# Evaluation of Serodiagnostic Assays forMycobacterium

Authors: Jeffrey T. Nelson; Kathleen A. Orloski; Audra L. Lloyd; Mark Camacho; Mark A. Schoenbaum; Suelee Robbe-Austerman; Bruce V. Thomsen; S. Mark Hall
Journal: Veterinary Medicine International
 (2012)

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## Abstract

In 2011, the United States Department of Agriculture conducted a project in which
elk (Cervus elaphus spp.), white-tailed deer (WTD)
(Odocoileus virginianus), and reindeer (Rangifer
tarandus) were evaluated by the single cervical tuberculin test
(SCT), comparative cervical tuberculin test (CCT), and serologic tests. The
rapid antibody detection tests evaluated were the CervidTB Stat-Pak (Stat-Pak),
and the Dual Path Platform VetTB (DPP). Blood was collected from presumably
uninfected animals prior to tuberculin injection for the SCT. A total of 1,783
animals were enrolled in the project. Of these, 1,752 (98.3%) were
classified as presumably uninfected, based on originating from a captive cervid
herd with no history of exposure to TB. Stat-Pak specificity estimates were
92.4% in reindeer, 96.7% in WTD, and 98.3% in elk and were not
significantly different from SCT specificity estimates. Using the DPP in series
on Stat-Pak antibody-positive samples improved specificity in the three species.
Thirty one animals were classified as confirmed infected, based on necropsy and
laboratory results, and 27/31 were antibody positive on Stat-Pak for an
estimated sensitivity of 87.1%. The study findings indicate that rapid
serologic tests used in series are comparable to the SCT and CCT and may have a
greater ability to detect TB-infected cervids.


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## Body

## 1. Introduction

Mycobacterium bovishas been detected sporadically in captive
cervids in the United States and is enzootic in free-ranging white-tailed deer (WTD)
(Odocoileus virginianus) in a small geographic area of Michigan
[1, 2]. A multistate outbreak of M. bovis involving 37
captive cervid herds occurred in the United States during 1990–1999 [3]. The cervid species involved in this outbreak
included elk (Cervus elaphusspp.), red deer
(Cervus elaphus), fallow deer (Dama dama), and
sika deer (Cervus nippon). M. bovis was detected
again, beginning in 2009, in elk, red deer, and fallow deer in 4 herds located in
Nebraska and Indiana [4, 5].Testing captive cervids forM. bovis in the United States is
conducted as part of an official disease eradication program administered by the
United States Department of Agriculture (USDA). The single cervical tuberculin skin
test (SCT) was first evaluated for use in elk in the United States in 1991 [6]. Captive cervids were not routinely tested
for bovine tuberculosis (TB) until these species were officially brought into the
federal tuberculosis program in 1994 [7].
Currently, testing for TB in cervids is performed using tuberculin skin tests. The
SCT is the initial test and consists of the intradermal administration of
bovine-purified protein derivative (PPD) tuberculin in the midcervical region [8]. Animals with any detectable responses are
tested by the comparative cervical tuberculin skin test (CCT), in which balanced
bovine and avian PPD tuberculins are injected, the pre- and postinjection skin
