FILED

DALLAS COUNTY
8/19/2015 12:55:03 PM
FELICIA PITRE
DISTRICT CLERK

DC-15-09369
CAUSENO. ____________- -

SOUL CIRCUS, INC., d/b/a UNIVERSOUL

CIRCUS, a Georgia corporation,

Christi Underwood

IN THE DISTRICT COURT

Plaintiff,
DALLAS COUNTY, TEXAS
v.
THE CITY OF DALLAS and DALLAS COUNTY
HEALTH AND HUMAN SERVICES, a Munjcipal

- - - JUDICIAL DISTRICT

Corporation,
Defendants.

PLAINTIFF'S ORIGINAL PETITION AND APPLICATION FOR TEMPORARY

RESTRAINING ORDER AND TERMPORARY AND PERMANENT INJUNCTIONS
TO THE HONORABLE JUDGE OF SAID COURT:
Plaintiff, SOUL CIRCUS, INC. d/b/a UNIVERSOUL CIRCUS ("SCI"), by and through its
undersigned counsel, files this Original Petition and Application for Temporary Restraining
Order and Temporary and Pennanent Injunction against Defendants the City of Dallas (the
"City") and the Dallas County Health and Hwnan Services Department ("DCHHS ," and
collectively, "Defendants"), and allege the following:

I. DISCOVERY CONTROL PLA.N AND RULE 47 STATEMENT

Plaintiff intends to conduct discovery pursuant to a Level 2 Discovery Control Plan as set
forth in Texas Rule of Civil Procedure 190.3, except as modified by Plaintiff's Motion for
Expedited Discovery filed contemporaneously with this Petition. Plaintiff seeks monetary relief
of $ 100,000 or less and non-monetary relief.

Page 1 of 13

II. PARTIES
1.

Plaintiff SCI is a corporation organized under the laws of the State of Georgia

with a principal place of business at 230 Peachtree Street, N.W., Suite 2000, Atlanta, Georgia
30303.
2.

Upon information and belief, Defendant City is a municipal corporation of the

State of Texas and may be served with process by serving the Dallas City Secretary's Office
Attn: Rosa A. Rios, 1500 Marilla Street, Room 5D South, Dallas, TX 75201-6390.
3.

Upon infonnation and belief, Defendant DCHHS is an agency of Dallas County,

Texas, where the City is located and may be served with process by serving the Dallas City
Secretary's Office Attn: Rosa A. Rios, 1500 Marilla Street, Room 5D South, Dallas, TX 752016390.

III ..mRISDICTION AND VENUE

4.

Jurisdiction is proper in this Court because the amount in controversy meets the

minimum jurisdictional limits of this Court and all parties are subject to personal jurisdiction in
Texas.
5.

Venue is proper in Dallas County pursuant to Tex. Civ. Prac. & Rem. Code

15.002, because all or a substantial part of the events giving rise to this claim occurred in Dallas
County.

IV. GENERAL STATEMENT OF THE CASE

6.

SCI, a nationally renowned circus, hereby brings this special proceeding pursuant

to the Tex. R. eiv. Pro. 680 and Tex. Civ. Prac. & Rem. Code, 65, and challenges the

Page 2 of 13

Defendants' decision to deny SCI a permit to exhibit two elephants, Betty and Bo, at SCI's
upcoming performances in Dallas, Texas.
SCI and its agents are duly and properly licensed by the federal government, as

7.

well as many state governments, including Texas, to exhibit elephants and other exotic animals.
SCI has been holding circus performances featuring elephants for the past 20

8.

years. SCI contracts with various vendors to provide its elephants and other exotic animals.
SCI's elephant vendor routinely tests its elephants for tuberculosis, complies with the most
recent Guidelines for the Control of Tuberculosis in Elephants, and has never exhibited an
elephant that poses a public health risk.
Defendants allege that Bo and Betty cannot be exhibited because they tested

9.

"reactive" to tuberculosis.

Defendants allege that they received information regarding these

results, but never provided any documents to SCI.

Consequently, it is Defendants' position that

the elephants pose a public health risk.

10.

The Defendants' position, however, is neither legally nor scientifically justified

and in fact is wholly arbitrary. In the event Defendants are relying on the DPP Vet TB Assay for
Elephants (the ''DPP Test") from December 2014, this test serves only as a screening test to aid
in the diagnosis of active tuberculosis. The DPP Test is not definitive and often gives false
positives. In fact, the only USDA-recommended diagnostic test to detect TB in live elephants
that is considered to be definitive is a mycobacterial culture of trunk: wash samples (the "Trunk
Wash Test"). See the supporting materials for the use of the Chembio Diagnostic Systems, Inc.
DPP Vet TB Assay for Elephants (the "DPP Test Supporting Materials") attached hereto as

Exhibit 1,

~3,

and the Affidavit of Dr. Michael D. Stafford (the "Stafford Affidavit") attached

hereto as Exhibit 2, ~11.

Page 3 of 13

11.

Following the DPP Test in December 2014, which indicated that Bo and Betty

were Hreactive" to certain antibodies, the elephants underwent the more extensive and definitive
Trunk Wash Test on January 2,3, and 5,2015. The results of the Trunk Wash Test indicated that
Bo and Betty are not infected with or carriers of tuberculosis and that their December 2014 DPP
Test results were in fact false positives. See Stafford Affidavit, ~12.
12.

Betty and Bo underwent another Trunk Wash Test on June 12, 14, and 15, 2015.

The results of this Trunk Wash Test indicate that Betty and Bo are not infected with or carriers of
tuberculosis. See Stafford Affidavit,

~12

and also see the June 2015 Trunk Wash Test attached

to the Stafford Affidavit as Exhibit A.

13.

Additionally, in accordance with the 20 I 0 Guidelines (defined below), Betty and

Bo had Trunk Wash Tests performed every three months in 2011 and every six months
thereafter. Each Trunk Wash Test result has consi.stently been negative for tuberculosis. See
Stafford Affidavit,

~M!8-9,

and Exhibit A to the Stafford Affidavit (which is a copy of aU the

Trunk Wash Tests).

14.

When SCI shared the aforementioned information with Defendants and asked

Defendants to reconsider the decision to deny permits for exhibition of Bo and Betty, Defendants
maintained their prior decision. See a copy of an E-mail from Sarah Ausherman attached hereto
as Exhibit 3.
15.

SCI now brings this proceeding to challenge Defendants' position that SCI cannot

exhibit the two elephants that tested reactive to tuberculosis, despite definitive and
uncontroverted scientific and medical testing and analysis that definitively establishes the DPP
Test was a false positive and the elephants do not have tuberculosis. SCI hereby requests that
Defendants' denial of the permit to exhibit Bo and Betty be enjoined vacated or annulled on the

Page 4 of 13

grounds that such Defendants' denial of such permit was arbitrary and capricious, an abuse of
discretion, and/or contrary to applicable governing law, and issue an injunction requiring the
City to issue a permit for Bo and Betty to perform.

V. RELEVANT FACTS

16.

Since its inception, SCI has been in the business of promoting and performing

circuses throughout the United States.

17.

Throughout its history, some of SCI's most popular acts have involved the use of

elephants.

18.

SCI is scheduled to promote and perfonn its circus from August 19, 2015 through

August 30, 2015 in Dallas, Texas. Each of SCI's scheduled performances inv01ve the elephants
Betty and Bo, and SCI has advertised and promoted the exhibition of Betty and Bo.
19.

Despite

scr being authorized to

exhibit elephants, including Betty and Bo, in the

State of Texas, SCI received an email from the City on July 27,2015, stating that the City denied
permission for SCI to exhibit Bo and Betty. Attached to the email was a letter written by Zachary
Thompson, Director of DCHHS.

The letter was sent to three agents of the City and

recommended that the City deny SCI permission to exhibit the elephants because they tested
reactive for tuberculosis in the December 2014 DPP Test. A copy of this communication is
attached hereto as Exhibit 4.
20.

In response to the aforementioned email, SCI sent the City additional information

regarding the two elephants, including information evidencing that the elephants do not have
tuberculosis and that the DPP Test was a false positive.
attached hereto as Exhibit 5.

Page 5 of 13

A copy of this communication is

21.

The City forwarded the information to DCHHS, who reviewed the information

but maintained their earlier recommendation to the City, who denied SCI permission to exhibit
Bo and Betty. A copy of the letter from DCHHS regarding reconsideration is attached hereto as

Exhibit 6.
22.

DCHHS explained that allowing the exhibition of elephants that tested "reactive"

to tuberculosis posed a threat of potential trarurnission of tuberculosis from the elephants to the
public. The fear that any elephant that has ever tested "reactive" to tuberculosis poses a threat to
the public is misguided.
23.

DCHHS did not provide any scientific explanations for its decision, nor did its

agents speak with any large animal veterinarians or experts with experience in tuberculosis
testing, diagnosis, or transmission in elephants. Upon information and belief, the City and
DCHHS failed to consider the 2008 Guidelines for the Control of Tuberculosis in Elephants (the
"2008 Guidelines") or the 2010 Guidelines for the Control of Tuberculosis in Elephants (the
"2010 Guidelines"). Instead, DCHHS ignored the best scientific and medical evidence without
any basis.
24.

On August 17, 2015, a representative of SCI, Alan M. Briskin, sent a letter to the

City explaining that SCI followed the standards of care, testing, diagnosis, and treatment of Bo
and Betty as set forth in both the 2008 Guidelines and the 2010 Guidelines. See the letter from
Alan M. Briskin to the City which is attached hereto as Exhibit 7.
25.

The aforementioned letter from Alan M. Briskin also evidenced that the 2010

Guidelines are more stringent than the 2008 Guidelines, and that SCI's elephant vendor and, of
course, 80 and Betty, met the standards of care, testing, diagnosis, and treatment as set forth in
the 2010 Guidelines.

Page 6 of 13

26.

The 2010 Guidelines provide that elephants testing reactive or positive to a DPP

Test, but also having a negative culture from a trunk wash and no known exposure to TB fall

under Group 3C. The 2010 Guidelines prescribe how often elephants that fall under Group 3C
should be tested. The 2010 Guidelines specifically state that elephants should be "[mJonitor[ed]
by culture (triple sample method) every 3 months for one year, then every 6 months for life."
I

The 2010 Guidelines further state that elephants should undergo "repeat MAPINDPP testing
every 3 months for the first year, then every 6 months for an additional 2 years if elephant
remains . . . reactive. If all cultures remain negative after 3 years annual serological testing may
resume as described in these guidelines." The 2010 Guidelines then go further to state that "the
elephant may be able to travel and have public contact after 6 months of successful documented
USDA approved treatment." See a copy of the 2010 Guidelines attached hereto as Exhibit 8.
27.

In accordance with the 2010 Guidelines, Bo and Betty were tested. by Trunk. Wash

Tests every three months for one year, and have been tested by Trunk Wash Test every six
months thereafter. All Trunk Wash Tests have come back negative. Bo and Betty also repeated
the MAPIAlDPP testing

28.

as prescribed by the 2010 Guidelines. See Stafford Affidavit, W8-9.

Based upon the foregoing, Bo and Betty meet all of the requirements of exhibition

for the United States Department of Agriculture (''USDA''), the Animal and Plant Health
Inspection Service ("APHIS"), the United States Animal Health Association ("USAHA "), and
the State of Texas. See Stafford Affidavit,

29.

~13.

Additionally, Betty and Bo's testing and care was provided and supervised by

Michael D. Stafford, D.V.M., who fully complied with the most restrictive protocols related to
the elephants. See Stafford Affidavit.

Page 7 of 13

30.

Despite the overwhe1ming evidence that Bo and Betty do not have tuberculosis

and do not pose a threat to public health, Defendants' refused to reconsider their position.
Defendants provided no scientific evidence or expert opinion to explain their refusal.

FIRST CAUSE OF ACTION - DECLARATORY RELIEF

31 .

SCI repeats and realleges all factual allegations as if fully set forth herein.

32.

Despite the clear language of the 2008 Guidelines and the 2010 Guidelines

regarding the screening role of DPP Testing and the definitive nature of Trunk Wash Testing,
Defendants have denied SCI pennission to exhibit Bo and Betty at SCI's Dallas, Texas
performances.
33.

SCI adhered to the standards set forth in the 2008 and 2010 Guidelines for caring,

testing, diagnosing, and treating tuberculosis in elephants. Additionally, SCI adhered to the 2008
and 20 10 Guidelines when it detennined that the elephants did not have tuberculosis and were
not a public health risk.
34.

Despite SCI's compliance with the 2008 Guidelines and the 2010 Guidelines,

Defendants have denied SCI permission to exhibit Bo and Betty at SCI's Dallas, Texas
performances.
35.

SCI denies that there are any other standards upon which it is required to adhere

regarding the control, care, testing, diagnosis, and treatment of tuberculosis in elephants.
36.

Pursuant to Tex. Civ. Prac. & Rem Code 37, et. Sec., SCI is entitled to an order

declaring that SCI adhered to the 2008 Guidelines and the 2010 Guidelines, thus Defendants are
required to permit the exhibition of Betty and Bo at SCI's upcoming performances in Dallas,
Texas.

Page 8 of 13

37.

Pursuant to Tex. R . Civ Pro. 37.009 Plaintiff seeks the recovery of its reasonable

attorney's fees in connection with obtaining this declaratory judgment.

SECOND CAUSE OF ACTION - APPLICATION FOR TEMPORARY RESTRAINING

ORDER AND TEMPORARY AND PERMANENT INJUNCTION
38.

SCI repeats and realleges all factuaJ a11egations as if fully set forth herein.

39.

SCI seeks injunctive relief under both equitable and statutory principles, Tex.

R.Civ. Pro. 680-693, and Tex. Civ. Prac. & and Rem.Code 65.011.
40.

Defendants have refused to permit SCI to exhibit Betty and Bo at SCI's upcoming

performances in Dallas, Texas, although Betty and Bo do not have tuberculosis and are not a
public health risk.
41.

Defendants have not provided any explanation for their decision, except for their

unfounded fear that the elephants are a public health risk. Defendants have refused to consider
any evidence to the contrary.

Accordingly, Plaintiff can demonstrate a probable right of

recovery in this action.

42.

There is an immediate threat that SCI will be irreparably harmed by Defendants'

acts and omissions, and SCI will continue to be irreparably harmed if it is W1able to exhibit Betty
and Bo as part of its 2015 performances in Dallas, Texas.
43.

SCI has no adequate remedy at law and the calculation of damages that may be

suffered by Plaintiff is not readily quantifiable or measureable.

44.

Prior to commencing this proceeding, SCI has not sought injunctive or similar

relief for its claims against Defendants in this Court or any other court.
45.

SCI is entitled to injunctive relief directing Defendants to pennit SCI to exhibit

Betty and Bo, andlor restraining Defendants from unlawfully interfering with SCI's exhibition of
Page 9 of 13

elephants as long as SCI continues to comply with all applicable rules and regulations governing
the exhibition of the elephants, including the 2010 Guidelines.
46.

Specifically SCI requests the issuance of a temporary restraining order, with or

without notice to Defendants, and immediate injunctive relief be awarded that will restrain and
enjoin Defendants and their applicable agents, servants, employees, attorneys and those persons
in active concert with them as follows:
a.

Enjoining Defendants from prohibiting the exhibition of elephants Betty and Bo;

b.

From interfering with the lawful exhibition of elephants Betty and Bo;

c.

