
Lung
Kidney
Lung
Kidney
Lung
Kidney
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Lung
Kidney
Lung
Lung
Lung
Lung
Lung
Lung
Lung
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
N/D
Negative
N/D
Negative
N/D
Negative
N/D
Negative
N/D
Negative
N/D
Negative
N/D
Negative
N/D
Negative
N/D
Negative
Negative
N/D
Negative
N/D
Negative
N/D
Cases 62, 63, 64, 65 are captive vultures that were experimentally fed diclofenac-treated
buffalo. IBV, infectious bronchitis virus; AI, avian influenza virus; WNV, West Nile virus; +,
positive for β-actin RNA; Negative, no viral RNA detected; N/D, analysis not done.
9
Supplementary Table 4 Acetylcholinesterase activity results on brain samples
Case No.
15
33
34
35
36
39
40
41
42
44
45
53
54
55
56
57
59
60
61
62
63
64
65
66
67
68
70
71
72
73
74
75
2
12
14
28
32
46
47
49
50
51
52
58
Gout
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
No
No
No
No
No
No
No
No
No
Diagnosis
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
None
Trauma
CNS signs
Lead toxicity
Normal
Diarrhea
Trauma
Trauma
None
None
Diarrhea
Diarrhea
Organophosphate
Brain values
7.7
5.0
7.9
7.2
7.9
13.3
7.5
6.9
13.2
9.6
7.7
10.0
15.9
11.4
9.4
18.6
23.7
16.7
14.4
13.2
10.8
11.8
22.2
13.9
14.3
11.3
11.7
10.8
9.5
9.4
20.7
13.6
12.3
11.5
20.9
10.4
5.5
9.2
7.5
5.2
6.5
14.0
8.2
2.0
ACE activity ( mol/g/min)
Reactivation (1.5 hrs)
Reactivation (24 hrs)
N/D
N/D
9.4 (47%)
8.1 (38%)
7.5 (-5%)
11.8 (33%)
7.0 (-3%)
9.6 (25%)
8.5 (7%)
9.7 (19%)
N/D
N/D
9.4 (20%)
8.0 (6%)
8.2 (16%)
7.4 (7%)
N/D
N/D
10.0 (4%)
10.0 (4%)
8.0 (4%)
7.7 (0%)
11.0 (9%)
10.0 (0%)
N/D
N/D
11.6 (2%)
9.5 (-17%)
11.0 (15%)
12.0 (22%)
18.4 (-1%)
16.2 (-13%)
23.5 (-1%)
20.2 (-15%)
16.7 (0%)
14.3 (-14%)
14.2 (-1%)
12.3 (-15%)
13.6 (3%)
11.8 (-11%)
11.0 (2%)
9.4 (-13%)
11.8 (0%)
10.3 (-13%)
22.4 (1%)
18.9 (-15%)
N/D
N/D
N/D
N/D
N/D
N/D
N/D
N/D
11.2 (4%)
9.6 (-11%)
N/D
N/D
N/D
N/D
N/D
N/D
N/D
N/D
11.8 (-4%)
10.3 (-16%)
11.5 (0%)
10.1 (-12%)
20.9 (0%)
10.1 (-52%)
9.8 (-6%)
9.2 (-12%)
N/D
N/D
10.0 (8%)
10.0 (8%)
7.7 (3%)
6.8 (-9%)
7.0 (26%)
6.3 (17%)
6.9 (6%)
7.1 (8%)
14.0 (0%)
12.7 (-9%)
9.9 (17%)
10.0 (18%)
2.0 (0%)
1.8 (-10%)
Cases 62, 63, 64, 65, 66, 67, 68, 70, 72, 73 are captive vultures that were experimentally fed diclofenac-treated
buffalo. Reactivation is the increase in ACE levels after incubation at 37oC for the indicated time; percent
change relative to original activity shown in parentheses. N/D, analysis not done.
10
Supplementary Table 5 Liquid chromatography and mass spectroscopy analysis for
organophosphate and carbamate pesticides
Case
No.
Gout
Tissue
Post mortem
Interval
Residues (g/g)
16
20
21
33
34
35
36
40
40
44
45
45
53
55
55
56
57
59
60
61
62
63
64
65
71
12
28
46
46
47
47
49
49
50
50
51
51
52
52
58
58
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Liver
Kidney
Kidney
Liver
Liver
Liver
Liver
Liver
S.C.
Liver
Liver
S.C.
Liver
Liver
S.C.
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Brain Cortex
Brain Cortex
Liver
S.C.
Liver
S.C.
Liver
S.C.
Liver
S.C.
Liver
S.C.
Liver
S.C.
Liver
S.C.