thickness is measured, and the measurements are plotted on a graph [8]. Areas of the graph classify the animal as
negative, suspect, or reactor. In cervids, the CCT must be administered within 10
days or after 90 days of the SCT tuberculin injection.Limited and at times conflicting information is available regarding the performance
of TB skin testing in captive cervid species. A comprehensive evaluation of cervid
TB testing estimated the individual animal specificity of the SCT and CCT used in
series to be 87.1 and 90.4% for deer and elk, respectively [9]. Individual animal sensitivity of the SCT
and CCT in series could not be estimated; however, 14 elk from 12 infected herds
detected between 1991 and 1996 were CCT test negative and subsequently confirmed to
be infected with M. bovis. More recently, 25/28 confirmed infected
Nebraska elk had a false negative result on the SCT [4]. The estimated specificity of the CCT in reindeer (Rangifer
tarandus) is significantly different than nonreindeer cervids, with
reindeer approximately four times more likely to test false positive on the CCT
[10]. Eleven experimentally infected
reindeer tested positive by the CCT at three and eight months after infection [11]; however, estimates of sensitivity of CCT
in naturally infected reindeer are not possible as TB is extremely rare in this
species.In addition to concerns regarding TB skin test performance, animal handling
challenges resulting in animal morbidity and mortality are not uncommon. With TB
skin testing, captive cervids may be required to be captured and restrained for
testing up to four different times depending on test results. Serologic testing
offers the advantage over skin testing of reduced animal handling, with a reduction
in the associated morbidity and mortality. An additional advantage is eliminating
the subjectivity of interpreting the skin response at the tuberculin injection site.
For these reasons, there is potential for improved surveillance in captive cervids
using a serologic test with adequate performance.In 2009, the CervidTB Stat-Pak lateral flow test (Stat-Pak, Chembio Diagnostic
Systems, Inc., Medford, New York) was licensed for use in elk and red deer. The
Stat-Pak is a rapid point-of-care test that utilizes single-directional lateral-flow
serological antibody detection technology and a cocktail of recombinantM.
bovisantigens [12]. In addition
to the Stat-Pak, Chembio has two additional antibody detection assays for TB, the
multiantigen print immunoassay (MAPIA) and the recently developed dual path platform
VetTB (DPP) assay [13]. The MAPIA is
performed in the laboratory as a confirmatory testing method for samples with
antibody-positive results on the Stat-Pak. The DPP is a new-generation point-of-care
test format that offers improved specificity compared to the Stat-Pak assay with
similar sensitivity in red deer, elk, and fallow deer [4, 14].The primary objective of this study was to evaluate the Stat-Pak as a primary test
for use in the diagnosis of TB in captive and free-ranging elk, WTD, and reindeer.
In addition, the DPP was evaluated as a followup test for Stat-Pak antibody-positive
samples, and the two serologic test results were compared to TB skin test
results.