From interfering with or prohibiting the display or exhibition within the City of

Dallas, circus elephants who have been tested for elephant tuberculosis by the Trunk Wash Test
in accordance with USDA, USAHA and APHIS guidelines and whose test results were negative;
d.

Enjoining Defendants from altering, deleting, removing or writing over in any

respect any documents, computer files (including, but not limited to, emails, hard drives, disc
drives, zip drives), communications, data d.rafts

Of

other things relating in any way to the

permitting or the denial of permitting for SCI to exhibit elephants in the City of Dallas.
WHEREFORE, SCI demands judgment as follows:

1.

On the first cause of action, declaring that a circus which abided by the 2008 and

2010 Guidelines for the Control of Tuberculosis in Elephants and whose elephants tested
negative for tuberculosis in the Trunk Wash Test is entitled to exhibit the elephants in the City of
Dallas;
2.

On the second cause of action, enjoining Defendants from interfering with SCI's

use of its elephants during its performances in Dallas, Texas,

Of,

in the alternative, directing

Defendants to permit SCI to exhibit Betty and Bo upon the condition that SCI continues to
Page 100f13

comply with all applicable rules and regulations pertain1ng to the care and maintenance of
tuberculosis in elephants, as set forth in the 2010 Guidelines;
3.

On all counts, the costs of this suit and reasonable attorneys' fees; and

4.

For such other and further legal and equitable relief as this Court deems just and

proper.
RespectfuUy submitted:
FERGUSON, BRASWELL & FRASER,

By:

P.e.

lsi John D. Fraser

John D. Fraser
State Bar No. 07393550
Danise A. McMahon
State Bar No. 00796148
2500 Dallas Parkway, Suite 501
Plano, Texas 75093
Phone: 469-726-3070
Facsimile: 972-378-9115
E-Mail: jfraser@dallasbusinesslaw.com
E-Mail: dmcmahon@daUasbusinesslaw.com

ATTORNEYS FOR PLAINTIFF

Page 11 of 13

LOCAL RULE 2.02 CERTIFICATE

Pursuant to Dallas LR 2.02 The undersigned counsel has provided opposing counsel at
least two (2) hours' notice before this Application for TRO and accompanying proposed order
were presented to the Court for decision.

/s/ John D. Fraser

John D. Fraser

LOCAL RULE 1.06 CERTIFICATE

To the best of the undersigned's knowledge, the case in which this application for TRO is
presented is not subject to transfer under Local Rule 1.06

/s/ John D. Fraser

John D. Fraser

Page 12 of13

VERIFICATION

SlATE OF GEORGIA

)
)

COUNTY OF FULTON

I, Cedric Walker, the PresUient of Soul Circus, Inc., being first duly sworn, verifies that the allegations
set forth in thls Original Petition and Application for Temporary Restraining Ottler and T~pora.cy and
Permanem Injunctions, ex.cepr those made upon infonnatioo and belief, are, withio my personal knowledge
and to the best of my knowledge and belief,

By.

Cedric Walke!

Its:

Presidetlt

truE:

SworD to and subscribed before me this

and accurate.

day of August, 201 S.

CATALOG 1/ 6!-9110-0

2 Te&t Kit

HEMBIO
DIAGNOSTIC

~nrEMs,

3661 HOrl7eblock Road

Medford, Ncw York 11763
U,S. Veterinary License No. 645

INC.

Mycobacterium bovis - Mycobacterium tuberculosis

Antibody Test Kit
D pp VetTB Assay for Elephants
A Rapid lmmunochromatographic Test for the Detection of Antibodies to Mycobacterium tuberculosis and
Mycobacterium boviJ in Elephant Serum, Plasma or Whole Blood
FOR IN VITRO VETERINARY DIAGNOSTIC USE
READ INSTRUCTIONS FOR USE CAREFULLY BEFORE PERFORMING TEST

INTENDED USE

PRINCIPLE OF TEST

The DPP VetTa Assay is a single use Immunochromatographic rapid test for the detection of IIntibodies to
M,ycobacterjwn tJlberculo.r/.! and Mycobacterium bovl.s In
serwn, plasma or whole blood from African elephants
(Loxodonta alr/eana) and Asian elophllllts (Elephas
nUTXlmus) . Tho test is used as an aid in the diagnosis of act! ve
tub~rculosls (TB) in conjunction with othot diagnostic
method8,

The Chembio DPP YetTB Aasay is bll.Sed 00 immunochromatographic technology. The test employs two
recombinant antigens, an M. fl<bercu/w/s and an M bovls
antigen, whloh ate separately immobilized on lhe membrane
solid phase. rt alBo utilizes recombinant Proteln AlO conjugated
to colloidal gold plll1lcles for antibody deteGtion. The DPP
VerrB Assay uscs ~erum, plasma or whole blood. The sample
Is applied to tho SAMPLE+BUPPER well with the buff6r.
After thc sample and buffer have migrated onto the test strip
additional buffer 15 added 10 the BUFFER well. Thc buffer
facJUtates the latoral flow of tho released products aod
promotes the binding of antibodies to the antigens. If present,
the antibodies bind to the gold conjugllted antibody binding
protein, in a reactive sample, the dye cor\.iugated-immune
complex migrates on the nltroc~llulose ml:lllbrane and is
captured by the antigens immobilized in the TBST (1 2) area
producing 8 plnkJpurple line. In tho absonco of detectable
antibody, no specific immune complex would be fonned on the
lest llna. and, therefore, no pink/pw-p\e line would appear In Ihe
TEST (I 2) area. Unbound conjugated gold particles continue
to migrate olong the membrane and produce a pink/purple lin"
in the CONTROL (C) ar~. This proudursl control serves to
demonstrate lhat the reagents have been properly applied and
have migrated through the device.

If specific antibodies are present in the sampl5, the expected

test result is reactive. A rmactlve result is suggestive of active

TB, In the absence of antibodies, the expected test rosult is

nonreactive.

SUMMARY AND EXPLANATION

Tuberculosis (1"8) In elephanlli Is 8 re-emerging zoonotic
disease caused priml!l'ily by Mycobacterium IlIberculosls IIDd,
In some cases, by Mycobacterium bovis [I J. The only USDArecommended definitive diagnostic test to detoet TS In live
elophants i8 mycobacterlal culture of trunk wash samples.
However, this method hIlS poor diagnostic sensitivity, as It
can only Identify animals with extenslvlI shedding of the
organism that typically occurs late in the course of disease
(2J. Rapid detectlon of lnfected elephants is 1\ crucial
pre~ulslto for more effective control of TB, 55 early
dlll.gIlosls a1lows timely isolation and/or in iliation of
chemotherapy [1-J].

MATERIALS PROVIDED
Each kit contains the following iterru:
2 DPP VetTB test devices

DPP VetTB buffer via! (6mL)

Product lnsert

Serological methods constitute an attractive alternatlve as

they are relatively simple, inexpensive, non-invasive, and
they do not depend on detection of mycobacteria (3-4 J. Nono
of the ex:lstlng TB tesl8 alone is sl)fficient to diagnose
disease. Therefore, new TB diagnostic algorithm.>! are being
developed, In which sorological assays may play ao important
1'010 [3-5) (see PERFORMANCE CHARACTERlSTICS
below).

Additional Material Required But Not Provided

Clock, watch or other Uming dllvice

Disposable gloves

Biohazard disposal container

Collection d~vices for specImens

Plpettor capable of delivering SilL of sample

The uae of CIIembio DPP VetTB AssBy is an effective

approach for rapid animal-side identification of elephants
infec10d with M. tllberculosl3 or M. bovls, as this test is
highly accurate, user-friendly, 80fe, and easy to perform.

Exhibit 1
10-6294-0 Rev 2
Nov.mb..,. 2012

i>"oS" I of4

STORAGE AND ST AB]LITY

TEST PROCEDURE

The DPP VetTB Assay should be stored at 2 to 30'C in the

original sealed pouch. The diluent should be stored in the
originsl vial at 2 to 30C. The kit is stable until the date
printed on the box label andlor pouch.
NOTE: Do not u~e expired test kits.
CAUTION: Do not freeze test kIts.

I.

2,

PRECAUTIONS
I.

2.

3.

4.

S.

The lest is designed POR IN VITRO DIAGNOSTIC

USB only. Use the test only In accordance with
Instructions supplied with the kii.
Handle all specimens 88 recommended fol' any
pOTentially in~ctious serum or blood specimen in the
CDC-NIH manual, 8Jruajery In Microbiological and

3.

Figure 1
Teal WIndow wllh

Biomedical Laboratories, 4'h 6d.. J999.

BUFFER--+nJ!:-'

Use suitable protective clothing (gloves, lab coat, safety

glasses) when handHng samples OT test devices after
samples have been applied. Avoid any contact between
hands, ey~s, nose or mouth during specimen collection
and testi ng.
Do not pipette any metedal by rnouth. Do not smokt:, eat
or drink in areas where specimens or kit materiel Ble
kept.
All testing ~ould be performed at a remperature of 18 to

Well 2

7.
8.

._~~t-

SAMPLE

+ BUFFER

WeUl

4.

Using a 10~ dIsposable

laboratory plpettor (for 5
the speci men carefully
SAMPL.E+8UFFER Well

5.

Once the specimen has been IIpplied 10 the

SAMPLE+BUFfER Well I, remove the cap, invert the
buffer
bottle,
hold
It
vertlcolly
over
the
SAMPLB+BtJFFER Well I, and IIdd :l drops (-65 ,..1) of
the buffer slowly Into SAMPLE+BUFPBR well. (Sec
FIgure 3)

6.

Wlllt S mlnl!tt:!l, and Ihen edd 4 dl'Ops of the buffer to the

square BUFFER Wo1l2. (See Figure 4 below.)
NOTE: The blue and greon colored Hnes should have
disllppearBd from the reotangullU' TEST and CONTROL
window. If not. discard the test device Bnd repeat thB
procedure with n new DPP test device.
Flgyre 4

pipette (for whole blood) or a

f.L1 of serum or plasma), release
to the center of the round
I. (Sex. Figure 2 below)

After the oompletlon of the as~oy, carefully dispose of

materIals troatlng them as blohiIZBIdous waIlte.
Do not use expired test kits. Do not freeze test kits.
Do not mix reagents from different kit lots.

SPECIMEN COLLECTION
The DPP VetTH Assay can be
serum or plasma.

p~lformed

on whole blood,

Whole Blood: Collect whole blood Into tubes containing

heparin or EDT A. Be sure to thorollgh ly mix whole blood by

Inverting capped tube several times just prior to testing.
Follow test procedure Instructions.
Serum: Serum Is obtained from whole blood collected
aseptically by venJpuncture Into a clean tube without
anticoagulant. Allow the blood to clot at room temperature,
centrifuge lit 2000 rpm for 10 minutes at room temperature,
18 to 30C, and soparato the serum from the clot wlthin 24
hours of blood collection ..
Pill, rna: Collect whole blood with anticoagulant (heparin,
BOTA or sodium citrate), centrifuge at 2000 rpm fur 10
minutes at room temperature, 18 to 3lte, and irolate the
plasma supernatant.

Samples perform best when tested immediately after

colleccion. Specimens should be immediately refrigerated at 2
to goC following collection and can be used up to 3 dll.ys. If
testing within 3 days Is not possible.. the specimens should be
frozen at 20C or colder until use. Avoid repeated ftcezing
and thawIng. DO NOT FREEZE WHOLE BLOOD.
~:

If specImens ara ro be shIpped, they should be

packcd in compliance with r~gulaliol18 covering the
transportation of etiologic agents. Venous whole
blood, serum and pllW1lR specimens should be
shipped refrigerated with cold packs or weI ice.

IO~294 0 R.av 2
Novcrnber 20 12

PaRe 2 of4

two

blue llnetl and one

green line

~r"l1~~~~

30D C.

6.

1f test samples ere refrl gera.ted, remove them frorn the

refrigeratol' and allow them to come to a temperature of 18
to 30"C bofore testing.
Remove tbe required number of DPP YetTa Assay
devices from their pouch~ and place the devices on II flat
8urface area. rt is not necessary to remove tbe desIccant
/Tom the package.
NOTE: If desiccant packet is missing, DO NOT USE,
discard the test device and II new lest device
should be used.
Label test units with sample names andlor identification
numbers. (see Figure I below)

7.

8.

Read the test result 15 minutes after the addition of the

buffet illto the BUFFER Well 2. In st)me cases a test
line may appear in less than 15 minutes; however, 15
minutes III' needed to report a non-reactive resulL Do
not read result!! after 2S minutes from addition of
Sample+Buffer to Well 1.

Nonreactive Rerult
Only a pink/purplo CONTROL (C) line is visible. The sample
contains no detectable BI1Iibody to bolh TB and
mycobacteriosis antigens. A nonreactive result does not
preclude the possibility ofTB infection,
NO n,ST (I 2) Line,

After reading aod recording test results, discard the used

test devices and MY other test materials into 8 biohazard
waste eonla [nero

CONrn.OL (e) Uno

QUALITY CONTROL
A pink/purple colored line should Blwa)'s appear in
CONTROL (C) area if the tesl has been perfonned correctly
and the device Is working properly. It serves as an Internal
test procedlll'al control.

INTERPRETATION OF RESULTS

Iuv!lIid Result
A pink/purple Bne should always appear In the CONTROL (C)
area, whether Dr not II line appears in the TEST area. If there is
no distinct pink/purple line In the CONTROL (C) area., the test
is invalid and should be repeated using a new devtee,

Reactive Result
I , Three pink/purple lines. one line In the CONTROL area,
one line in the TEST (1) area Ilnd ono line In the TEST (2)
area indicates a reactive result. Thl" suggests that the
satnple is reactive fur TB.

No CONTROL (el Li ,.
LIne

2. A pink/purple TEST (2) line and a pink/purple CONTROL

line are
for TB.

vlsibl~.

This suggests that

th~

sample is reactive

LIMITATIONS OF THE PROCEDURE

I, The Chembio DPP VetTB Assay is desJgned for detecting
antibodies against M 'Ubl!rcl(/o9is and M. bovis only frOID
elephant plasma, serum or whole blood. Any other body
fluids or pooled samples or specimens from other than
elephant species should not be used.
2. Test results must be read between 15-20 minutes after the
additIon of the buffer to the square BUFFER Well 2.
], Do nol usc hemolyzed blood samples.
4, Blood specimens must be thoroughly mixed just prior to
testing.

3. A pink/purple TEST ( I) line and Ii pink/purple CONTROL

Une are visible. This sugge.<lts that the sampl6 is reactive for
TB or mycobacteriosis.
TllST(I) Line

CONTROL (C) Lino

NOTE: Intensities of the TEST and CONTROL lines may

vary. Test lines are considered reaetlvCl regardless of
intensity.

Do not open the ~ealed fall pouch until Just prior to use.
Do not use kit contents beyond labeled expiration date,
7.
Read r~ults in a well-lit are!!..
8. A reactive result using tbl': Chembio DPP VetTD AlIsay
suggests the presence of antibodIes to M. ,uberr:u/O.f/J andJor
M boy/so The Chemblo DPP VetTS Assay is intended as sn
aid In th~ diagnosis and treatment of TB in elephants.
9. For a reactive result, the intensity of tho to.!! IInc does nol
necessaril y correlate wi lh the titer of antibody in the
specimen.
10. A non-reactive result does not preclude tne pass ibliity of
cxposure to TB or lnfectlon with TB. An antibody response
to recent CXP08UI'e may take several mootbs to reach
detectable levels.
II . In treated elephants, interpret rellults with caution:
a. Treatment against TB may reduce antibody responses,
thus resultIng In non-reactive results in some cases.
5.
6.

