Abstract
Mycobacterium tuberculosis has been known to infect elephants for approximately 2,000 years and currently infects approximately 3% of elephants housed in captivity in North America.5 The United States Department of Agriculture (USDA) has set and updated guidelines for the control of tuberculosis in elephants covered under the Animal Welfare Act.8 Diagnosis at this time is based strictly on a single required test, Mycobacterium tuberculosis culture. This is markedly different than diagnosis in humans which includes screening of high-risk populations or individuals with clinical signs using tuberculin testing, thoracic radiographs, and sputum cytology which is followed by culture.3
Intradermal testing in Asian elephants (Elephas maximus) has had poor correlation with culture results.6 Thoracic radiographs in a 6,000–12,000-pound animal are not realistic. Sputum cytology is recommended in the USDA guidelines but not required. Additionally, nucleic acid amplification tests (NAAT) currently used in humans are used to diagnose M. tuberculosis complex (the mammalian pathogens), which includes M. tuberculosis, M. bovis, M. africanum, and M. microti. These different species are all included in the reported 95% specificity of NAAT when compared with acid-fast sputum smears and culture. These would not be applicable in the specific diagnosis of the species M. tuberculosis in elephants. Additionally, NAAT is currently only used in human medicine in combination with cytology.3 ELISA testing in combination with gamma interferon testing is promising but not currently validated. It can be costly and is only run in a few laboratories across the country.4
An evaluation of standard blood work was undertaken to determine if there are any values that could be identified as consistently different between culture positive elephants and clinically normal elephants (Tables 1, 2). These values, though not providing a definitive diagnosis of M. tuberculosis, may function to flag affected animals within a population.
Table 1. Hematologic and serum biochemical reference ranges for Asian elephants (Elephas maximus) with a negative TB status
|
|
n
|
Mean
|
Standard deviation
|
Reference range
|
|
A:G ratio
|
31
|
0.75
|
0.12
|
0.6–1.1
|
|
Gel A:G ratio
|
31
|
0.69
|
0.12
|
0.45–1.0
|
|
Total prot (g/dl)
|
31
|
8.1
|
1.11
|
5.4–10.1
|
|
Albumin (g/dl)
|
31
|
3.4
|
0.36
|
2.6–4.4
|
|
Globulin (g/dl)
|
31
|
4.6
|
0.88
|
2.9–6.3
|
|
Pprot (g/dl)
|
17
|
8
|
0.66
|
6.7–9.3
|
|
Ael alb (g/dl)
|
31
|
3.3
|
0.43
|
2.1–4.4
|
|
ALPHA1 (g/dl)
|
31
|
0.6
|
0.11
|
0.4–0.9
|
|
ALPHA2 (g/dl)
|
31
|
0.5
|
0.16
|
0.3–0.9
|
|
BETA1 (g/dl)
|
31
|
1.4
|
0.46
|
0.6–2.6
|
|
BETA2 (g/dl)
|
31
|
1.3
|
0.25
|
0.9–2.0
|
|
GAMMA (g/dl)
|
31
|
0.9
|
0.31
|
0.5–1.4
|
|
Na (mEq/L)
|
20
|
125
|
6.32
|
109–141
|
|
K (mEq/L)
|
20
|
4.5
|
0.34
|
4.1–5.3
|
|
Cl (mEq/L)
|
20
|
90
|
3.17
|
83–97
|
|
TCO2 (mEq/L)
|
20
|
24
|
1.86
|
20–28
|
|
Anion gap
|
20
|
15.5
|
4.4
|
7–24
|
|
BUN (mg/dl)
|
20
|
10.8
|
2.81
|
5–16
|
|
Creat (mg/dl)
|
20
|
1.4
|
0.2
|
0.9–1.6
|
|
Ca (mg/dl)
|
20
|
10.2
|
0.88
|
8.2–12.0
|
|
Glucose (mg/dl)