## 2. Methods

Blood samples from two sources were tested as part of the project: blood collected
prospectively during triennial herd TB accreditation testing and banked serum. The
species evaluated included elk, WTD, and reindeer. Banked serum was from the TB
serum bank located at the National Veterinary Services Laboratories (NVSL) in Ames,
Iowa.Banked serum samples were derived from animals sampled during 2008–2010 and
included samples from elk, WTD, and reindeer with corresponding SCT results. Most
banked samples were from captive cervid herds with no history of exposure to TB with
the exception of one infected elk herd that was detected and depopulated in 2009.
Some samples reported by Waters et al. [4]
were retested for the sensitivity portion of this project. Prospectively enrolled
animals were sampled during calendar year 2011. Captive cervid producers were
recruited to enroll animals in the project through producer organizations. In
addition, free-ranging cervids being TB tested (such as for wildlife restoration
projects) were enrolled. The animals sampled prospectively came from 51 separate
premises, including 16 for elk, 3 for reindeer, and 32 for WTD herds.Accredited or regulatory veterinarians collected blood samples. Blood was collected
into 10 mL serum collection tubes or serum separator tubes on the day of
tuberculin injection for the SCT. An exception to these methods occurred with the
infected elk herd; SCT testing was conducted three months prior to the animals being
euthanized and necropsied, and blood collection was done immediately after
postmortem at necropsy. Blood samples were centrifuged, separated, and shipped to
NVSL. Alternatively, unseparated blood was cooled and shipped overnight to NVSL,
where the serum was separated. Copies of the completed SCT results were submitted
with blood or serum [8].Prospectively sampled animals that were antibody positive on the Stat-Pak were
further evaluated by the CCT and were later necropsied. However, for some cases, the
owners chose not to have their animals CCT tested and euthanized. Animals that
responded to the SCT test were administered a CCT according to current USDA TB
program requirements, regardless of Stat-Pak results (Table1) [15]. The USDA TB
program requires that CCT-positive animals are euthanized and necropsied, with
tissue samples taken for histopathology and bacteriologic culture; CCT-negative
animals are released from quarantine.Table 1
Decision algorithm for supplemental testing.
Single cervical tuberculin (SCT) test
result
Stat-Pak result
SCT negative
SCT positive
Negative
No additional testing
Follow USDA TB program regulations [15]
Positive
MAPIA and DPP, purchase with indemnity
MAPIA and DPP, follow USDA TB program regulationsTissue samples from necropsied animals were divided, and the two sections were
submitted separately in 10% buffered formalin and a saturated sodium borate
solution for histopathology and bacteriologic culture, respectively. Head and
thoracic cavity lymph nodes and any lesioned tissues were submitted from necropsied
animals. No followup was conducted on animals testing negative on the Stat-Pak,
although some reindeer sampled in 2011 were subsequently slaughtered and inspected.
Histopathology and bacteriologic culture were conducted at the NVSL.Tissues submitted in 10% buffered formalin were initially evaluated for lesions
and the presence of acid-fast bacilli by histopathology. Tissues were diagnosed as
compatible with mycobacteriosis when granulomas were identified that contained
acid-fast bacilli. Tissues for culture were prepared as previously described [16]. All submitted tissues were cultured using
at least one liquid media system (BACTEC 460 or MGIT 960) and 2 tubes of a modified
Middlebrook 7H11 supplemented with calf serum, hemolyzed blood, pyruvate, and
malachite green.The Stat-Pak was performed at NVSL per the manufacturer’s instructions.
Serologic and TB testing in the infected elk herd have been previously described
[4]; however, in this study, serum samples
from this herd were tested by the Stat-Pak at NVSL. All sera with an
antibody-positive result on the Stat-Pak were further tested via the MAPIA and DPP
at Chembio, Inc., in Medford, New York as described previously [12–14].Animals that are necropsied as a result of official TB testing or antibody-positive
Stat-Pak results and have microscopic lesions that are compatible for
mycobacteriosis, and/orM. bovisis isolated from tissue, were
classified as TB infected. Animals were classified as presumably uninfected if they
were from herds with no history of exposure to TB and resided in states declared
free of TB in cattle. Animals that did not meet the case definition for confirmed TB
infected but were from herds with infected animals were classified as exposed and
not included in the analysis.The sample size estimates were derived from Greiner and Gardner [17]. Sensitivity was defined
as test positives/test positives + test negatives using the sample of animals that
met the case definition for confirmed TB infected. Specificity was defined as test
negatives/test negatives + test positives for animals meeting the case definition
for presumably uninfected. Ninety five % confidence intervals for proportions
were calculated using the F statistic function in Excel (function = F.INV.RT,
Microsoft, 2010). Tests of statistical significance were performed using the
chi-square, Fischer exact and mid-p exact, and were calculated online using Open Epi
(http://www.openepi.com/OE2.3/Menu/OpenEpiMenu.
htm).
Results were considered statistically significant for P values <
0.05.