5 days
2 days
7 days
< 1 day
< 1 day
>9 hours
>12 hours
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
< 1 day
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
None Detected
11
Aldicarb (0.5), Aldicarb Sulfone (0.5), Aldicarb Sulfoxide (0.5), Ametryn (0.2), Atraton (0.2), Atrazine (0.2),
Baygon/Propoxur (0.5), Bromacil (0.2), Butachlor (0.2), Carbaryl (0.5), Carbofuran (0.5), Chlorpropham (0.2),
Chlorpyriphos/Dursban (0.2), Coumaphos (0.2), Cycloate (0.2), Demeton-S (0.2), Diazinon (0.2), Dichlorovos
(0.2), Dimethoate (0.2), Diphenamid (0.2), Disulfoton (0.2), Disulfoton sulfoxide (0.2), EPTC/Alirox (0.2),
Ethoprop (0.2), Famphur (1.0), Fenamiphos (0.2), Fensulfothion (0.2), Fenthion (0.2), 3-Hydroxycarbofuran
(0.5), Merphos (0.2), Methiocarb (0.5), Methomyl (0.5), Metolachlor (0.2), Methyl Paraoxon (0.2), Metribuzin
(0.2), Mevinphos (0.2), Molinate (0.2), Naled (0.2), Napropamide (0.2), Oxamyl (0.5), Parathion Methyl (0.2),
Pebulate (0.2), Phorate (0.2), Prometon (0.2), Prometryn (0.2), Pronamide (0.2), Simazine (0.2), Stirofos (0.2),
Sulprofos/Bolstar (0.2), Terbacil (0.2), Tebuthiuron (0.2), Terbufos (0.2), Tokuthion (0.2), Triadimefon (0.2)
Cases 62, 63, 64, 65 are captive vultures that were experimentally fed diclofenac-treated
buffalo. The specific compounds tested for, with the detection threshold in μg/g in
parentheses, are indicated in the box. The post mortem interval, which may affect the
recovery of organophosphate and carbamate residues, is also shown. S.C., stomach
contents.
12
Supplementary Table 6 Liquid chromatography and mass spectroscopy analysis for
organochlorine pesticides and polychlorinated biphenyls
Case No.
Gout
Tissue
Residues (g/g)
15
15
16
16
33
34
35
36
39
44
62
65
75
2
12
14
28
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
No
Liver
Fat
Liver
Fat
Brain
Brain
Brain
Brain
Brain
Brain
Brain
Brain
Brain
Fat
Brain
Brain
Fat
None Detected
Lindane (0.35), DDD (0.35), DDE (1.7), DDT (0.10)
DDE (0.24)
Dieldrin (0.06), Lindane (1.7), DDD (1.1), DDE (5.4), DDT (0.07)
None Detected
DDE (0.04)
None Detected
DDE (0.036)
None Detected
None Detected
None Detected
None Detected
None Detected
Lindane (16.00), DDD (23.00), DDE (61.00), DDT (15.00)
DDE (0.07)
DDE (0.09)
Lindane (0.52), DDD (0.55), DDE (4.3), DDT (0.11)
Aldrin (0.03), Chlordane (0.10), Chlorneb (0.25), Chlorobenzilate (0.50), Chlorthalonil (0.08), CisPermethrin (0.50), Dieldrin (0.03), Endosulfan I (0.04), Endosulfan II (0.04), Endosulfan Sulfate (0.05),
Endrin (0.04), Endrin Aldehyde (0.05), Etridiazole (0.40), hexachlorocyclohexane alpha (0.03),
hexachlorocyclohexane beta (0.04), hexachlorocyclohexane delta (0.03), hexachlorocyclohexane gamma
(Lindane, 0.03), Heptachlor (0.04), Heptachlor Epoxide (0.06), Hexachlorobenzene (0.02),
Methoxychlor (0.20), PCB 1016 (1.0), PCB 1221 (1.0), PCB 1232 (1.0), PCB 1242 (1.0), PCB 1248
(1.0), PCB 1254 (1.0), PCB 1260 (1.0), pp’ DDD (0.06), pp’ DDE (0.03), pp’ DDT (0.30), Propachlor
(0.40), and Trans-Permethrin (0.50)
Cases 62, 65 are captive vultures that were experimentally fed diclofenac-treated buffalo.
The specific compounds tested for, with the detection threshold in μg/g in parentheses,
are indicated in the box.
13
Supplementary Table 7 Virus isolation passage protocol and results
Case
No.
Gout
Tissues Tested
15
15
16
20
20
21
21
33
33
34
35
36
44
60
62
75
2
2
12
12
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
No
Kidney, spleen, lung
intestine
Kidney, spleen, lung
Kidney, spleen, lung
intestine
Kidney, spleen, lung
intestine
kidney
Spleen, lung
Kidney, spleen, lung
Kidney, spleen, lung
Kidney, spleen, lung
Kidney, spleen, lung
Kidney, spleen, lung
Kidney, spleen, lung
Kidney, spleen, lung
Kidney, spleen
intestine
Spleen, lung
intestine
Cells Used (# passes)
DEF (4), CEF (4), PEF (3)
DEF (2), CEF (3)  PEF (2)
DEF (4), CEF (4)
DEF (4), CEF (4), PEF (3)
DEF (2), CEF (3)  PEF (2)
DEF (4), CEF (4), PEF (3)
DEF (2), CEF (3)  PEF (2)
DEF (4), CEF (4)
DEF (4), CEF (4)
DEF (4), CEF (4)
DEF (4), CEF (4)
DEF (4), CEF (4)
CEF (3), VERO (3), DEF (3)
CEF (3), VERO (3), DEF (3)
CEF (3), VERO (3), DEF (3)
CEF (3), VERO (3), DEF (3)
DEF (4), CEF (4), PEF (3)
DEF (2), CEF (3)  PEF (2)
DEF (4), CEF (4), PEF (3)
DEF (2), CEF (3)  PEF (2)
Result
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Mycoplasma
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Case 62 is a captive vulture that was experimentally fed diclofenac-treated buffalo. DEF,
duck embryo fibroblasts; CEF, chicken embryo fibroblasts; PEF, Peregrine Falcon
embryo fibroblasts; Vero, African Green Monkey kidney cell line. Number of passages
per cell culture type indicated in parentheses. Arrows indicate using the last passage in
previous cell type to start series of passages in the new cell type.