I().62940 Rov 2
Novembor 1012

PlSO j of4

tubercli/osis infwlion among captive Asian elephants in a

Swedish zoo. Vel. R~. 156: 171-175 . Lyashc~enko, K .P.,
ct ai., (2006) Tuberculosis in elephants: antibody

b. A reactive result may persist in Infected and treated

elephant!! for months IU1d years even (f an alephan! (s
considered cured.

responses to defined an1igcns of Mycobacferilim

tuberculosis, potential for early diagnosis. tlnd otonitoring
of treatment. Clinical And Vaccine Immunology 13 : 722-

PERFORMANCE CHARACTERISTICS

732.

Highly specific lind sensitive antibody binding antigens are

u.~ed In the DPP VetTB Assay. The diagnostic performance
WIIS compared to the standard USDA-recommended method,
trunk wash culture, and the D?P VorTB Assay dlagnostlc
performance was found to be superior (4-5),

3.

Further, it was shown that both Asian and Afi'ican elephants

Irlfected wldl M. tubercliloJ/s or M bov/s could be detected
by DPP YetTB Assay up to several years prior to finding
positive culture io trunk washes [4-5J.

Sensitivity

4.

Specificity

lli1d

Sensitivity of the DPP VetTB As~ay was determined by

testing 40 culture positive elephants . All 40 SIlmp[e,g were
rellctlve (Table I).

FOR MORE INFORMATION. CONTACT:

The specificity of the DPP VeITB Assay was determined by

testing serullI, pla.sma, andlor whole blood samples collected
from 147 trunk-wash culture negative elephants without
history ofTB . All 147 samples were non-reactive (Table 2).

CHEMBIO DIAGNOSTIC SYSTEMS, INC.

3661 HORSEBLOCK ROAD
MEDFORD, NY 11763 USA
U.S. Veterinary License No. 645
Tel: (631) 924-1135
Pax: (63 1) 924 -603 3
Email: i.nfQ@chembio.com
Web Site: www.chembio.i:lom

Table 1.
Diagnostic seusltlvU), 01 DPP VerrB AJny

DPP VetTe

Mycobacterial
species isolated
M. tuberculosis
M bovls
M. I1.Iberculo.Yls

Elephant

species
AITicen
Aftlcan
Asian

reactive
6/6
III
33/33

ORDERING INFORMATION
Cat II
65-9110-0
65-9111-0

Tobie 2.

Speclfic!1}' studies ofDPP VetTB Assay

Elephant

DPP YetTB Assay

~pecies

non-reactive
79179
68/68

African
Asian

Greenwald, R., Lyashchonko, 0., 8sf8ndiari, J., Millel',

M., Mlkota., S., Olsen, J.H" Ball, R., Dumonceaux, a"
Schmitt, D., Moller, T., Payeur, J.B ., Harris, B., Sofranko,
D., Waters, W.R., and Lyashchenko, K.P. (2009) Highly
accurate antibody assays for early and rap id detection of
tubcrculosi~ in A frJCtln and Asian elephants. Clinical And
Vacclna Immllnology 16 :605-612 .
LyBshchenko K.P., Gr~nwilid R., Bsfundiari 1., Mikota
So, Miller M., Moller T., Yogelnest t~, Gal rhe K,P.,
Robbe-AU.'Iterll'llln S., Gai J., and Waters W.R. (2012)
FIeld application of serodiagnostic:! to identify elephants
with tuberculosis prior to case confirmation by cultul'e.
CII". Vaccine Immunol. 19(8): 1269-75 .

65-9112-0

1'nInk wash
culture negative

79179
611/68
I'./.TI ""
uh,ll.,j

."\lIlIoll'

REPRODUCIBILITY STUDIES
Reproducibility was evaluatoo at three independent
laboratories using two serials of DPP VetTB Assay. A
roference pane! of 40 blindly-coded $8mples representing
negatl ve, weakly reactive and strongly react! ve sera wel'C
tested 3 times on 3 different days . The complied results fi'om
3 laboratories demomrtraled 98.6% accuracy.

REFERENCES
I.

2.

Mlkota, S.K., Peddle, L., Peddle, J . Isala, R., Dunker,

F., Wost, G. Lindsay, W., Larsen, R.S., Salmon, M.D.,
Ch8tterJee, D., Payeur, J., Whipple, D., Thoen, C.,
Davis, D. S., Sedgwick, C., Manta It , R.I., Ziccardi, M .
tlnd Maslow, J. (2001) Epidemiology snd diagnosis
Mycobacterium tuberculos/J in captivo Asian elephants
(Elepnas maximus) , J. Zoo Wlldl. Med. 32: 1-16.
Lewenn, S.S~ Olsson, S-L., Eld, K., Rllken, S.,
Ghebremichael, S., Koivulo, T., K~lIenlus, G., and
BlIlskc, G. (2005) Outbreak of Mycobacterilim

10-62940 Rov 2
November 20 \ 2

Page 4 of4

Product
DPPYctTB 2 Test Kit
DPPYetTB 5 Test Kit
DPPYetTB 20 Test Kit
Antigens licensed from
Statens Serum Institut
www.ssi.dk

CAUSE NO . _ _ _ _ _ _ __

SOUL CIRCUS, INC. d/b/a UNTVERSOUL

CIRCUS, a Georgia corporation,

IN THE DISTRICT COURT

Plaintiff,
DALLAS COUNTY, TEXAS

v.
THE CITY OF DALLAS and DALLAS COUNTY
HEALTH AND HUMAN SERVICES, a Municipal

Corporation,
Defendants .

AFFIDAVIT OF DR. MICHAEL D. STAFFORD

1.

My name is Michael D . Stafford and I am competent in all respects to testify

regarding the matters set forth herein. I am a veterinarian, and I have personal knowledge of the
facts stated in this Affidavit. This Affidavit is given volWltarily.
2.

I am licensed to practice in the State of Missouri, and have been practicing since

3.

I have been the veterinarian for Larry Carden's elephants, Betty and Bo, since 2011

1989.

and have reviewed all their records and diagnostic testing back to 2008.
4.

Both Betty and Bo have been tested using the Trunk Wash test and Stat-Pale test

since 2008.
5.

The Trunk Wash test is a diagnostic test used to determine whether an elephant has

tuberculosis by taking three samples from the elephant's trunk on three separate days.

Page 1 of 3

ExHIBIT

2
.0IIII

6.

The

Stat~Pak test

is a rapid blood test that was used to deteImine whether antibodies

in an elephant's blood exist that would suggest a risk for tuberculosis.

7.

The

Stat~Pak

test became unavailable in 2012 and we started using the DPP Vet TB

Assay for Elephants (the "DPP Test"), which was approved as the replacement to the

Stat~Pak

test

in 2013.

8.

Both Bo and Betty have been MAPIA and

Stat~PaklDPP

reactive since 2011 (Betty

was reactive in 2009). As such, we followed the procedures set forth in the 20 10 Guidelines for
treatment. That is, we tested both Bo and Betty every three months using both the Trunk Wash
Test and DPPlMAPlA tests for the first year, then every six months thereafter.
9.

Each Trunk Wash Test came back negative. The DPP tests came back reactive as

well as the MAPIA tests; however, the MAPIA tests improved after Bo and Betty were treated
using a three-drug cocktail suggested by the 2008 and 2010 Guidelines.
10.

In 2013 and 2014 the DPP tesis from ChemBio were very scarce, which did not

allow us to perform. the test twice those years.

11 .

The OPP Test serves only as a screening test to aid in the diagnosis of active

tuberculosis. The DPP Test is not definitive and often gives false positives. The only

USDA~

recommended defmitive diagnostic test to detect TB in live elephants is the Trunk Wash Test.
12.

Following the DPP Test in December 2014, which found that Bo and Betty were

"reactive" for tuberculosis, as always, the elephants underwent the more extensive and defmitive
Trunk Wash Test on January 2,3, and 5, 2015. The results of the Trunk. Wash Test indicated that
Bo and Betty do not have tuberculosis and their December 2014 DPP Test results were false
positives, which means that they only showed antibodies were present.

Page 2 of 3

13.

B6 and Betty ~urr6Iltly meet all of the reqUirements for exhibition prt'lscrlbed by

the United States Depart:b1ent of Agriculture (UUSDA ")t the Animal fl!ld Plant Health Inspection
Service ("APHIS"), the United States Anitn.al Health Association ("OSAHA'1, and the State of
Texa.s. Moreover. Dr. Sidwa. the Trow State Public :Health Veterinarian, who is in charge of
approviiIg whether an animal is safe fur traval in the

Stftte of TexfUl. apptoved Bo and Betty for

travel and agreed that thoy both met the 2010 Guidelines.

~A~~
.
.
.

/~1D. Stafford
Sworn to and sUbset:1bed

. .
bofote me this

~ ,

lq- day

of AuguS"t 2015

Notary Public

My Commission'E~les:

Pego 3 of3

EXHIBIT A

TRUNK WASH TESTS

Date ofTests

Bo

Betty

Result

Negative

X
X
X

Negative
Negative
Negative
Negative

2010
December 20,21,22,2010
2011
--_.
March 17, l8, 19, 20!1
June 10,11, 12, 2011
August 18, 19,20,2011
December 13,14, is, 2011
2012
May 7, 8,9,2012
May 23,24, 25,2012
August 22, 23, 24, 2012
December 18,19, 20,2012
2013
June 25,26,27,2013
December 11, 12, 13,2013

X
X

Negative
Negative
Negative
NegatIve

X
X

Negative
Negative

X
X

X
X

Negative
Negative

Negative

2014
June 16, 17,18, 2014
Janua ry 2, 3, 5, 2015
i 2015
June 12, 14, 1S, 2015

"-

 iv'iA R. I 6. 20 11' ! 2: 04PM

TS LAB NAT'l JEWISH/DENVER, CO

'0 Natilonal Jewish

' 12 HeEllth'
S~lin~o

Trans1ormlno lifo'

!>.FB SMEAR AND CUVrURE

SPECIMEN SOURC'B

ACTUAL COLLECT D~TE

?ATISNT'$ PHYSICIAN
HOSP LAB PHONE NO
HOSP LAB FA:i. NO
SMEAR EXA.HIWJl.TlON

AFB

CUUI1J1U~

AfB CULTURE COMMEN1

NO. 77 61

P. j

1400 Jcr.i(!on 51"W

DetlVl:r. Color$\do 80206
www N) Lab! ,ots

ADx
TEST RESI)L TS

clinrcD~b,@..,jc,Dta

TRONT< WASH
12/20/10
DmNIS SCHMITT
417 833 3588
U 7 833 9141
(NOAFBl
No AFB found
12/29/10
ONE LARGE SMOOTH COLONY RECOVERED FROM
BI PLATE
01120/2011
fout units of me6iwn are inoculated for

recovery of the organism.

Aoid fast

bacilli (ArB) a:ce read at 1,3, and 6

weaks,

Default

~ulture

result iB

"P'&NDING" and should be inte~preted

06 NEGA~rV8

until a positive result

is entered or the final ne~at!ve
result is entered at 6 weeks. Eight
waeK LJ cult~re will be reported
only if positive.

IDENT EY HPLC 12)

MYCOLIC ACID PROFILB MOST CLOSELY

RES~BLES MYCOBACTERIUM FORTUITUM
CHELONAE COMPLEX OR MYCOSACTERIUM
SMEG~~TlS GROUP, SPECIES
IDENTIF1CATJON IN PROGRESS.
1/26/2011

MYCOBACT~RJAL

ID 2

IDENTIFICATION

CARDEN C!:RCUS,BO

MYCOBACTERIUM rORTUITUM IDENTIP'IED BY

1 PAGE

CONTrNtr~D

Advanced Diagnostic Laboratories

NATION~~ JEWISH HEALTH
l~OO uackson Street
DENVER, CO 80206
Client Serviceal 800-550-6227
CAP #21789{)1

Legend: H;i High L" Low @:r Orltlcal '

03/15/2011 WflD 1~1 00

ellA #0600644301
LAB 108 (B.08)
(JOB NO.

88B]

~QH

MAR. 16. 2011

. . 12 :04 PM
(:).

.Q

T8 lAS NArL JEWISH/DENVER,

Na~~ona' Jewrsh

Sol BnUII Traosformlng LIfe

P. ')

1400 Jnck30n Srre~t

Col()<'&do ~0206

OeO~/,

www.NJL~b . orl(

cIlnrefl~ba@l'Ilc'01g

(CONTXNUED)

16S
2/3/11

CARDEN CIRCUS,BO

NO. 7761

ADx
TEST RESULTS

Hearth'

MYCOBACT8RIAL ID 2

co

rDN~

SEQueNCING,

2P)'>.GE

reND OF REPORT

Advanced Di~gnoatic Laboratorie~

NAT!ONAL ~EWIS~ HEALTH
1400

Jac~Son

D~NVER,

co

Street
80206

Client Services! 800-550-6227

CAP #21769-01

CL \A #06D0844307
LAa 106 (B.OB)

Legend: H a High L:>" Low @ .. Crlt10el .

03/16/2011 \>/80 13100

r JOB

NO.

8U5)

filJ002

frS. 16, 2011 6:02PM

T8 LAB NAT ' l

JEW1SH/DENVE~

o National Jewish
Q

NO, 6356

P. 1/20

1400 Jncll~o n Strw

I1nnvcr, f:r>lnmo"

ADx
TEST RESULTS

Health'

Scl~nce

CO

1'ra nafol mlng Lire

RO)I)~

W\IIW,N,n ,~b&.O'!g

cllnl'I!)P,bi@IIJc ,rlr!;,

,I'
,

! .

. GEORGJ:i
:.

~RDEN

3901 WEST HWY

.'

, ..

CIRCD'S

SI?RINGFlELP, MO 65803

'

16172]

COLL: 12/21/2010 08:00

REC: 12/28/2010 11 : 52 ?HYS: G~ORGB CARDEN C

AFB St-iE.h.R AND COLTURE

SPECIMEN SOURCE

TRUNK WASH

ACTUAL COLLBCT PATE

PATI8NT'S PHYSICIAN
HOSP LAB PHONE NO

12/21/10
SCHMrTT
417 833 3588
417 633 9141
No AFB to~nd
12/29/10
DE~lS

HOSP LAB 'FAX NO

SMEAR EXAMINATION

(NOAFB]

CULTURE NBGATIVE FOR

AFB CULTURE

MYCOBACTERI~

0.2 /12/11

Four units Of medium are inoculated for

recovery of the organism. Acid fast
pacilti (~FS) are read at 1,3, and Q
we~ks. Detault cu~ture result is

AF.B CULTURE; COMMENT

"FBNDIN(j" Q,nd should be inl;Elrpreted

JeNNY GRAHAM

as NEGATIVE until 8 positive ra~ult

is ente~6d or the final neg~tive

9UFERV!SOR

result 15 entered at 6 weeks. Eight

week LJ culture will be report eo
only if positive.