## 3. Results

A total of 1,783 animals were tested by the Stat-Pak, including 873 elk, 725 WTD, and
185 reindeer. Of these, 1,752 (98.2%) were classified as presumably uninfected,
and 31 animals (elk from one affected herd) were classified as confirmed TB
infected.


### 3.1. Presumably Uninfected Animals

Of the samples from presumably uninfected animals, 52/1752 (3.0%) were
antibody positive by the Stat-Pak assay, including 14/842 elk (1.7%),
24/725 WTD (3.3%), and 14/185 reindeer (7.6%) (Table2). Twenty six of 853 (3.1%) samples
from banked serum and 26/899 (2.9%) samples collected prospectively were
antibody positive by the Stat-Pak assay. Of the 26 antibody-positive animals
sampled prospectively, 9 were elk, 13 were WTD, and 4 were reindeer. Of these, a
necropsy was completed on 7 elk, 4 WTD, and 1 reindeer with no evidence of
M. bovis infection. For the remaining 14 animals, the
owners declined to have the animals euthanized for necropsy. One reindeer and
one elk had a histopathologic diagnosis of microgranuloma. Two elk had a
histopathologic diagnosis of lymphoid hyperplasia. Mycobacterium
intracellulare and an unidentified atypical mycobacterium were
isolated from the elk with lymphoid hyperplasia; M.
intracellulare was isolated from an elk with no significant
findings on histology.Table 2
Stat-Pak results in presumably uninfected animals.
Species
Sex
Stat-Pak Negative
Stat-Pak Antibody Positive
Total Tested
Percent Positive
95% Confidence Interval
Elk
Female
428
7
435
1.6%
0.6–3.3%
Male
338
3
341
0.9%
0.2–2.5%
Not known
62
4
66
6.1%
—
Elk Total
828
14
842
1.7%
0.9–2.8%
Reindeer
Female
138
12
150
8.0%
4.22–13.6%
Male
33
2
35
5.7%
0.7–19.2%
Reindeer Total
171
14
185
7.6%
4.2–12.4%
WTD*
Female
391
17
408
4.2%
2.4–6.6%
Male
233
6
239
2.5%
0.9–5.4%
Not known
77
1
78
1.3%
—
WTD Total
701
24
725
3.3%
2.1–4.9%
Grand Total
1700
52
1752
3.0%
2.2–3.9%
*WTD = white-tailed deer.The estimated specificity of the Stat-Pak by species is 98.3% in elk,
96.7% in WTD, and 92.4% in reindeer in this study (Table2). Specificity was significantly
different for the three species (P
=
0.00008, uncorrected chi-square). SCT data from USDA TB
Program official testing during FY 2009 provided an estimated specificity of
98.5% for elk, 97.4% for WTD, and 82.7% for reindeer, from a
sample of over 6,700 presumably uninfected animals (Table 3). For elk and WTD, the estimated specificity of the
Stat-Pak compared to the FY 2009 SCT was not significantly different
(P
>
0.05, mid-p exact). In reindeer, significantly fewer
animals were antibody positive by the Stat-Pak compared to responders on the SCT
(P
=
0.024, mid-p exact). The USDA TB Program testing
requires that SCT responders be administered the CCT test. During routine USDA
TB Program testing during FY 2009-2010, 5/123 (4.1%) elk were positive on
the CCT, and 118 were negative. For WTD, 6/119 (5.0%) animals were positive
on the CCT, and 113 animals were classified negative. In reindeer, 19/19 CCT
tested animals were negative. The specificity of the SCT and CCT tests in series
could not be calculated.Table 3
Single cervical tuberculin test response fraction in elk, WTD and
reindeer in federal fiscal year (FY) 2009.
Species
Total Tested
Responders
Percent Responders
95% Confidence Interval
Elk
3,223
47
1.5%
1.1–1.9
WTD*
3,421
89
2.6%
2.1–3.2
Reindeer
81
14
17.3%
9.8–27.30
Total
6,725
150
2.2%
1.9–2.6
*WTD = white-tailed deer.USDA has reported the estimated specificity of the SCT and CCT used in series to
be 87.1% (95% CI 84.5–89.4) in deer and 90.4% (95% CI
87.4–92.9) in elk and red deer [9]. This was significantly lower than the estimated specificity of the
Stat-Pak and DPP used in series for WTD and elk (P
<
0.0001, mid-p exact). A second study by Norden et al.
[18] reported higher specificity