|
20
|
80
|
13.92
|
63–124
|
|
PO4 (mg/dl)
|
20
|
4.3
|
0.82
|
3.3–6.1
|
|
Total bilirubin (mg/dl)
|
20
|
0.16
|
0.06
|
0.1–0.2
|
|
Triglycerides (mg/dl)
|
20
|
52
|
54
|
18–160
|
|
Alk phos (IU/L)
|
20
|
116
|
37.19
|
53–204
|
|
AST (IU/L)
|
20
|
13
|
4.6
|
7–25
|
|
GGT (IU/L)
|
20
|
8.6
|
3.36
|
5–18
|
|
RBC (x106/µl)
|
20
|
2.9
|
0.4
|
2.1–3.6
|
|
HGB (g/dl)
|
20
|
12.3
|
1.26
|
9.6–15.3
|
|
PCV (%)
|
20
|
35.7
|
3.79
|
27–44
|
|
MCV (fL)
|
20
|
125
|
7.42
|
111–138
|
|
MCHC (g/dl)
|
20
|
34.7
|
0.58
|
33.4–35.7
|
|
RDW
|
18
|
16.8
|
1.08
|
14.9–18.7
|
|
PLT (x103/µl)
|
18
|
513
|
98.43
|
342–719
|
|
WBC (x103/µl)
|
20
|
13.6
|
2.7
|
8.6–18.2
|
|
Fibrinog (g/dl)
|
20
|
400
|
137.17
|
100–600
|
|
Bands/µl
|
20
|
0
|
0
|
0.00–0.00
|
|
Segs/µl
|
20
|
3180
|
820.59
|
1800–4300
|
|
Lymphs/µl
|
20
|
2650
|
1456.64
|
990–5000
|
|
Monos/µl
|
20
|
7140
|
2751.96
|
1600–12600
|
|
Eos/µl
|
20
|
220
|
172.94
|
0–580
|
|
Basos/µl
|
20
|
50
|
60.88
|
0–150
|
Table 2. Hematologic and serum biochemical reference ranges for Asian elephants (Elephas maximus) with a positive TB status (n=5)
| |
Mean
|
Standard deviation
|
Reference range
|
|
A:G ratioa
|
0.56
|
0.04
|
0.5–0.6
|
|
Gel A:G ratio
|
0.66
|
0.11
|
0.52–0.81
|
|
Total prot (g/dl)
|
8.5
|
0.26
|
7.9–8.9
|
|
Albumin (g/dl)
|
3.2
|
0.3
|
2.7–3.5
|
|
Globulin (g/dl)
|
5.3
|
0.07
|
5.2–5.4
|
|
Gel alb (g/dl)
|
3.4
|
0.44
|
2.8–3.5
|
|
ALPHA1 (g/dl)
|
0.7
|
0
|
0.7 (all five animals)
|
|
ALPHA2 (g/dl)
|
0.7
|
0.04
|
0.6–0.7
|
|
BETA1 (g/dl)
|
1.2
|
0.47
|
0.5–1.5
|
|
BETA2 (g/dl)
|
1.5
|
0.46
|
1.1–2.1
|
|
GAMMA (g/dl)
|
1.1
|
0.22
|
0.8–1.4
|
|
Na (mEq/L)
|
122
|
20.77
|
86–150
|
|
K (mEq/L)
|
4.3
|
0.37
|
3.9–4.8
|
|
Cl (mEq/L)
|
93
|
3.67
|
89–98
|
|
TCO2a (mEq/L)
|
25.7
|
2.13
|
23–28
|
|
Anion Gap
|
15.9
|
8.03
|
7.5–25
|
|
BUN (mg/dl)
|
11.2
|
2.28
|
9–14
|
|
Creat (mg/dl)
|
1.4
|
0.32
|
1.0–1.7
|
|
Caa(mg/dl)
|
11.3
|
1.58
|
9.9–13.9
|
|
Glucose a (mg/dl)
|
65.6
|
11.76
|
52–83
|
|
PO4 (mg/dl)
|
4.7
|
0.64
|
4.1–5.8
|
|
T bili (mg/dl)
|
0.2
|
0.15
|
0.1–0.4
|
|
Glyceride (mg/dl)
|
40
|
33.18
|
14–86
|
|
Alk phos (IU/L)
|
109
|
24.34
|
86–138
|
|
AST (IU/L)
|
16.4
|
4.62
|
10–22
|
|
GGT (IU/L)
|
9.4
|
1.34
|
8–11
|
|
RBC (x106/µl)
|
3.1
|
0.19
|
2.85–3.30
|
|
HGB (g/dl)
|
12.68
|
1.65
|
10.5–14.7
|
|
PCV (%)
|
36.4
|
5.03
|
30–42
|
|
MCV (fL)
|
120.2
|
7.92
|
110–129
|
|
MCHCa(g/dl)
|
33.9
|
0.57
|
33.4–34.8
|
|
RDW
|
17.24
|
1.5
|
16.0–19.2
|
|
PLTa(x103/µl)
|
616.6
|
48.11
|
540–658
|
|
WBC (x103/µl)
|
16.48
|
6.09
|
10.0–25.9
|
|
Fibrinog (mg/dl)
|
460
|
230.22
|
200–700
|
|
Bands/µla
|
88
|
85.26
|
0–170
|
|
Segs/µl
|
4712
|
3475.08
|
2300–10620
|
|
Lymphs/µl
|
1564
|
934.2
|
670–2850
|
|
Monos/µl
|
9690
|
2474.68
|
6690–12000
|
|
Eos/µla
|
434.2
|
206.91
|
200–700
|
|
Basos/µl
|
52
|
116.28
|
36894
|
aIndicates values that are statistically different (p<0.05).