MV~OIJACTERIOLOGY LAB

T61724

COLLI 12/22/2010 08100

REC :

12/28/~010

11 : 52 PHYS ;

G~OROE

CARDEN C

AFB Sl":(EAR AND CULTURE

SPECIMEN SOURCE
ACTUAL COLLECT DATB

12/22/10

HOSf! LAB PHONE NO

DEtmIS SCHMITT
417 833 3586

PAT!~NT'S ~HYSICIAN

CARDEN CIRCUS/BO

'I'RONK WASH

lPAGE

CONTINUED

Advanced DiagnoStic Laboratories

NATIONAL JEWISH HEALTH
1400 Jackson street
D~NVER,

CO

Client services I
CAP #21781:/01

~egend: H '" High L"

80206
800-550-6~27

Low @" OrWe1l1

ellA #o6D0644S07
LAB 106 (B.08)

02/16/2011 WHD l81~7

[JOB NO.

88111

~HO

fEB, 16, ,011

6:02PM

T8 lAB NAT'l

JfWISH/DENVE~

6356

P.

8/20

Denver, CoIClJ1o.lo 1102('16

ADx

Health'

NO.

1400J~o;hon SI"'N

"0. National Jewish

CO

www.NJII.lb!.ol.(\
cljruelHlb,@nic.ul')l

TEST RESULTS

Solanoe Tnln&lormlllg lIle'

GEORGE CARDEN C1RCUS

3901 WEST HWY 0
SPRING~I~LD,MO

_ _ .... _

...

T61724

,
I

,.

...

_ ..... 1 _ _

',.

. . . . ; _ . _ .. _

COLL: .12/22/2010 08:00

AFB SMEAR AND CULTURE

HOS)? LAB FAX NO

SMEAR
AF'~

55B03

~INATION

REC: 12/29/2010 11:52 PHYSI GEORGE CARDEN C

(CONTINUED)
417 933 9141

No AFB found

(NOAPB}

12/29/10

COLTURB

CDLTURE NEGATIVE FOR MYCOBACTERIA

02/12/11

AFB CULTURE COMM!!:N'l'

Four units of medium are inoculated for

recovery of the organism. Acid fast
bacilli (ArB) are read at 1,3, ~nd 6
weeKs. Default culture result is
"PENDING" and should be interpretecS
as NEGATIVE until a positive r esult
is entezed o~ tne final negative
result is entered at 6 weeks, ~ight
week LJ culture will be reported
only it ~ositive.

JENNY GRAHAM
SUPERVISOR

MYCOBACTERIOLOGY LAB

CARDEN CIRCUS,BO

CAP (1.21789-01

2PAGE

END OF REPORT
Advanced Diagnostic Laboratories
~ATIONAL JEWISH HEALTH
1400 Jackson Street
DENVER, CO 80206
Client Servicesi 800-550-6227

eLlA #OSDOG44307
U\8 105 (B.OB)

Legend; H:: High L = Low @" Crltloal

02Jl6/20tJ. W'ElD 18157

r JOB

NO.

~811)

~Olt

MAR, 24. 20 II 12: 27PM

J[WISHIDfNVE~

T8 LAB NAT'l

D~~ve:,

AOx
TEST RESULTS

Health'

Setenol Trenmrmlng Ute'

If;;".~_~:

NO. 8088

P. I,

I4()O JaakICldt:l8Cf

O Natlonal Jewiah

CO

CnI.ot'tdu 80206

WW\V.NJ~J.O'lr

cllntcllabi@nle,OXX

j;

. " ~~!!'!!2:~~~~~L.:..::J..~

'.",~II"~-:r-r--:-::-Yn'\lr'TIT":"T'I~~~~~-"r-'---r--;r~1

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"

I
I

"

, .. SARAHtLIJASETHTontN

3.90l WEST UWY 0

SPRtNGFIELP,MO 65603

417 833 3588

. ',.'

~ICR?f3W~O.G~T(~SCP)
.

"

T~i'~"~Ottt-~~trhtrlft'81lt ~~tet'!inBriOW:t'lIi~~pH1S't ~~G~C~~~'c' -AF.a SMEAR AND CULTURE

SPECIMEt\ SOURCE

ACTUAL COLLECT DATE

PAT~ENTJS PSYSICIAN
HOSP LAB pHONE NO
HOSP W FAX NO
SMl!AR EXAMINATrON
AlI'B CUuruRE

I
f

TRONK WASH

12/20/10

FINAL REPORT

DENNIS SCHMITT

417 833 3588

417 B33 9141

No AFl3 found
12/29/1D

crruraRE

[NOAlI'B J

N~ATIVE

10R MYCOBACTERIA

02/12/11

APB CUL'L'ORB COMMEN'r

units of medium are inoculated for

recoVery of the o~qanism. A~id fa.t
bacilli (AFa) are read at 1,3, and 6
~eeks.
Oefault oulture ra9~lt is

~our

"PE:ND!NO' and should ba intexpreted

RS

NEGATIVE until

~ POSit~v6

result

is entered or'~e final negative

result is entered at 6 weeks, Eight
week LJ culture will be reported
only if Dositive.

CARDEN CIRCUS,BETTY

lPAGE

l!JND OF REPORT
Advanded Diagnostic Laboratories
NAfIONAL JEWISH HEALTH

CAP ~17l1901

1400 Jackson Street

D~, CO
80206
Client sarvioas:L80~~S0tl-6227
t.:ag&nd: H. tflSn L" oW ~. Crt! eal
03/24/2011 'l'RO 13120

C1.IA #0500844307
LAB 1~ (8.09)
{JOB NO.

U07)

/ilJOlD

MAR. 24. 20 II 12: 28PM

T8 lAB NAT'l JEWISH/OENVER, CO

~~ Natiol1f.1l Jewish
Q' Hes/th'

NO. 8088

f.!OO JlcUoa Sttll3t

Deaver, C%r9.do SOUl6
1>'9IW.l'U u.b l,org
clintelltb8@nIG.otg

AOx
TEST RESULTS

. 6o/enu& '1'ranifQrmln" Ufe'

p, 16

.,

3!/01 WliiST HWY 0

SPRrNGFIELD,MO 65803

,"
, ,

'417 ' 633 3588

,
I
I

A!'B .SMEAR AND CULTUl\!

SPECI..MBN SOOR~E

AC~OAL

COLLECT DATE

PATIENT'S PRYS!ClAW
H08~ ~

PRONE NO

. flOSP W>J3 FAX NO

Sl!EAR EXAMINATION

'nWNK W'ASH

1:2/21/10
D~IS

FINAL REPORT

BCHMI'l"1'

417 S33 3Sea

417 833 9141
No AFB found

[NOhl'EJ

lJ/29/10
AP'B COWURE

CUII1'UM NEGlITM FOR MYCOBACTERIA

AFB COUrURE COMMENT

Four units of mediUl'Q are inoeubted tor

02J12/1l

recove~ of the organism.

Acid fa~t
bacilli (APB) are read at 1,3, an~ ~
W8eks. Default culture result 19
"PENPING" an~ Should be interpreted

I
I

as NiGA~IVE until a posit~ve result

is eneered or the finAl negative
result ~S entered at 6 weaks. Eight
week LJ culture ~ill be reported
only if positive.

!
,'

CARD~

CIRCUS, BETTY

1 PAGE

END OF REPORT

Advanoed Diagnostic Laboratories

NATIONAL JEWISH H2ALTH

1400 Jackson street

DENVER, CO

90206'

Client Services, BOO-950-6227

CAP #21789-01

Legend: H.. HIgh L = Low @ ~ Orftloal

el.IA ~600844307

/.AB 105 (8.08)

MAR. 24.2011 12:28PM

18 LAB NAT'l

JEWISH/DENVE~

~. N~lonal' Jewl,h

CO

NO. 8088

1400 Jaduol1 Stt~t

DGJTVt, ColO1'lldo 80206
WW';V,NJLt'bs.ore
ollnn.lltbe@njc.otg

AOx
rEST Rf:SUlT8

Q , .Health"

8~18nclI 'nantiormlnB Llta

p, 17

I
j

'

..
AFB SMEAA MID CuurURE

SPECIMEN SOURCE

TRUNX

12/22/10
DENNIS SCHMITT

SMEAR fruMINA'l'ION

No ArB .found

DATE
PATIENT'S PH1SrCIAN

FJNAL REPORT

~SH

~CTU~ COLLEC~

1.2129/10

CULTURE

{WOAFB)

NSG~rlVE

fOR

02/12/11

Four units of medium

MYCOBACT~RIA

a~e ~nooulated

reclovery of the organism.

for

Acid fast

bacilli /AFB) are r.ea6 at 1,3, ~nd 6

weeke. Pefault culture result is

19 entered or the final negative

6 weaks. g1ght
be reported
only if positive ,

PENDING " and Bhoul~ be int~reted

as NEGATIVE until a positive result

res~lt ia entered at
week LJ cult~ra will

CARD~ CIRCUS,B~TTY

lPAGi

END OF REPO~T
Advanoed D~agnoat1c Labo~Atories
NATIONAL JEWlSH HEA!Jl'H:

1400

J~ck8on

DENVER, CO

Street
80206

Client Services: 800-550-6227

CAP #217a9~1

Legend: H" Hlah III Low @ .. critIcal

~~J"'/~~ I '

"'IHT "120

eLtA NllBC0644S07
LAB 105 (8.08)
{JOB NO.

&t071

LAB ~AT L .JEVJ!S:J/DENVR, CO

o Nntlo~CJ.I Jewish
Q 1-lef.1lth
Solen~o TrumlormlllQ

ADx
TEST RESULTS

We

AFB SMEAR AND CULTURE

Sp~CnIEN SOURC!!!
ACTUAL COLLECT DA~u

PATIID-l'r'S

NO, 06l0 -p, 1/6 .--

1400 J~,l!on Stml

CoIWl(io 1!O206

f)el\\'~f,

""""",NJbb!.("r.
djn(tChb1@njc,otg

'rRU1\l/( WAS;!

03/17/1 1

PHY~!CIAN

DENt,r!S SCIiMITT

PHYSICIAN'S PHONE
PHYSrCX&~'S F.~ NO

BCD 861 9572

BOO ad9 0869

COMfyfENTS
SMEAR E.'(}J.nN~TIO~

?LEASE ID lLl\lD DO SUSC H' ?OSIT;rVE "

[NOJ>.!.i'B)
3/24/20 11
11

No AYe fO'.1nd

CULTURE NEGATIVE POR MYCOBACTERIA

04130/11

Four units of medium are tnOculated for

recovery of the org~nig~, Acid fase

AFB CULT ORE COltll'rENT

bacill i (AFB) are read at :,3, and 6

DeE~ult cultur e result is
"PENl)WG" and shou ld be intel."oreted
as NEGATIVE until a positi ve re6ult
.\S entere d or the final negati ve
tee~lt is entered at 6 weeks. ~~ht
week~ ,

';~I {~!y GRAHM\t~

~3urr:RV!80R

/,:,"J':; M ,'r-';'!OIO ! O~Gv I 111"2

<"',',vlL .nl f~
.J) 1wJ';~

\~ee)( fJ.J Cl\lt\lr e Ni 11 be reyorte rl

only it positi ve.

W2712

COLL: 03/iS/2 011 Oll~OO

Rt:::C: 03/23/2 011 10:44 PHYS: GEORGE CAROEN C

Ai"B SMEM AND CULTURE

SPECI}mN SOlJRCE

ACTUAG COLLECT D~Te

PATIENT'S PHYsrcr~~
CAR~F,N

CI~CU8,EO

TRUNK NASH

03/18/1 1
DENNI.9 SCHMITT
CONT1;NUlW

ll?AGE

Advilnce.;i Diagn o s ti c Labo);'a tor i es

NAT;1:0NAL JEWISH HBAL'l'H

1400 Jacks on Stree t
DE~R, CO
80206
clien t Servi ces: 800-5 50-62 2?

CAP #2178901

Lagend: H" HIgh L: Low @ '" Critical

ellA 110600844307
LAB 105 (e.OBl

v National Jewish
52 Health'

\400 JckSOIl Stlm

_ ..,

. _

\Vww NjfAbl,o,.,.

TEST RESULTS

='.' .

eli n..e:)o bIl1fill\G. Of\(

I'''' '" 'fiijd~'n 1'4 ' f 'l1o

'. i , ,- r, "i" ' \. - ....~;~ .. .- . ~ -. " ,' -- ~.~,."\:.- :' - ... . - ~-.--....--- ' ''-.
) , ,.' ~1 '
1\
, 1 .. . , ; ,r l j' , ' . ' . .. ' ' . "W'~fi':if.
' f' I atl). ~Q fI.l1ltlld ,
~'.\ "I; ).

~I _I'_"

,. '

t:,;Y;!\~".

C)UIDEN

p.\~''' .'

" I~

f' '\,~:;

I' f . 'w~
":
,

I,

[,'1"; '

}'/

' :;~ '.,::,

, . ,; .'

.:

.2

. - --

---

'04/30/2011 16.1S
I

...

_.'.J!

" :
.
.
', '. . '

""

~"

,I

3901 WEST HWY 0

SPRINGF'IEl:D,MO 65803

"": "l
4'7 8"\3 3588
t
~)~ I.I~ a.: ,.JJ~~.J,..'_~.~. (~\:'-"~~':'::~.! :~"'~'''' ~'J"..~~~:'':-.!../.'.1';"~ 4.':'':'':':'-i t,t. '.:........ ~!..!.(
W2,12

""\/

'

'.

GEOltGE CAROEN CIRCOS

'l':':"
k /' '.\

_.

f::Jo'~
""
~
~
::,.4:ecHf
'~
~
~
' . "', ,' .!l ' t"" "..
., I' .' . .:. ihneRopo~'P.r.11119
I ' ,
:~\' :
24Y ,
QS~05 60952
' l001427031 :: 'c~ {,. ,: ,' .. . .
"
r

,~(.'
~\ M
~ ' ~I"Vr '

c:m cus, BO

,',

i:'/i:l" ;.

t.

80W~

Dtnvet, Cnlo(ado

ADx

Sclonce 1'l'~nsfo(mlno Llle'

, , ., - ,""'-"t -;;,.___ ,

2/6--~~

NO, 06IQ-P.

TB LAS ~Ar L J t'IIISH/OE~YR, CO

MAY, 4. 201 I \ 1:06AM

..