estimates for the SCT and CCT in series of 98.3% (95% CI
96.0–99.5) in deer and 99.5% (95% CI 95.4–98.6) in elk
and red deer, in which animals classified as “suspects” (weak
positives) on the CCT were reclassified as negative. Comparing the 1997 SCT and
CCT in series results to the current study, the specificity estimates for the
Stat-Pak and DPP in series were not significantly different for WTD but were
different for elk (P
=
0.000036, mid-p exact), with the Stat-Pak/DPP
combination having a higher specificity than the SCT/CCT. Test performance for
reindeer was also reported in Norden et al. [18], where 29/29 reindeer were negative when tested in series by the
SCT and CCT, compared to 182/185 Stat-Pak/DPP test negatives in the present
study.Nearly 62% of the animals evaluated were female (993/1608). The highest
percentage of female animals occurred in reindeer (150/185 female) (Table2). Gender was not reported for 144
animals. When stratified by species, test performance was not significantly
different between females and males in this study (P
=
0.575, elk; P
=
0.969, reindeer; P
=
0.282, WTD; mid-p exact).Samples collected during 2011 were used to compare the performance of the
Stat-Pak with the SCT (Table4). A
total of 879 animals had results for both the SCT and the Stat-Pak. For elk,
7/344 (2.0%) animals were SCT positive, while 9/344 (2.6%) were
antibody positive on the Stat-Pak. For WTD, 13/494 (2.6%) and 13/494
(2.6%) animals were SCT positive and Stat-Pak antibody positive,
respectively. SCT results were available for 41 reindeer. Of these, 9/41
(22.0%) and 4/41 (9.8%) were SCT positive and Stat-Pak antibody
positive, respectively. Three reindeer and two elk were SCT positive and
antibody positive on the Stat-Pak. In all other cases, the Stat-Pak
antibody-positive animals were different individuals than the SCT-positive
animals. CCT tests were administered to 11 Stat-Pak antibody-positive animals.
Three elk, four reindeer, and two WTD were CCT negative. Two WTD were classified
as positive on the CCT.Table 4
Comparison of single cervical tuberculin skin test (SCT) and the
Stat-Pak, presumably uninfected animals, by species in 2011.
SCT Result*
Stat-Pak Negative
Stat-Pak Positive
Total
Elk
Negative
330
7
337
Positive
5
2
7
Elk, total
335
9
344
White-tailed deer (WTD)
Negative
468
13
481
Positive
13
0
13
WTD, total
481
13
494
Reindeer
Negative
29
2
31
Positive
7
3
10
Reindeer, total
36
5
41
Total
852
27
879
*Includes only animals sampled prospectively during 2011.Fifty two samples antibody positive on Stat-Pak were tested by the DPP and MAPIA
(Table5). Both assays use an expanded
panel of antigens in addition to the antigens included in the Stat-Pak, to
improve specificity. The largest improvement in specificity was observed in elk,
where 13/14 samples (each antibody positive on Stat-Pak) were negative by the
MAPIA, and only one sample was positive (Table 5). In WTD, 13/24 (54.2%) antibody positive on the Stat-Pak
were negative on the MAPIA. In reindeer, 11/14 (78.6%) antibody positive on
the Stat-Pak were negative on the MAPIA. The DPP had similar results to the
MAPIA. The increase in specificity estimates from the Stat-Pak only to the
combination of the Stat-Pak and DPP tests used in series were 98.3% to
100.0% (842/842, 95% confidence interval (CI) 99.6–100.0) in
elk; 96.7% to 99.3% (720/725, 95% CI 98.4–99.8) in WTD;
92.4% to 98.4% (182/185, 95% CI 95.3–99.7) in
reindeer.Table 5
Results of multi-antigen print immunoassay (MAPIA) and dual-path platform
VetTB (DPP) for Stat-Pak antibody positive samples.
MAPIA Results*
DPP Results
Elk
Reindeer
WTD
Total
Neg
Pos
Neg
Pos
Neg
Pos
Negative
13
1
10
1
13
6
44
Positive
0
0
0
3
0
5
8
Total
13
1
10
4
13
11
52
*Neg = negative, Pos = positive, WTD = white-tailed deer.