Approximately 50 Asian elephants were routinely evaluated over a 2-year time period. Appetite, behavior, and physical condition were recorded daily. Routine veterinary physical examination including blood work was performed annually for healthy animals and on an as needed basis for sick animals. A data set of healthy animals was established. Any animal that showed any signs of illness including decreased appetite, weight loss, clinical disease, or significant changes from baseline blood work was excluded from the data set. The data set of 20 healthy elephants was then compared to samples taken from five culture-positive Mycobacterium tuberculosis elephants when they were actively shedding organisms. Parametric values were compared using a Student’s t test for two groups. Nonparametric values were compared using a Wilcoxon rank sum. Albumin:globulin (A:G) ratios were further evaluated for sensitivity and specificity using a receiver operating characteristic (ROC) curve.2 Albumin was determined using a bromcresol green method and total protein was determined using a pyramolol red method on a Hitachi 911 (Boehringer Mannheim, Indianapolis, Indiana, 46250 USA). Globulin was calculated by subtracting the two. Protein serum electrophoresis of the sample was performed using an appraise densitometer system (Beckman Coulter, Inc. Fullerton, CA 92634 USA).
Cytochemical staining of the leukocytes revealed that a bilobed cell type unique to elephants is lightly chloroacetate esterase positive and strongly peroxidase positive. This is consistent with recent previous literature.1,7 This cell is therefore classified as a monocyte and is reported as such in the tables above. Misidentification of these cells may result in underreporting of monocytes and increased reporting of lymphocytes.
Values that were significantly lower in positive animals (p≤0.05) included A:G ratio, mean cell hemoglobin concentration (MCHC), and glucose. Values which were significantly higher in positive animals (p≤0.05) included platelets, band neutrophils, eosinophils, calcium, and bicarbonate (TCO2). Two values, red blood cell numbers and segmented neutrophils, were equivocally higher.
The A:G ratio is a measure of general inflammation in the body. It was therefore of particular interest in these animals and was further statistically evaluated. A ROC curve is a plot of sensitivity versus specificity over all possible decision thresholds. A ROC curve describes compromises in sensitivity and specificity independent of the prevalence of disease.6 When the A:G ratio, determined using the Hitachi 911, was analyzed with a ROC curve, the value of 0.7 was determined to be the most effective decision threshold. This value had a 100% sensitivity and a 77% specificity in the population examined. The area under the curve (AUC) is considered to be the overall summary measure in a ROC curve. An AUC of 0.5 is considered to be a useless test while an AUC of 1.0 is considered to be a perfect diagnostic test. The A:G ratio determined using the Hitachi 911 had an AUC of 0.91. The gel A:G ratio had an AUC of 0.55. Additionally, animals that were initially negative all had a decreased A:G ratio at the time of positive trunk wash in comparison to baseline A:G ratio values for those animals.
Literature Cited
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