'

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/1 "

'

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,

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_,.:_.;. :':'''__ ';' I ~:'''~: ~. }. .I~.~ ~J.~ ~
':l~J~.;... _
1

, ' . _ _

COLL: 03/161Z011 08:00 REC : 03/23/201! 10:44 PHYSI GEOROg CARDEN C

AFB SMEAR AND ClP.JTURB

(CON'l'INUED)

800 861 9572

800 B~9 0869
"PLFJ>.SE In AND DO SUSC IF ?OSITIVE "
No ~FB found
(NOAFS)

PHYSlCrAN'S PHONE
PHYSICIAN'S FAX NO
CO~tMENTS

SMEAR &XAMINATIOM

J/24/2011
CULTU~E

AF1:J CULTURE

NEGATIVE FOR MYCOBACTERIA

04/30/11

Four units of medium are inoculated for

AFB CULTURE COMMENT

recovery of eha organism, l\cid fast

bacilli lAFB) are read at 1/3, ~nd 6
week Q Default culture rssult is
"PWDING" ~nd should be interpl:ated

~s

NEGATIVE until a ~06itive result

is entered or the final negative
~esult ~~ enesred at 6 we~ks, Eight
\~eek W cult\.Jre will be reported
only if positive,
W2114

COt.L: 03/19/2011 09 100

).U'S SMElIR

A1~D

RSC: OJ/2J/2Qll 10:46 PIll'S, GEORGE CARDEN C

CUI/l'lIRE

SPECIMEN SOURCE
ACTUAL COLLECT DATE
PATIENT'S PHYSICIAN
PHYSICIAN'S PHO~E
PHYSICIAN'S FAX NO
COMM8NTS

CARDEN CIRCUS/BO

TRUN"t!. WASH

()3/19/11
DENNIS SCH..\11.T'I'
SOD 861 95'12

000 849 0869

".?LEASE In AND DO SUSC !F POsrTIVE"
com'lNUED

~pAGE

Advanced Diagnostic Labor;l.tories

NATIONAL JEW!SH HEALTH
1400 Jackson street
DBNVER, CO 80206
Client Services: 800-550~6227
CAP #21789-01

Leg~ncl:

H c HI[lh L .. Low @" Crillcai

WA #QeOOB44307
LAB 1M (p. nIt\

IS LAB

MAY. 4,21)11 II:OJAM

~Al ' l

()6 Na1ional Jewis h

Healtll

NO. (\610 -

JEWISH/OENVEk, CO

10100 )~,~wr. Slr~1

ADx

OtJWC"1 (()}(lr~d(l ~I)2D~

1I'W\v.NJuh"org

TeST RESULTS

S"ivnce ThMslorml~g lIle P

p:~':!Jlif;h~"iii". ~:;: ";7 '. ";;;"":"~ ':""'::":';~"- ';;'T;':(~

t i<{'1

j
ih:.:~~,
,\

;.~:'-.'-;;;.: ~;.,

CARDEN CIRCU S/BO

'': :?:'

["(;,,,,,1--1","

('~;-i:;-;~'
~ . ;...

'.,

I.: ;':

~,
!l:":( . ;"',',;Iff} j . , ..'., ..../ .:, :.. ",, ,:'",'I.,_,"A'6b!.,!/j. '~,

:.:',

.::

24Y

<:'

tUnrcOftbl@Qjc.o1ff

-:-d,j;~ .;'ii:PliOl;d'--

l~:

)'<>;,

nr<

...-.

. 04/30/ 2011 16: 18

.:.) 1 .

'

OS-0560962"l.1:0014~703~

.'

1' 1" "' . '

,

.'lliTIv,REI\:)Qr.UlrJ(lftl,

' "

GEORGE C.~~Dl!:N CIRCUS

r. 3/6-, , - -

"
~

.. ,

'. I

"

" . ,

3901 WEST HWY 0

.,

"

.. '

SPRINGFIELD,HO 65803
. ' , '.'."', ' ", ' , "', ':' ' . " I
I,:J;~; ,
417 83 3588
,, ' " ,;"",'
!.~....:..!.~,,:/;.rl_" ,.. ~.-!_,,\~ .... ,..#-~j '~~" ., ..:~~r,.~J~_.A. ...,..:~;J ....i-:.I..'~"l . r l~-,;.:...~!c-";'~I, I.' .....-!. ....\ _
,~'._-;: .~'.:",:.'. :::~ ... :.~ ~ ~~. !'..!. . ' ::..! ~.. ,~,~:..'.:. . I -...!
1

.... :

W2714

COLL:

' ':' "

'

.. ' . ; , ,

'

03/19/20~l

J>.,FB SMEAR AND CUl-TORE

SM~AR EX~INAT!ON

08:00 REC; 03/23/2012 10146 PHYS: GEORGE CARDEN C

(CONTlNUFD)

11)0 fJ.PB founci

[!~OAFBI

3/2~/20!1

CU~URE

AFB CUI/fUNS

AFE CULTURE

COMME~f

NtGATIVE

04/30/1 1

~OR

MYCOBACTBRIA

Four units of medium are inocul ated for

:ecovery Of the organism. Acid fest

bacill i (AfB) ~re read at 1,3/ a~d 6
weeks, Default cultur e result is
"PENDINO' and should be interp reted
as NEGA~IVB u~til ~ positi ve result
is an~ered O~ the final negative
result is entere d at 6 ~)l9illl<s ,

Eight

week LJ cultur e will be re'9orteci.

only it positi ve,

,JENNY GHAH~,M

'I

CARDEll) CIRCUS/BO

SUPSfN~SOH
VC08A8Tj~H!OL.OGV

LA2.

3PAGE

END OF REPORT

Advanced Di~gnostic Labo ratori es

N'ATIONAL JEWI SH HEAL,T'!1

:400 JackSO n Stree t

DENVeR, co 80206
clien t Servi ces] 800-5 50-62 27
CAP #2'1789-01

Lagend: H a HIgh l" Low @" erllical

ellA 1I06D0644307
LAB 108 /S,OR\

4. 2011 II:07AM

MAV.

TB lAS NAT ' l

JEWIS~/ntNVfR)

~'Notlonal Jewish

12

Health'

ACTUAL COLLECT DATE

PATIENf'3 ~SICI~
PHYSICIAN'S PHONE

P}{Y'SICIAN I S FAX NO

1400 JQr~IOO SlICCI

Dennl, Calorodo 8~6

ADX

~.NJLAh.5 , nrg

TEST RESULTS

Solenca Trunsfotmlng Uta'

AFB S11EAA AND CULTURE

SPECrMEN SOURCE

NO.0610- -Po 4/6 ---

co

rllnrdl~ba@nlo.OIIl

JENNY GRAHAM

SUPERVISOR
MYC08ACTERIOLOGY LAB

TRUNK WASI{
03/17{ 11
DENN1S SCf{MIDl'

BOO 861 9572

800 B49 0869
PLEi~SE

mo

COMMENTS
SMEAR BXAMINATION

No

AFB CULTURE

3/24/2011
CULTQRE NEGATtVE FOR MYCOBACrERIA

AFB CULTURE COMMENT

W2698

APe

J.D

fOUlld

DO SUSC IF POSITIVE"
[NOAFBJ

04/30/ 11

Four units of mediUm are ~noculate~ for

recovery of the organism . Acid fast
Qacill i (AFB) are ~ead at 1/3/ and 6
weeks, Defau lt cultur e r~eult ia
"PENDrNG~ ~nd should oe i.rtte~reted
as NEOAT!VE until a positiv B result
ie entere d or the final negati ve
result is entere d at 6 weeks. Eight
week LJ c~lture will be report ed
only if positi ve.

COLLi 03/18/2011 08,00 REel 03/23/2011 10132 Ph~S: GEORGE CARDEN

C

ArB SMlWt AND CULTURE

SPB:ClMEN SOURCE
AClJ'aAL COLLECT DA'l'E

PArrENT'S PHYSICIAN

CARDEN C!RCUS,BETTY

'.\~~lNY GI~AHAM

TRUNK WASH

SUPERVISOR

03118/11
DENNIS SCHMITT

.: ::. ~\ASTr:R!OLOGY LAB

lPAGB

CONTINUli:D
Adv~ced Diagn ostic Labo ratori es

NATIONAL
l~OO

J~W!SH

HRALTH

Jacks on stree t
DENVER, CO 80206
Clien t servi ces; 800-5 50-62 27
CAP#217S9-01

Leg~nd : H:I Hlgtl l.;: low @ -= Orltlcal

ellA #0600!J4.4301
LAA WI IA M\

t ~on JIcuno Sh:tOl

O' National Jewish

12 Health'
Sclen~e

NO. 0610- P. 5/6---

TB LA9 HAT'l JEW!SH/OEHVER,CO

MAY, 4,2011 \1 :07AM

n.ava., ('.olotlldQ 1l(12()6

ADx

"""' ~br.or!J

tEST RESULTS

Tra ~!lormln9 ~Ife'

ellnrdllb1@n!a,Q,

.
,
"
,

W2698
AlI'1l

':

"

1"

J'

:', .

'l'

'

I,.',

'

"

COLLe 03/1B/2011
S~

AND

cm,TTJRE

(CONTINUED)

SOo S61 9572

800 849 0869
PLBASE ID AND DO BOSe IF POSITIVE"

PHYSICIAN'S PHO~
PHYSICIAN'S FAX NO
COMMENTS

[NOAF~)

SHEAn EXAMINATION

No "FB fOlmd
3/24):aOll

AFB CUL'rURB

CULTURE NEGATIVE fOR

04/30/11

AFt! CUIII'U'Rl!:

MYCOBACTERI~

Four units of medium ate ino~u16ted tox

reoovery of the organism. Acid east
bacilli (AFB) are reaQ at 1 , 3, and 6
weeks, Default culture result ~a

CO~'I'

~,::; ~~lY GRAHAM

"PIflJDINO"

~:V:-'';.HVfSOR

"' ~) ': :\')'1 ~RlOLOGY LAO

W2703

"
I

'

~nd

should be interpreted
until a posi. ti ve result
is ent$red or the final negative
result ie entered at 6 weeks. ~ight
week LJ oulture will be reported
onl~ it ~ositive.
~B NEGATIVE

COuL: 03/19/201L 08;00 REe, 03/23/2011 10:34 PRYS:

AP'B SMl!M AND CULTURE

SP1!:CIMEN SOURCE
;\C'l'OAL COLLlei' DATB

PATIENT'S PHYSICIAN
PH'! SICIl\.N' S PHONE
PHYSIClk~IS r~

NO

COMloOOlTS

CARDEN C1RCUS,pETTY

TRUNK WASH
03/19/11
DEtfflIS SClIMITT
BOO BH 9572

800 849 0869

"FLBASE l:D AND DO SUSC IF l?OSl't'IVE I
2PAG~

CON'l'I~D

Advanced Diagnostio Laboratories

NATlONAL JEWISH HEALTH
1400 Jackson Street

DENVER, CO

80206

Client Services: 800-550-6227

CAP #2178901

legend: H" HIgh Lc Low @" critical

ellA #06006<44807
I All. 1M IA

11~\

MAY, 4, 2011 11:08AM

, -No, 0610' -p,

TB LAS NAT'l JfWISH/OENVER, CO

o NEltlof1sl JewJsh
J2 Health'

t.wo J;el:.OlI Sacot

De'IVlIr, Colol'llcio &0206

AOx

'I'FII'YNJlAb.. otg

TEST RESULTS

Selano's Transtormlhg Ufa'

6/6r--~

t.llwao.l@oJc,~

..

.'

"

.~=::t.!.!..2b::;:u~:i:::i~:::::::::::~~~=::!J.!:-=:=~~~~" , "', ' ' '~.::,.' ,:.,I.. ;, " ' . ." '" ':
"

"

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I I

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:" ~J....!....., .~~'t../o"' '':'' -''''oohl 4.. ~:.:_lh 4-"'" " ::""'~'. w~

W2103

CO~I

AFB SMEAR

03/19JJOll 08100 REC:

CUL')'UlU!

SMEM WMJ,."'1IATION

AFB CULTURE

03/23/201~

10:34 PHYS: GEoRGE CARDEN C

(CONTINUED)

No AFB found
mOAPB J
3/24/2011
CQLTURE NBOA~lVE FOR ~CO~ACTtRIA
04/30/11

lIPB CULTURE COb!ME1fr

Four units of medium are ino~lated for

recovery of the organism, Acid fast

~cil1i (ArE) are re~d at 1,3, and 6

weeks, Default ~ulture result is
"PENnlNGQ and should oe intel~~eted
BS NEGA.'l'IV1!: until a. posit.ive result
ill entez'eO or the final negative

result is entered at 6 weeks,

Eight

week LJ cultura will pe reported

only if positive.

" ,~\~NY GRAHAM

SUPERVISOR

~08I\CTERIOLOGV LAB

CARDEN CIRCUS,BETTY

~ND

OF

3PAGE

~~POR~

Aevanoed oiagnostic Laboratories

NATIONAL JEWISH HEALTH
1400 Jackson atreet
DENVER/ CO 80206
client se~ice8i BOO-550~6227

CAP #21789.01

Legend: H ~ H1Sh L" Low @" Crl1lO81

ellA #06Doe4~O"
I .11:1

~1'I1'

,0 1'10'

:Ul, 25,2(HI :C: 56AM

NO, 4616

I"00 J<I(~!on SI't(

0 f'Jational Jewish

H31951

ADX

Heall'h'
Sr.'o~r.~

? (/3

n.,\VOf,

TEST R.ESU LTS

TrenstollllioQ lIlo'

COLL: 06/l0120H 06:00

Cohmd(. NlW6

w\V\\',NJL.b.I , Ol'~

cllnan,b.@,IIMCS(

REC: 06/1612013. lO:H PHYSI GEORGE O.RDE~ C

AFB SMEAR AND CUi,T{JRE

SPECIMEN SOURCE
COLLECT DATE

TR\1N:< WJ\SH

ACTU~

06/10/11

?ATIENT'S PrtYSICIAN
PUY$ICINlI ' S PHONE

DBNNIS SCHMITT

~HYSIC1AN'S fAX NO
SMEAR ~XAMJNATXON

800 849 OBli9

No AFB foun<i

417 861 9572

6/16/2011

iNOAt1B)

J\F:3 CULTlffiS

CULTURE NHGA'H'lE rOR

AFB CaLTlB.8 COHNBN'l '

Pour units of me6i\Ul1 are inoclllat:.td Eor

l-ecovery of t 'ne orQllnism, Acid f~st

1l~1
f.';31953

bacilli

(A~BI

\veekg,

Oefaul t cult-llt'e result is

REe: 06/16/20H 10:45 ?HYS: GEORGE CARDEN C

SPECIHE)1 SOURCE.

':'RiJ~!(

06/11/11

DENNIS SCHMITT

l?HYSICIA.~'S

417 861 9572

C~RCUS,BO

~nd

a6 NEGATiVE ~~~11 ~ positive result

is eotered or the fin~l negative
resl\lt is entered CIt 6 weGks, Eighr.
1I1eek LJ culture will be reported
o"ly it positive.