### 3.2. Serology Test Performance in a TB-Affected Elk Herd

A total of 34 animals from a known TB-affected Nebraska elk herd were evaluated
for the project. Thirty one animals met the case definition for confirmed TB
infected and had a serum sample available for inclusion in the project. Of
these, 27 (87.1%) were antibody-positive by the Stat-Pak, and followup
testing resulted in 26 and 27 antibody positive samples by the MAPIA and DPP,
respectively. Twenty of the 31 confirmed infected animals were SCT tested with
negative results three months prior to being necropsied. Among these 20 SCT
negative animals, 18 (90.0%) were Stat-Pak antibody positive, and 2 were
negative. Three animals from the infected elk herd did not meet the definition
for confirmed TB infection; these animals were considered exposed to TB and were
excluded from the analysis. Lesioned tissues from these 3 animals were
compatible for mycobacteriosis by histopathology, butM. bovis
was not isolated. For these 3 animals, two serum samples were negative, and one
was antibody positive on the Stat-Pak and the DPP.


## 3.1. Presumably Uninfected Animals

Of the samples from presumably uninfected animals, 52/1752 (3.0%) were
antibody positive by the Stat-Pak assay, including 14/842 elk (1.7%),
24/725 WTD (3.3%), and 14/185 reindeer (7.6%) (Table2). Twenty six of 853 (3.1%) samples
from banked serum and 26/899 (2.9%) samples collected prospectively were
antibody positive by the Stat-Pak assay. Of the 26 antibody-positive animals
sampled prospectively, 9 were elk, 13 were WTD, and 4 were reindeer. Of these, a
necropsy was completed on 7 elk, 4 WTD, and 1 reindeer with no evidence of
M. bovis infection. For the remaining 14 animals, the
owners declined to have the animals euthanized for necropsy. One reindeer and
one elk had a histopathologic diagnosis of microgranuloma. Two elk had a
histopathologic diagnosis of lymphoid hyperplasia. Mycobacterium
intracellulare and an unidentified atypical mycobacterium were
isolated from the elk with lymphoid hyperplasia; M.
intracellulare was isolated from an elk with no significant
findings on histology.Table 2
Stat-Pak results in presumably uninfected animals.
Species
Sex
Stat-Pak Negative
Stat-Pak Antibody Positive
Total Tested
Percent Positive
95% Confidence Interval
Elk
Female
428
7
435
1.6%
0.6–3.3%
Male
338
3
341
0.9%
0.2–2.5%
Not known
62
4
66
6.1%
—
Elk Total
828
14
842
1.7%
0.9–2.8%
Reindeer
Female
138
12
150
8.0%
4.22–13.6%
Male
33
2
35
5.7%
0.7–19.2%
Reindeer Total
171
14
185
7.6%
4.2–12.4%
WTD*
Female
391
17
408
4.2%
2.4–6.6%
Male
233
6
239
2.5%
0.9–5.4%
Not known
77
1
78
1.3%
—
WTD Total
701
24
725
3.3%
2.1–4.9%
Grand Total
1700
52
1752
3.0%
2.2–3.9%
*WTD = white-tailed deer.The estimated specificity of the Stat-Pak by species is 98.3% in elk,
96.7% in WTD, and 92.4% in reindeer in this study (Table2). Specificity was significantly
different for the three species (P
=
0.00008, uncorrected chi-square). SCT data from USDA TB
Program official testing during FY 2009 provided an estimated specificity of
98.5% for elk, 97.4% for WTD, and 82.7% for reindeer, from a
sample of over 6,700 presumably uninfected animals (Table 3). For elk and WTD, the estimated specificity of the
Stat-Pak compared to the FY 2009 SCT was not significantly different
(P
>
0.05, mid-p exact). In reindeer, significantly fewer
animals were antibody positive by the Stat-Pak compared to responders on the SCT
(P
=
0.024, mid-p exact). The USDA TB Program testing
requires that SCT responders be administered the CCT test. During routine USDA
TB Program testing during FY 2009-2010, 5/123 (4.1%) elk were positive on
the CCT, and 118 were negative. For WTD, 6/119 (5.0%) animals were positive
on the CCT, and 113 animals were classified negative. In reindeer, 19/19 CCT
tested animals were negative. The specificity of the SCT and CCT tests in series
could not be calculated.Table 3
Single cervical tuberculin test response fraction in elk, WTD and
reindeer in federal fiscal year (FY) 2009.
Species
Total Tested
Responders
Percent Responders
95% Confidence Interval
Elk
3,223
47
1.5%
1.1–1.9
WTD*
3,421
89
2.6%
2.1–3.2
Reindeer
81
14
17.3%
9.8–27.30
Total
6,725
150