ACTUAL COLL~C~ DATE

PATIENT'S ~HYSICrAN
?MNE

are read at 1,3,

___--'""... D1NG" ll1ld shall 1d be inter.preted

COI,:-: 06(11/2011 09:00

M'E SMEAR A.ND CULTURE:

ClLQDBN

~YCOBAC1'ER:rA

0, n2 III

WISH

CONTINUBD

1 PAGE

Advanced DiagnoBt,J,C Laborfltories

NATIONAIJ JEWISH HEALTH

1400 Jackson Serest

DENVER, CO 80206
Clieot Services: 800-550-6227
CAP 1121769-01

L~gend;

H" High L" Low @ c Oritlcal

CIoIA fl0600644307
LAB '(05 (6,08)

)Ul, 25, ]O! I II,:: S7AM

TB LAC ~ArL ..JEWISl{/ONv~~, CO

0- National JGw'sh

H31953

We'

COLL; 06/11/2011 08:00

ArB SMEAR AND CUL7URg

P~YS1CIAN'S ~AX
S~EAR

REC! 06/16/2011

101~5 PH~S:

P, 2/ 3

1.j00 .IR~"O"n SCfctr

C,)I01'l'ldo t020!)
IVWW,NJUtll.ot'l:

De~ycr,

ADx
TEST RESULTS

Health"

Solellc~ Tr~Mlorm/ng

NO. 4616

cUnr~flr.b!@nic.ocg

GgoRGE CARDEN C

(CON'l'I~UBD)
SOO 8d9 OB6~

NO

E)W'{INATION

NO lIF3

found

(NOAFJ3)

6/15/2011

CULTURE NEGATIVE FOR

07122/11

l\FB CUurORE
AFB CULTURE Co.~

1\ \

)~

H31955

-y;1

Four: units of medium a~e inoculated for

recovery of the orqanism. Acid tdst

bacilli (APBI are read at I,], and 6
weeks. Default culture result is

(I
vl

"PENOlNG' and should be interpreted

as NEG~T!VE ur.:il a poaitive result

is e~cered or tbe final negative
result is entered at 6 weeks, Eiaht
week LJ culture will be reported
oply if positive,

COLL; 06/12/2011 06 : 00

AYe SMEAR AND CQLTORE

SPBC!NEN SOURCE
ACTUAL COLLECT DATE
PATIENT'S PHYS~CIAN
~HYSICIAN'S PHONE
~nSICIA.N I

S fAX NO

SMEAR EX}.J'11NATION

MYCOBACTERr~

REe: 06/16/Z0l1 10:46 ~HYSl GlORGE CA~EN C

TRUNK WASH

06/12/11
DE..tomtS SCHM':TT

417 S61 9572

800 849

086~

NO ~FB found

{NOAFBJ

6/16/2011

CARPEN

cn.cus

BO

CDNTXNUED

Advanced Diagnostic L~boratories

N'Alf'IONAL JEWISH HB:P,LTE
1400 Jackson Street
DENVBR, CO 60206
Client services: 800-550-6227
CAP #2178901

Legend: H" HlfJh L:t Low @" Critical

2PAGl

ellA #0600644307
LAS 105 (8.08)

Hil. h

n II

10 : 57AM

:)

L A~ ~AT ' l ,JWI S H/ DENv~~,

0' I\llltional Jew;sh

CO

TEST RESULTS

Solonco TMns(Dr{lllnij Ufo I

~31955

COLL i 06112/2011 OB : OO

MB Sl'1BAR, AND CULTURE

lIP'S CULTURE

ArB CCLTURE COMMENT

06/16/2 011 10:46 PHY,'3l GEORGE

R~l

(CONT'rWED)

CULTUF3

3/3

\."'w,NJI.~"of8
ctlnr~f1~b,@nic.ot~

CAl'tD~N

FOR MYCOBAcrERIA

0112~/11

Fovr units of medi~m are inoc~lated for

recovery of the organism. Acid fast
bacill i (AFB) ate read at 1.3, a~d 6
week9, Defaul t cultur e result is
"PEl'IDING" and should be inter);'Jreted
as NEGA~IV2 unt il a positi ve result
is a~tered or the final negati ve
{e8ult ie entere d t1t G weeks, Eight
week LJ cvltur e will be reporr.ed
ol~ly

CARDEN CIRCUS,BO

NBG~TIV3

?,

J4QO J~ 1t'OJ) Srrtt/

D,,"'t(, Coloruuo 802(k;

AOx

Health'

NO, 46! 6

if positiv e,

3PAGE

END OF REPORT
~dvnnced

Diagn ostic

Laboretorie~

NATIO NAL JE 1f1ISH 'HEAL1'H

1400 Jacks on Stree t

DENVER, CO

CAP #2178901

80206

BOO-550 - 62~7
Legend: H" High L" Low @" Crltlc!'>i

Clien t servi ces:

ellA #06D0644301
LAB 105 (8.08)

JUL. {5, 20 II 10 :55AM

TB LAB NAT ' t JEWISH/PENVER, CO

()I NatIonal Jewish

12

~O.

ADx

HealM
sofanC8 Tranlfofmlng LIfe"

TEST RESULTS

4615

p, I

1406 JQCQon SIRO'

DCIlVe:r, Co \o!td~ BOZC~
wI\I7./llJlAb,.ora
clinr~&bl@njc.O'il:

G~ORGE

II

CARDEN CIRCUS

3901 WEST HWY 0

SPRINGFIELD,MO 65803

H31937

COLL: 06/10/201 1 08 : 00

ArB S!!t!AA AND ClJLT'ORE

SPECIMEN aOO'RC~
ACTUAL CO~wECT DATE

PATIENr ' S PHYSICIAN

PHYSICIAN'S PHONE
pHYStCIAN'B FAX NO
SMEAR EXAMINATION
AFB CUTlNYRE

I
,

REC: 06/16/2011 10;39 PHYS ; GEORGB CARDEN C

~RUW<

WASH

06/10/11
DENNIS SCMITT
417 861 9572
BOO 949 0869
No ArB tound

(NOArB)

611612011

clJi,Tt]R~ ~EG'\'l'lVE

FOR MYCOBACTERlll

07/22/11

AFB C01IrURE CO)IMENT

Four units of meoium are inOdulated for

racovery of the organism , ACid fast
li (AFB) are rea~ at 1,3, ~d 6

w~eks,

Default culture result'ia

"PENDlNG' and should be interpreted

as NIDGATrvE until a positive ~esult
is entered or the final negativB
result ia entered ~t 6 W!ekS, Eight
week LJ culture will be reporteQ
only if pOBitive,
HJ1939 CO~Ll 06/11/2011 08:00 REC: 06/16/2011 10 ;39 PHYS: GEORGE CARDEN C
AVB SMEAR AND CULTfJR E
Spg.CIHEN

SOURCE

ACTOAL COLLECT PATE

PATIENT'S PHYSICIAN
PHYSICIAN I S PHONE

CARDEN CIRCUS/BETTY

TRUNK WASH

05/11/H
DBNmS gCHMITT
61, 833 9141
CONTINtlED

lFAOE

Advanoed Diagnos t ic Laooratories

NATIONAL JEWISH HEALTH
1400 Jackson Strset
PENVER, CO 80206
Client Services: 800-550-6227
CftP #217811-01

Lagend: H "High L I< Low @" Crlilcal

ellA #OBDOB~4307
LAB 1D5 (8.08)

JUl.25.201110 :56AM

TB LAB NAT'l JfWISH/OENVER, CO

I~OO

i). N(ltio(1al Jewish

12

NO. 4615

TEST RESUlTS

Selene, Tt'Ilnsformlng tlfe'

J,dcaoa Stmr

D~"tl,

ADx

Health'

p, 2

Colot.do 80206

\lOWW.N)LJb 1 .0I8
clJnrcD.oh.t@lIlC. OIll

GEORGE cARDEN CIRCUS

3901 WEB'!' HWY 0
SPRINGFIELD/Me 65603

i
I

I
R31939
AHB

COL]:,: 06/11/201-1 08rOO

~AR

AND CULTU'ltE

PHYSIC~AN/S

SMEAR
}I.P'B

FAX

~O

KXAMIN~TIO~

CULTURE

ArB C!JlII'ORS COMMENT

REC; 06/1612011 10139 PHYSI aE'RGB CARJ)EN C

(CONT1NUEDl

800 849 0869

No AFB found
[NOJ\FB]
6/16/2011
CU~ITRE NBGATIVB FOR MYCOBACTERIA
01/22/11

~our

units of medium are inoculated for

of the organism. Acid fest
baoilli (AFB) are read at 1,3, and 6
Weeks. Default culture resul~ ie
reoo~ery

"PENDING" and I1hould be lnt.et-preted

as NEGATIVE until ~ Dositive result

i~ entered or the final negative
rellult if! entered ~t 6 wee)c,:/. 'Eiqht
week 'IN cultllte will be reported
only 1f

H3i943

~ositiv~.

COLLI 06/12/2Cll 08100 agel 06/16/20l1 10 141 ~HYel GEORGE CARDEN C

AFl1 SWEAR AND COU'I'URE

SPECINEN SOURCE
ACTUAu COLLZCT OATE
PA~rENT's PHYSICIAN

TRUNK ~SH
06n2/11

PHY SlCI)IJJ' S PHONE

PENNIS SCHMITT
417 861 9572

PHYSICIAN'S ?AX NO
SMEAR BXAM!NAT!ON

No AFB found

CARDEN CIRCOS/BETTY

800 869 0869

(NOAFS)

6116/2011

2PAOE

CONTINUED
Advanced Diagnostic Laboratories

NATIONAL JEWISH
1400

HEAL~H

J~ckson St~eet

DENVER/ CO 80206
Client. Services: 800-550-6227
CAP #21789-01

Lagsnd: H" H!llh L = Low @'p= CrfUoal

ellA (106005807
)..AB lOS (8.08)

JUl, 25. 20 j/ 10; 56AM

TS LAB NAT'l JEWISHIDEHYER,CO

NO. 4615

P. 3

tWO JtClt!QII S~~I

(). National Jewish

Q Health"

Donvcr, Colntldil S02l)d

AOx
TEST RESU/...TS

Sclenoe Transformino Ufv

~.NJJ,.h"0f!l

dUu:62Jbl@nle.0l8

I
J

R31943

!
i

COLLI 06/12/201l 08:00 Rmc! 06/16/2011 10:41 PBYSI GRORGB CARDEN C

Al'B SMEAR AND COLTt.mE

AF1l CULTURE

(CCNT INiJEi))

CUUTURE NEGATrvE FOR MYCOBACTERIA

07/22/11

PJl'B CoIIl'URE COMMENT

CARDEN

CIRCUS,BE~TY

Four units of medium are inooulat~ for

recovery of the organie~. Acio fast
b~Cilli (ArB) ~re read ~t 1/3, ~d 6
weeks. Default culture result is
'PENDING" and should be intertlreted
as NEGATTVE until a positive result
is entered or the tinal negative
(ssult is entered at 6 weeks. Eight
~eek LJ culture will :Os reported
only if po~itive.

END OF
Advance~

.3P]l.GE

REPO~T

Diagnostic

NATIONAL

Labo~atoriea

~EWlgH

HEALTH

1400 Jaokson street

DENVER, CO

90206

ctient Services: 900-550-6227

CAP'21789.QI

legend: H'" HIgh L" low @. Cr/Ucal

eLlA iIOeD0844~07
LAB 10S {8.0S)

AUG. 24. 20' 1 I: 46PM

T8 LAB im'l .JfWISH/ONV ER, CO

NO. 622 5

p, 4/ 6

IoIUO J"kl(l~ Srreol

Vl'Ilil1;ional Jewi!fh
Heolth'

DClI~II',

ADX

12

TEST RESULTS

Sclenne Trlll&/ormlng life'

Colol'1do 81)206

\"""".NJT.1bl.Ol~

ellrutlbbl@nic.o"

GEORGE CARDEN CIRCUS

3901 WEST HWY 0

SPRINGFIELD,MO 65803

T9496

COLLi

08/18/2Q~1

08:00

REC: 00/23/2Q11 16:06 9HYS: GEORGE CARDEN C

l\FB SMEAR .t\ND C(]L'l'URE

SPECIMEN SOURCE
ACTUAL COLLECT DATE
PATIENT'S PHYSICIAN
?HYSIC~&~'S PHONE
~HYSICIAN'S FAX NO
SMEAR EXAMItIlF\T:;:ON

TRUNK Wl\SH

APB CO!.rTURE
APB CULTURE COMMENT

PENDING
Four units of meiurn are inoculate~ fo r
recovery of the or~anisro, Acid fast
bacilli (AFB) ~re ,ead at 1/3 / and 6
weeks , Default culture result i9
"PE:NDING' and should be int~rpreted
as NEGATIVE until ~ positLve result
is en t ered or ~hB final neg~t i ve
result is entere~ at n waaks. Eight
week LJ culture I"ill be reported
only if positive.

T949B

COL~:

SPECIMEN SOURCE
Ar.rOAL COLLECT DATE
S PH'(SICIAN

PHYSICIAN'S PHONE
PHYSICIAN'S FAX NO
CARDEN

DBNNIS

SCHMI'r ~1

417 852 957:1

800 849 0869

~o AFB found
B/2~J201.1

(NOAfEI

08 / 19 / 2011 08rOO REC : 08/23/2011 16 :08 PHYS; GEORGE CARDEN C

AFB SMEAR AND CUL'l'ORE

P}l.TIE~IT'

08/18/11

CIRC~S,BO

TRUNK WMH
08 / 19 / 11
DK,l\INIS SCHi'lITT
417 861 9572

800 849 0869

lPAGE

CONT I N'(JED

Advanced Diagnostic Laborator.i.es

NATIONAL JEWISH HgALTH
1400 Jackson Street
DENVER, CO

" r " . ,'

'."""'. <', ;f "

~"I'(mWt~
t '1l~

~t.! ~,rd .A' .: . ~

"]",,. /1 . "
~ ~~1\ 1 " .. ,j

" -,&f
I!'l lt.'j'"
f~ .

80206

Client Services: 900 - 550-6227

CAP W78S-01

Legend: H" High L c; Low @" Crlllc~1

ellA #0600644307
LAB 105 (8.0a)

AUG,

n 2011

1:46PM

CULTUR~
~XAMrNATION

,V\,w.NJI.nbM/&

TEST RESULTS

COLL; 08/19/2011 08:00

SMEAR

D<m\"tJ, Cni()Y1I(\o e02IJ6

ADx

SciBnce Trenstolmlnll Lila'

AF8 5MBAR AND

5/6

?,

, 400 J,,\(son StL'tOt

'V' National JewIsh

12 Health'

T949B

NO, 6225

1B tAB NAl'l mV ISH/OENYER, CO

REC:

08/2~/2011

(CONTINUED)
No AlB found

clio!o fl tbloj C.OtS

16 : 08 PHYS: GEORGE CARDEN C

(NOAFBj

8/2(/2011
A~B CULTURE
AP'B CULTURE COlolMENT

P!tNDING

FO\lr unite M medium

~re

inoculata<l (or

:ecovery of the organism. AGio fast

bacilli (Ara) are read ~t 1,3, ano 6
weakS. Default culture result J.s
"PENDING" and J.!hould be interpreted
as NEGATIVE until a positive reBult

is enterect or the final

l~ entered at
week LJ C\,lltVl"e will

result

Dnl~

'l'950Q

COLI, : OS120/20H 06:00

AFB SMEAR AND CULTURE

SP~ClMEN SOURCE
ACTUAL COLLE~ DATE
PATIENT'$ PHYSICIAN
PHYSICIAN'S PHONE
PHYSICIAN ' S FAX NO

SMEAR

EX~INATION

APB CULTURE
APB CULTURE

CAAD~N

COMM~NT

C.! RCUS J BO

ne~ative

6 weeks. Eight
Pe rspottea

if positive .

RgC: 081n/201l H:09 PH'lS: GtORGE CARDEN C

BRONCHIAL WASHLNGS
08/20/11
DBNNr S SCHMITT

417 861 9572

800 849 0669

No AF8 found
8/24/2011

(NOAFBJ

PE'.!'1D!NG
(!'Ollr lIl'li ts of lOediu!\\ eire inoculated for

recovery of the organism,

Acid East
APAGE

CONTINUED
Advanced Diagnostic Labor&tories
NATIONAL JEWXSH HEALTH
1400 0ack~on Street
DENVER, CO

CAP #21789-01

)yJr-:::, ': " ":I~'

-IV,'.

l,:wllfr.:~

" . ,iJ,8'i

80206

Cl~ent Servioes: 800-550-6227

lS991ld: H" HI(l1'I L" low @ = Crilical

ellA #00D0644307
LAB 106 (6,08)

AUG. 24. 2011

1:47PM

fB lAB t(AT ' L ,IEWISH/DENVER, CO

()- Natronal jewish

Q Health'
ScIence 'Trftn.(ormlog u,~

T9500

AOx
TEST RES ULTS

NO, 6225

P. 6/6

1400 Jocklon SaccI

DenY4T. C:DI.m~~ 80206
'''''"\II'.NJU\ bLor!:
(Unl1;J1~lQ1lnj, ,nrg

COLL: 08/20/2011 08:0D R~C; 08/23/2011 10;09 PHYSi GEORGE Ck~DEN d

AFB SMEAR AND CtJI.fiURE

(CONTINUED)
bci11i (AFB) ara read at 1,3. and 6
Default c~lt~re result is
" PENDING" ano should be interpreted
as NEGATIVE unti l a positive reault
is entered or the final neg~tive
result is entered at 6 wee~s . Eight
l\feek W culture will be reported
only if posit ~ ve\

we~s.