2.2%
1.9–2.6
*WTD = white-tailed deer.USDA has reported the estimated specificity of the SCT and CCT used in series to
be 87.1% (95% CI 84.5–89.4) in deer and 90.4% (95% CI
87.4–92.9) in elk and red deer [9]. This was significantly lower than the estimated specificity of the
Stat-Pak and DPP used in series for WTD and elk (P
<
0.0001, mid-p exact). A second study by Norden et al.
[18] reported higher specificity
estimates for the SCT and CCT in series of 98.3% (95% CI
96.0–99.5) in deer and 99.5% (95% CI 95.4–98.6) in elk
and red deer, in which animals classified as “suspects” (weak
positives) on the CCT were reclassified as negative. Comparing the 1997 SCT and
CCT in series results to the current study, the specificity estimates for the
Stat-Pak and DPP in series were not significantly different for WTD but were
different for elk (P
=
0.000036, mid-p exact), with the Stat-Pak/DPP
combination having a higher specificity than the SCT/CCT. Test performance for
reindeer was also reported in Norden et al. [18], where 29/29 reindeer were negative when tested in series by the
SCT and CCT, compared to 182/185 Stat-Pak/DPP test negatives in the present
study.Nearly 62% of the animals evaluated were female (993/1608). The highest
percentage of female animals occurred in reindeer (150/185 female) (Table2). Gender was not reported for 144
animals. When stratified by species, test performance was not significantly
different between females and males in this study (P
=
0.575, elk; P
=
0.969, reindeer; P
=
0.282, WTD; mid-p exact).Samples collected during 2011 were used to compare the performance of the
Stat-Pak with the SCT (Table4). A
total of 879 animals had results for both the SCT and the Stat-Pak. For elk,
7/344 (2.0%) animals were SCT positive, while 9/344 (2.6%) were
antibody positive on the Stat-Pak. For WTD, 13/494 (2.6%) and 13/494
(2.6%) animals were SCT positive and Stat-Pak antibody positive,
respectively. SCT results were available for 41 reindeer. Of these, 9/41
(22.0%) and 4/41 (9.8%) were SCT positive and Stat-Pak antibody
positive, respectively. Three reindeer and two elk were SCT positive and
antibody positive on the Stat-Pak. In all other cases, the Stat-Pak
antibody-positive animals were different individuals than the SCT-positive
animals. CCT tests were administered to 11 Stat-Pak antibody-positive animals.
Three elk, four reindeer, and two WTD were CCT negative. Two WTD were classified
as positive on the CCT.Table 4
Comparison of single cervical tuberculin skin test (SCT) and the
Stat-Pak, presumably uninfected animals, by species in 2011.
SCT Result*
Stat-Pak Negative
Stat-Pak Positive
Total
Elk
Negative
330
7
337
Positive
5
2
7
Elk, total
335
9
344
White-tailed deer (WTD)
Negative
468
13
481
Positive
13
0
13
WTD, total
481
13
494
Reindeer
Negative
29
2
31
Positive
7
3
10
Reindeer, total
36
5
41
Total
852
27
879
*Includes only animals sampled prospectively during 2011.Fifty two samples antibody positive on Stat-Pak were tested by the DPP and MAPIA
(Table5). Both assays use an expanded
panel of antigens in addition to the antigens included in the Stat-Pak, to
improve specificity. The largest improvement in specificity was observed in elk,
where 13/14 samples (each antibody positive on Stat-Pak) were negative by the
MAPIA, and only one sample was positive (Table 5). In WTD, 13/24 (54.2%) antibody positive on the Stat-Pak
were negative on the MAPIA. In reindeer, 11/14 (78.6%) antibody positive on
the Stat-Pak were negative on the MAPIA. The DPP had similar results to the
MAPIA. The increase in specificity estimates from the Stat-Pak only to the
combination of the Stat-Pak and DPP tests used in series were 98.3% to
100.0% (842/842, 95% confidence interval (CI) 99.6–100.0) in
elk; 96.7% to 99.3% (720/725, 95% CI 98.4–99.8) in WTD;
92.4% to 98.4% (182/185, 95% CI 95.3–99.7) in
reindeer.Table 5
Results of multi-antigen print immunoassay (MAPIA) and dual-path platform
VetTB (DPP) for Stat-Pak antibody positive samples.
MAPIA Results*
DPP Results
Elk
Reindeer
WTD
Total
Neg
Pos
Neg
Pos
Neg
Pos
Negative
13
1
10
1
13
6
44
Positive
0
0
0
3
0
5
8
Total
13
1
10
4
13
11
52
*Neg = negative, Pos = positive, WTD = white-tailed deer.