CARDEN C1RCUS,EO

3 PAGE

END OP REPORT

Advancea Diagnostic

La~oratories

NATIONAL JEWISH HBALTH

1400 Jackson Street
DENVER, CO 80206
Clien't Sarvicl!Is: 800 - 550-6227
CAP t/217Se-01

Legi!ll\d: H '" High L =Low @ =Cr1t1cal

:. ,;\!Wl!:~

. ' '~i!S'(
ellA lIoeD06443<l1
LAB 106 (8.0B)

12/22/2011 THU 14124

'A~ 4~1 833 '14L Georoo Carden Ctr~u8 Inc

~An .IWlS1/0flIV{~

18 LAB

~OV006

CC

HC, 201:
1400 J,~laon SJl~:

0" NatIonal Jewl.h

12

D:n\'<;1, Colol,u' 101Qd

Health'

1AI\I'W,"UlAbl,cI 6
duuen.bl@nj.'Olo..IIltv

Sell/lq. Trln,larlllll1lll.."

'f9'90

COLL, 08/18/2011 OS:OO

REel C8/23/2011 H,!)l PHYSl GBORGE

CARDEN C
,

AiB 5MBAR AND CiJUl'!JR!

SPECIlm.'l SO(JRCE:
COLLtcT D~TE

TRUNK If1I8H

P1.'l':ENT' B

PHYSICI~l

PKYSIC~'S
PWYSYC~AN'B

'

~:~~~~1 ~C~II'lT

~TU~L

PHONE
'AX ~O

~/NAL RF:POR: '

41 j 861 9Sn
800 849 C869

No AYB found

SNE"R EXAlUNATIOM

:NOA~~;

8/24/2011

CULTURE NR~trvs rOR MYCOBAC~~:h

10/06/2011
Four \,fn~la of madium Ull Lnoculae~d Ol"
recovery of the or'An~sm, "al~ !~st
baclll1 (AFB) sre r~ad a~ :,3, ~~d 6
weeks, DefaUlt culture reB~lt is

ArS C:UL'l';JRE

Af5 CJLTUR!

COMM~T

IJ

?WO!NG' and should 1;)9 interpreted

as NBaATlV! until a voa:tlve resul:

19 entered or the finlll negative
rusult La enterIC 4~ 6 w,.~g, 3~g~t
~Ieek 1...1 culture will be r3Po.rted
only it posit! VEl,

T9491

CO~:J

OB/19/2011 09;00

M'S SN"!XR MlD

nsc,

08/23/~Qll l610J

G~ORG! CARDE~

C~'IJl'!:RE

SPBC))IE.'; SOURCE

TRUNK W,GN

ACTUAL COLLECT PA'l'IC

08/19/11
oENNI S SCHMr'M'

PATIBNT'g

pHV9:

pK~SIClAN

p!(YSrCI1IN'S PHONE

CARDmn CIRCUS, BETTY

,I

411 861 9572

CO~TINUED

l~AGl

CAP lIa 1781!.Q I

DEC-2e-2911 14r29

SArv~cesl

a~o-5S0-6227

Ltgend: H a Hlg~ L" Low @ a erlUQlI

Froml 417 6J3 9141

IOtCF'IA NIAGARA

I
I

Advanoed OLagnog:ic ~aborator1e~

NA'l'IONAL J!lWISP. HBAIIl'i-{
1400 JacXJo~ Street
DENVER, co a~2~6
Clier.t

I!

ell> "06D~A~.:107
(.AB 105 (4,11)

ll/2Z/20Il fHU 1412'

DfC, 12.

,0 I!

PAX 411 B33 9141 a8o~~O Cordon

,: 3/PM

~OLLI

"

SHEA~ ~o
~HYS!C!ANIS

CO!1NJR!

FAX NO

GUZAR 'Exoom:rtOH

AI'B CUx.miRE
M'3 CUL,/,~ COOON':'

,.' ~\

'W, ~)):

p;) ,

... ~

': !'i.
I

f, 6

\.00 J",uen ~!Jetl

DOJIW', ColclJ~o S~100

AOX

\\'III\'.NJl..br,~'J
'l.'rlt'\)I@/Lbc&lI~.O "

TEST RSSUL.TS

REC! 08123/201l 15103 PHVS: GEOR~E C1\RO::;N C

08/19/2011 OBIOI)

PJ'B

00J5/0a5

on lAP HAT ' l JfWISHIOfUVR, CQ

'
rif 'l~n)'

o NatIonal JewIsh
Q Health'

':'9491

C1rcua Inc

(CONTINUED)
BOO 849 C86,
I~O AJ'S foun~

(:INAt AE'POA"
!l'IOAFB]

812412011
CUlIIURZ tnX1AT:VE POR
10/06/2011

lofYCOIlACfl~IA

Fou:- \,:n! tB of I\\ediul'l drs boc',:late<1 fo:,

:acove~y oi 'he orgJr,ism.
Acid fast
bacilli (ArB) ere reed at 1,), and 6
Wle~&.
Oefa~lt oulture re~ult ia
I'PSNDING' jl1ld should be interpret ed
AI N20~TIV~

until a

po.~t!ve

r ult

II ent,red or tr.o f~na: n,_ative

result 18 antare4 At 6 ~ee~8 , Ei~hc

week LJ oultu~e will be reported,
only if positive.
T9~92

~~B

C~L11

0&/20/2011 08,00 RSCI 08/2J/2011 lb,04 PHYSI GEORGE

CA~~N

Ii

SMEAR kND CULfURS

apgc:IiI!DI' SO[ffiC!
~CTI1AL- COLLEC'I' JAT:i
p~rIEt/'l'" S PRYBIC!AN

TRONK '/lASH

PKYBICtAH'S PHONE

H7 95'1 9Sn
800 949 0869
No ~~B ~our.d

PHYSICIAN) 8 F~ l'ID
Sl'IEAR !X.lY.INATlOr.r

C8/20/11

DENNIS aCHxITT
(~APB)

8/'34/2011

CARJBN CIRCUS, BETTY

DEC 22 2011 FH12: 14

HAGel

CO~TINUED

Advanoed Di~ostia L~r~toriea

NATtO~AL

JEWISH aEhLTH

1400 JaoKson street

OENVER, CO 80206
OA/> ~21789(J1

Client

SeTv~ce~1

Le;endi H Hlgh

800~S50-6~2?
@. Critical

r.." Low

12/22/2011 rIte 141 L'

OEC-22-2el1 14:2B

From! 417 833 9141

IOI CFIA

NH~GARR

eLlA .06006 4l3J7

LAS IQ6 (4.11)
( n l ~O'

H21)

~tH

J2/22/20 11 TRU 1&124

O[~.

f2.

20 J I

J1hll Gl7

an

91U 0"0':'18 Cudlln C1ro<u rr.o

I: 32P~l

T~492

: lOj )111,0.' StrW

v Health'

J2

rzJO~6/q06

~.

NlltJonai Jewish

lXnYCl, ::oillUl~o !~

ADK
TEST RESULTS

selenot itlNtulI1llng L~o'

1YGI\\,NJubl.Ol'B
~1l1'\l.O.b 1D:h' 1Iho 1&

COLLI OB(lO(20il 08,00 REel OB/21/l011 16104 PBYS: OtDRGB

C~RDE~

C
I

(CDWINUSD)

APS SMEAR A.\'D CtJI/rORE

COLTUIW

"'i!

cut,rUllB NEGATIVE
10/06/2011

FO~

MYCOBACTERIA

I
I

Four units of me~um ~e inocula ted for

recovery f t~~ organ~8m. A: ~ d ~~st

II FB CCUItJR I!: COl'lHBN1'

bac!l~i

(APB) are. read At 1,3,

~d

Dahlll~ au1t:l.1re result ill

'P!m11NGI and 8hou~d be lnterp~~~ed

....eGk.a.

I-lEG,\'Z'IVE I.ln~n A ~ot1t!ve ~e~\l:.t

is snbred :>r the final n~(1Ati va
re8~1~ l' e~e8red ac 6 ~eek&. Eight
weak LJ cu~ture wIll be =eportad
only if POf~t!Ve.
lUI

FINAl REPOf( .
OEC 2220Hp~12\14

CARDEN

~~D

C!RC~S,SE~~

ci'Jl *211BH1

OF

R~?ORT

Aovar.ced ~i&gnoetic L6Por~tori8.

NATIONAL JEWISK H!~LrH
1400 Jaokso n Stree t
OENV'R/ CO aO~06
Cl.hnt :: servio es I 800~S50622?
Lag~rld: H" High L GLow @ .. Crlllc::al
1~/22/2011

OEC-22-c8!1 14,21

Froml 417 833

91~1

!'HU 141

WICFIA NIAGAAA

~t

eLlA t/OBDDW3U1
!.AS 1;)6 (04.1')
I,:oll NO.

~H7'

~007

2/14/2012 8 : 24 AM

Pa.gs

of 7

. . . . . . .."\15 . . .".......

FINAL REPORT

Nlltion!'1 VotertnalY Services Laboratorlos

V(ll:\~-rJIl ~rr~t.'rvi("'8 PO BOK 844
, ..
"t\
' '''
Amaa, Iowa 60010
~\-t."~l. Phon.: 516.:l:l77514 Pax: 616.337-7938
;'''''' v..,.. ":\~~I_"" ~ FeDERAL RELAY SERVice (Volc&/TTY/ASCIl/SpBnlsh) 1-&00.87106339

:;r , . . ,

La be rntorv Tut Ropor!

....... This 1$ a oon(ldentilll repo/1lnlended

Ownor

offlolal U$B only. " ....

AC0&89Ion Number;

11-063398

i98120144ge

OreanA County MO

NFC Control Numb&r.

Date Collocled;
Oato FleOlllvoo :
Date Co mpletlld:

Submitter 269lle

George Carden Clrous

Sprlnglleld,

MO

Anlma( Location

(01

Collected By:

12/21/2011
02/13/2012
01. MIOhell1 Stal'(ord

Pair Grove Vel S8rYlce

Purpoa9:

TB10-4

PO BoXG
Fell Gre1/9, MO G664B
FAX #: 417-7697803
Phone #; 4177697900

RGlorrallRetaln Tllg Number:

Thle Ie II billable C880.

NOTIi: Condition 01 tho tample{e) wu IId9qUIII~ union otherwise r\oted.

Animal ID: 80 Case#: TB12-02037 Semple: 12/13/2011 Spealmen Typo: Trunk Washings Speclu
: Elflphanl, Aslan

Mycobaclerlal Culture ResuJl

No Isolal/on MS.de

Anlm.I 10: 80 Case #: T81202038 SamplG: 12114/2011 Specimen Typo: Trunk Washings Speoles
; Elephant, Asian

Mycobaclarlal Culture Reau/l

No Isolation Made

T81202039 SQm pie: 12/15/2011 Spealmen l'yP9; Trunk Washings Speoles

Mycobacterial Culture Resu/l

No Isolation Made

AnlmallD: Bimbo C(l6& #: TB1202040 Sample! 12/13/2011 8pocllTJon Typo: Trunk Washings
Species: E.lephant, Asian

Mycobacterial Culture Result

No Isolation Made

AnlmsllD: Bimbo CaS9 II: TB1202041 Sample: 12(14/2011 SpecImen Type: Trunk Washings
Speoles: Elephant, Asian

Mycobacterial CultUre Result

No Isolation Made

Poge 1 of 4

Data Generelsd:

2/13/2012

211Q/2012 8:24 AM
Accession:

1H03398

Owner:

Page
George Cardell Circuli

4 of ?
Rafarrlll Number.

An Imal to: Bimbo Case #: TS 1202042 Sam pte: 12/15/2011 Specimen Type: Trunk Washings
Sp4!c19s~ Elephant, Asian

Mycobacterial Culture Result

No Isolation Made

Anlm81lD: Janny Case #: T812-02043 Sample: 1211312011 Specllnen Wile: Trunk Washings
SpecleB: Elephant, Asian

Mycobacterial Culture Result

No Isolation Made

Animal 10: Jenny Caso #: T81202044 Sample: 12/14/2011 Speelmon TYpo: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result

No Isola lion Made

AnImal 10: Jenny Case #: lB12-Q2045 Sample: 12/1612011 Specimen Typo: Trunk Washings
Speolos; Elephant, Asian
Mycobacterial Culture Result

No lsolallon Made

Animal ID: Tory Case #: TB1202046 Sample: 12/13/2011 Specimen Type: Trunk Washings
Speoles: Elephant, Asian
Myoobacterlal Culture Result

No IsolaUon Made

AnlmallD: Tory CaS9 #: T91202047 Samplo: 12/1412011 Specimen Type: Trunk Washings
SFWoles: Elephant, Asian
Mycobacterial Culture ResuU

No Isolation Made

Animal ID: Tory C~M #: TB12-02048 Saml'le: 12115/2011 Specimen Type: Trunk Washings
Speolas: Elephant, Asian
Mycobaclerlal Culture Result

No 180lallon Made

Animal ID: Liz Case tI: T81202049 Sample: 12/13(2011 Speclman lYpa: Trunk WashIngs Species
: ElepMnt, AsIan
Mycobacterial Culture Result

No Isolation Made

Animal 10: Liz CalHI #: TB12.02050 Sample: 12114/2011 Speolmorl TYpe; Trunk Washings Speoles

: Elepha.nt, Asian

Mycobacterial Culture Result

No Isolallon Made

AnlmallD: LIz Cau #: T812.02051 Sample: 12/1512011 Specimen Type: Trunk Washings Species
: Elephant, Asia/)
Mycobacterial Culture Result

No Isolation Made

Page 20(4

Date Gentllllted:

2113120 I2

2/14/2012 8:24 AM
Accession:

11-o83S98

Owner:

P~ge

5 Of 7

George Carden eire us

RelElrrnl Number:

AnlmsllD: Janice Case lJi T812-02052 Sample: 12/1312011 Specimen Type: Trunk Washings
Speoles: Elephant, Asian

M ycobacler!ai Culture Result

No Isolallon Made

AnlmallD: Janice CSge #: T812-02053 Samplo: 12/14/2011 Speclrr19n Type: Trunk Washings
Spades! Elephant, Asian
Mycobaoterlal Culture Result

No Isotallon Made

AnlmallD: janice Cass #: TB1202054 Sample: 12/15/2011 Specimen TYpe: Trunk Washings
Species: Elephant, Asian

Mycobacterial Culture Result

No

I~olatlon

Made

.----------------------~------------~
AnlmsllD: Cindy Case #: T81202055 Sample: 12(13/20'11
Specimen Type: Trunk Washings
Speclu$: Elephant, Asian

Mycobacterial Culture ResuU

No Isolallon Made

AnImal 10: Cindy CaBe #: TB1202056 Sample: 12/14/2011 Specimen Type: Trunk Washings
S eoles: Elephant, Asian
Mycobacterial Culture Reeull

No Isolation Made

Anlma.IID: Cindy CSBe #: TB12 02057 Sample: 12/15/2011 Specimen Type: Trunk Washings
S oolos: Elepharn, Asian

Mycobaoterlal Culture Result

No Isolallon Made

Animal 10: Vicky Case #: T8120205$ Sample: 12113/2011 Specimen TYP9: Trunk Washings
Speoles: Elephant, Asian

Mycobacterial Cullure Resull

No Isolation Made

AnImal )0: Vicky Case #: T812-02059 Sample: 12/14/2011 Specimen Typ.a: Trunk Washings
SpGclos: Elephant, Asian

Mycobacterial Culture Result

No Isolelion Made

Anlm<lllD: Vicky Caile #: TB1202000 Sample: 12.11512011 Specimen Type: Trunk Washings

Species: Elephant, Asian

Mycobacterial Culture Result

No Isolatlon Made

AnlmallD: Belly Case #: TB12-02061 S~mple: 12/13)2011 Specimen Type: Trunk Washings
Speoles: Elephant, AsIan

Mycobacterial Cu!lure Result

No Isolation Made

Page 3 or 4

Date Generewd:

2/1312012

2/1~/2012

Accession:

11.063398

8:24 AM
Owner:

8 of 7

Page

OeO/'g8 CardM Clrous

Refs/1lI1 Number:

Animal 10: Betty Cllse #: TB1202062 Sample: 12/14/2011 Specimen IYP9: Trunk Washings
Spooles: Elephant, Asian
Mycobacterial Culture Rasul(

No Isoletion Made

Animal 10: Betty Ca8~ #: TB12-020e3 Sample: 12/15/2011 Specimen Type: Trunk Washings
SpeclQs: Elephant, Asian

Myoobacterlal CuHurA Resul!