## 3.2. Serology Test Performance in a TB-Affected Elk Herd

A total of 34 animals from a known TB-affected Nebraska elk herd were evaluated
for the project. Thirty one animals met the case definition for confirmed TB
infected and had a serum sample available for inclusion in the project. Of
these, 27 (87.1%) were antibody-positive by the Stat-Pak, and followup
testing resulted in 26 and 27 antibody positive samples by the MAPIA and DPP,
respectively. Twenty of the 31 confirmed infected animals were SCT tested with
negative results three months prior to being necropsied. Among these 20 SCT
negative animals, 18 (90.0%) were Stat-Pak antibody positive, and 2 were
negative. Three animals from the infected elk herd did not meet the definition
for confirmed TB infection; these animals were considered exposed to TB and were
excluded from the analysis. Lesioned tissues from these 3 animals were
compatible for mycobacteriosis by histopathology, butM. bovis
was not isolated. For these 3 animals, two serum samples were negative, and one
was antibody positive on the Stat-Pak and the DPP.


## 4. Discussion

The estimated specificity of the Stat-Pak from this study was not significantly
different from the SCT in elk and WTD and was higher in reindeer compared to the
SCT. This finding is similar to a study in which significant differences in SCT test
performance between cervid species were found [9]. A substantial improvement in specificity was observed when the
Stat-Pak was used in series with either the DPP or MAPIA. The increase in
specificity varied by species with the largest improvement occurring in elk and the
lowest in WTD. In this study, the specificity estimates for the Stat-Pak and DPP
used in series for WTD and elk are significantly higher than estimates of the SCT
and CCT used in series in deer and elk [9].
However, the 1996 estimate for deer may not accurately reflect test performance in
WTD, as multiple species were included as deer and could have included reindeer,
roe, fallow, and other species, in addition to WTD.In this study, the specificity estimates for the Stat-Pak and DPP were similar to
other reports. Waters et al. [4] reported an
estimated specificity of the DPP of 98%, while in New Zealand red deer, a
species closely related to elk, the estimated specificity was 98.3% [14]. A previous study of the Stat-Pak found an
estimated specificity in WTD of 98.9% [12].While this project was primarily focused on specificity because of the limited number
of samples from infected cervids, sensitivity is also an important consideration. In
this study, the sensitivity of the Stat-Pak was 87.1%; however, this estimate
was determined from a relatively small number of animals in a single herd. Buddle et
al. [14] reported a sensitivity for the
Stat-Pak and DPP of 75% each (not used in series) in naturally infected red
deer. Waters et al. [4] noted that the SCT
performed poorly in detecting TB, as only 3/28 confirmed infected elk were SCT
positive. The failure of the skin test to detect infection in elk has been
documented previously; the CCT was negative in 14 elk that were subsequently
confirmed to be infected with M. bovis [9]. In the present study, three months elapsed between the time
the SCT was administered and blood samples were collected for serologic testing. It
is possible that the SCT-negative results occurred in animals that were not yet
infected; however, at necropsy many animals in the herd had advanced clinical
disease [4], making this explanation less
likely. However, Norden et al. [9] described
that several of 14 confirmed infected elk that were initially CCT negative were CCT
positive when tested again at a later date.Samples from naturally infected WTD and reindeer were not available for this project;
however, published studies have reported Stat-Pak sensitivity ranging between
55% and 67% in naturally infected, free-ranging WTD, and 79% in
experimentally infected animals [12, 19]. The estimated sensitivity of the CCT
administered as the only skin test in experimentally infected WTD was 97%
[20]. An evaluation of 11 experimentally
infected reindeer found that M. bovis-specific antibody was found
as early as four weeks after infection [11].
No naturally infected reindeer have been detected in the US.Animals that were positive on the Stat-Pak were different than the animals that
responded to the SCT, with only a few exceptions. One possible explanation for this
finding may be the basic differences between these tests, with one measuring
humoral, and the other, cell-mediated immunity. Additionally, the serologic tests
use specific antigens, while PPD tuberculins are a complex mixture of many
cross-reactive antigens. This finding has important implications for choosing a
supplemental test. The CCT may not be an appropriate supplemental test to use in
series with the Stat-Pak, because some of the sensitivity gained using the serologic
test could be lost using the skin test. Alternatively, measuring different aspects
of immunity may be more robust at detecting infection than two tests that measure
the same immune response components. For example, the humoral immune response may
not be subject to anergy, which is known to cause false-negative skin test
results.The primary study limitation was that animals testing negative on the Stat-Pak were
not necropsied to confirm that they were not infected withM.
bovis, with the exception of slaughter inspection conducted in some
reindeer sampled prospectively. Given the recent findings of M.
bovis in several US captive cervid herds, there is a small risk that
infected animals classified as presumably uninfected were included in the study.
Additionally, the samples included in this study were a convenience sample of
animals being TB tested for routine purposes and subject to producers volunteering
to participate. For example, a number of producers that originally agreed to
participate in the study declined after learning of the requirement to euthanize
animals testing positive on the Stat-Pak and a maximum indemnity payment of
$3,000. Only 12/26 prospectively sampled antibody-positive animals were
necropsied because producers declined to have their animals euthanized and
necropsied. To avoid these limitations in the future, studies would need adequate
funding and industry cooperation to more thoroughly evaluate the disease status for
both test-negative and -positive animals.In addition to the potentially higher diagnostic accuracy of antibody detection
methods evaluated in the present study, point-of-care serologic testing by simple
and rapid assays offers the advantage over skin testing of reduced animal handling
and the associated morbidity and mortality and eliminates the subjectivity
associated with evaluating the tuberculin injection site for a response. It is
anticipated that growing acceptance of this approach by producers will result in
gradually expanded TB testing to further improve overall TB surveillance in captive
and free-ranging cervid populations.



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*Source: 563293-2012-06-25.xml*