No Isolal/on Made

AnlmallD: Judy CS80 #: TB1202064 Sample: 12/1312011 Specimen Type: Tfunk Washings
SpClcl9s: Elephant, Asian
Mycobacterial Culture Result

No Isolallon Made

~----------------------~---------~---------~--------------------.-------,
Animal 10: Judy C8S0 #: T81202055 Sample: 12/14/2011 SpBclm&n Type: Trunk Washings
Specills: Elephs1\t, Asian

Mycobaoterlal Cultura Result

No Isoletlon Made

Animal 10: Judy Case #: T812-02066 Sample: 12/15/2011 Specimen Type: Trunk Washings
Speoles: Elephant, Asian

Mycobacterial Culture Result

Results I'lutnorkeo by:

No Isolallon Made

Dr. BUslee Roboe-AlJetermlln, Sacllon Head, Mycobecterle lind Brucelill Secllon

NVSl MB General phol'le: tl1~37-~e8

page 4 014

Date

Gonereted:

211312012

'

--V3 __

FINAL REPORT

National Veterinary ServIces Laboratorlos

V~t.erlnary Services PO Box 644

~
~'
,
Ames, lowe 500iO

. . '1~~~

1'..!'.. .......,....:...""

"lIone: 516-3377514 Fu: 615-3377938

FEDERAL RELAY SERVICE (VoleeITTY/ASClllSpanlsh) 1800-877-8339
Tho USDA Is an equal opportunlly Pfovlder and employer.
laboratory Teet Report.
- . Th~ Is a conflden\lijl report IOlanded for official use only, 'w

owner

1201B552

Accession Number:

George Carden
Iowa City, IA
NFC Control Number:
Datu Collected:
Dab! ReceJved:
Date ComplEllDd:
Collected By:

00511112012
07/09/2012
Dr. Allnda Buckingham

Bright Eyes & Bushy Tails

3030 Nort/lgale Dr

Purpose:

TS 10--4

Iowa City, IA 62245

FAX It. 319--341-8445
Fhone #: 319-3514266

RoferrallRetel" Tag Number:

Animal Location
Johnson County lA

SubmlUer- 27437

19612036;379

T/ltR Is a billable calle.

NOTE: Condition of the samplers) Willi adaqu.rt& unl898 otherwise noted,

AnlmellD: 80 AVID# 036564056 Csse #: TB12-05790 Sample: 5/7/2012

ashlngs Species: Elephant, Asian

Mycobaoterial Culture Result

Specimen Type:

Trunk

No Isolation Made

AnimallD: 80 AVID# 036584056 Case #: TB1205791 Sampl&: 6/8/2012 Specimen Type: Trunk
Washings SpecIes: Elephant, Asian

Mycobacteria! Culture Result

No Isolation Made

Animal 10: 80 AVID# 036584056 Case #: T812 05792 Sample: 6/9/2012 Specimen Type: Trunk
Washings SpecIes: Elephant, Asian

Mycobacterial Culture Result

Results Buthorlzed by~

. No Isolation Made

Dr. Suelse Robbe-Auslermsn, Section Head, Mycobacteria and Brucella Section

NVSl MB General Phone: 6163377388

Help Ua H,lp you

(11110 "SON leoUon will be updaled petlodlClll1)i willi Up-' tor submitter ..)

To W(pedlte the Pl'ocessing of youl' lostlng. pleaae use youI' Submltlcl' IIlouu(ei./ althe top IGIl oryow' rtlPOli un IIOY ,
future submIssions, and please remain consistent wllh your submission Infol'l11ollon (I.e, addre.~s, phone, fax, cmBI!, ele,),
Page 1 of 1

Date Generated;

71912012

.7/31/2012 9113 AM

Page

3 of 4

...... . "."."%.,,. . ".~." NlltJonal VeterInary Senllces Laboratories

\(l:\':tll)lIry Sttlvi~

/"

't'

.....

~-~
_,..... ,,;,,

FINAL REPORT

PO 8o~ B44
Arne" Iowa 60010
Phone: 615-337-7614

Fait: 616-337.7938
FeDERAL RELAY SERVICE (VoJoe/TTYJASCIiISpanlah) 1-800.677.8339
The USDA IUh aqua( opponunHy provider andt)mp/oyer.

Labo rstory Toet Report

...... This Is II oonfldentlal repotf Intended [or o/frttal usa only. _ ...

Owner

12-021322

AC088elon Number.

George Carden Circus

.SpringReld, MO

19812039342

NFC Control Number:

Anlrnalloe.allon
Greene county MO

Oat. Colleoted:
Date Completed;
Col/ectad By:
Purpolle:

Submltt.r 26999

CJe/O 1120 12 .
07131/2012
Dr. Michael D. Stafford

Date Received:

Felr Grove Vet Service

PO Bow 9
Fair Grove, MO 85848
FAX#; 4177~7603
Phone #: 4177t191B03

T810-4

Referral/Retain Tag Numbtr:

This la 8

b/lfablll O.lIe,

NOTE: Condition oftl\e s.mp/e(8) was adeC/lfIlte unital! otherwise noted.

AnlmallD: Betty Catile #: TB1200108 8amplo; 5/23/2012 Spoclman Type: Trunk Washings
Specle8: Elephant
Mycobacterial Culture Result

No Isolallon Made

AnlmallD: Betty Case II: TB1200109 Sample; 5124/2012 Speclmliln Typo: Trunk Washings
Specla.: Elephant

Mycobacterial Culture Result

No Isolation Made

nlmallD: Betty Csse #: T81200110 Sample: 5/25/2012 Specimen Typa: Trunk Washings
Speclea: Elephant

Mycobacterial Culture Result

,haullS authorized

by:

No Isolation Made

0,. Buslee Robbe-Austerman, Section Heed, My(;obBct1!rie and Bruoella Sectlon

NVSL MB GaneraJ Phone: 515..3~773SB

~19 Uj tlQLR~

(TIl" new ~eD1lon "'" bl Up:!Olo!d p&rtodlOlify with l/pt for 9ubmftl&re.)
To oxp~/te the prOl"leMing DC yO\lr l681ing. pJeaso UJ& your

Submltl&r 1Ilocatod at

th6

top left of your rllpOl'1

Oil

any

fu~ 8ubmlsslollJ. IIlId plGUQ romaln coo.tlalonl with your subm/41iDD Informatioa (i,e. Iddr()4B. pIlona, fax. Dmall, etc.).

Page I 011

Date (3anarated:

7/31/2012

FINAL REPORT

Natlonol Voterlnary Servioes Laboratories

PO Box e~<\
Ames. Iowa 50010

Phone: 1516-337 76 t 4 Fax: 616-337 7938

FEDERAL RELAY SERVICE (VolcoITTY/ASCIlISpanlsh) 1-800e77-83'3g
The USDA Ie all oQllal opporlunlty prOYider and employer,
Laboratory Test Report
Thlels a connden~al (Spor! Inlended (or olflcialuse only, ......
OwnlH

A~cesllon Number:

12-033097

George Carden Circus

Springneld, MO
Animal Location
Greene Counly MO
Submitter' 26999

Fair Grove Val Service

NFC Control Number:

Oate Colleoted:
Date Received:
Dat& Completed:
Collected By:
Purpou:

19812052182
08/28/2012
10124/2012
Dr. Michael D. Slof(ord
TB104

PO 60x 9

Fair Grove. MO 65646

FAX #: 417759-7803
Phona #: 417-7597803

Reforrul/Relsln Tag Number:

Thle 19 a billable

0&1$.

NOTE: Condition of the Slimple(e) was adequate lInlees otherwlee not9d.

AnlmallD: 80 Case #: TB12-07936 Sample: 8/2212012 Spaclm9n Type: Trunk Washings Species:
Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

TB1207937 Sample: 812312012 Specimen Type: Trunk Washings Speclas:

Mycobacterial Culture Result

No Isolation Made

Animal ID: 80 Case II: T812-07938 Sample: 8/2412012 Specimen Type: Trunk Washings Speoles:
Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnlmallD: Betty Case #: TB12-07939 Sample: 8/22/2012 Specimen Type: Trunk Washings
Sp6clea: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnlmallD: Betty Cas9 #: TB12-07940 Sample: 8/2312012 Specimen Type: Trunk Washings

Species: Ell'>phant, Asian

Mycobacterial Culture Result

No Isolation Made

Page 1 of 2

Dele Generated:

10124/2012

Accession:

12033097

Owner:

George Cardan Circus

Relerrill

Number:

Animal 10: Betty Csse #: rB12-07941 Sample: 8/24/2012 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

Animal 10: Bimbo Cue #: TB12-07942 Sample: 8/22/2012 Speolmen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

Animal fD: Bimbo Case #: TB1207943 Sample: 8/23/2012 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

Animal 10: Bimbo Case f#: T912-07944 Sample: 8/24/2012 Specimen Type: Trunk Washings
Species: Elephant, Asian

Mycobac:terial Culture Result

Rellulbl authorl,.ed by:

No Isolallol1 Made

Dr. SUakie Robbe-Austermen. Section Hood, Mycobacterls end Brucella Sectlon

NVSl MB General Phone: 615337 7368

HeiR Ua Help You

(This new ,_oIlen wlU bo updottd p.nod!cally wi(h Up' (0'

.ubmlll.~ . )

To expedite the procc~lng of you I' testing, plc/lSe use your Suoml(ler ~ located at lhe top left of your repon on any
fuMe !ubmlsslons, Bnd pleDSC romain consls(onl with your submission informlllion (I.e. 6dclr~s, phol\e, fIIx, emoll, etc.).
Page 2 of 2

Date Generated:

10J2412012

\Ij)
\'.' !<.I IllMr
,

National Velorll\(lry Sc/vh;os

Sen' i, I', PO Box 844

Labot'~tor1cs

Ame3, 101"1'1 5UU10

,( . Ph",IO ' 51G~377G14
Fax : 51S-~3-1.79JS
'!': ':'1~1: :r:r:J7,Vl!~ Ft: I)EI~. Rf:I..A Y SGnvlc~ (V'>i,;eI1lYIi\SCII/$pR"(~IIJ '000 A77-03:iB
Tile IJSDA Ii< /lll cqllnl (\I\pOI~tJnlly pmvld cr
tllllploy",

. '..'{ ',

""ri

lailorainry TBSI' Report

" .~.,

Ownll'l

Thts Is a wn'idalllls( repolllnl(3nded for OmCllll use only . ,. ,

Accoss/on NUIYlller.

12060113

Gl)orge Car<J~1l
Sprlngneld, MO

Anlmol Loootion
Greene COl.lllly MO

Sullm/ttG r 26999

Fair Grove Vel Service

PO Box Q
Fair Grov6, MO 55&46

NFC Control NUlllb'H:

Dato ColioctotL
Date RGcelvpd:
Date Complot~d:
Colfoctod By:
Purpose:

19813013906

1212812012
03/05/2013
D,. Michael D. Sta/(ord

TB-104

R9ferra/IRstaln Tag Number:

FAX /I.: 417-71>9-7803

Phone #.417759-760:1
TIlls Ie a bllls.bl<l

CQ9\),

NOTE: CondltlOl1 of tho eample(s) WlH. acloquato unless otherwise noted.

Animal 10 : Betty Ca&e #: TB1210503 Sample: 12/18/2012 Speclmon Typo: Trunk washings
Spao/es: Elephant, As/an
Mycobacterial Culture Result

No Isolation Made

Anlma/lD: Betty Case II: TB12-1050<1 Sample: 12/19/2012 Specimen Type: Trunk Washings
SpecIes: Elepi1an~ Asian
Mycobacterial Culture Resliit

No Isola(lon Made

Animal 10: Betty Csse #: T81210505 Sample: 12120/2012 Specimen Type: Trunk Washings
Speoles: Elephant, Asian
MycobacterIal Culture Result

No Isolation Made

fAnlmallO: 80 Case #: T812-10506 Sample: 12/18/2012 Specimen TYPQ: Trunk Wa!?hings Speclss

~ Elephant, Asian

._ _ ._________

Mycobacterial Culture Resull

No Isolallon Made

G'm~i-jD:-BO CElB;;;;:
: Elephant, Asian

TB12-10507 Sample: 12/1912012 Specimen Type: Trunk Washings Spades

__ _ _ __

Mycobacterial Culture Result

No Isolation Made

Pag81 of 4

DctG GenerE1ted:

3/5/2013

Accession:

12.050113

Owner:

Gsorge Cardon

Relerl'lll Number:

Animal 10: Bo Case #: TB12-10508 Sample! 12/20/2012 Specimen Type: Trunk Washings Speoles

: Elephant, Asian

Mycobacterial Culture Result

No 19o1ation Made

AnlmallD: Cindy Case #: TB12-10609 Sample: 12/18/2012 Specimen Typo: Trunk Washings
Speoles: Elephant. Asian
Mycobacterial Culture Result

No Isolation Made

AnlmallD: Cindy CeIIe~: TB12-10510 Sample: 12119/2012 Speclmlln Type: Trunk Washings
Species: Elephant. Asian
Mycobacterial Culture Result

No Isolation Made

AhlrnallD: Cindy Case #: TB12-105,1 Semple: 12120/2012 Specimen Type: Trunk Washlnge
Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

Anlma/ID: Janice Caso #: TB12-10S12 Sample: 12/18/2012 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result
Animal 10: Janice Case 'II:
Spe~le8;

No Isolallon Made

TB12~10513 Sample:

12/19/2012 Specimen Type: Trunk Washings

Elephant, Asian

Mycobacterial Culture R&sult

No Isolation Made

Animal 10: Janice Cseo #: TB12-10514 Sample: 12/20/2012 Speolman Type: Trunk Washings
Speoles: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnlmallD: Jenny Ca~e #: TB12-10515 Sample: 12118/2012 Speolmen Type: Trunk Washings
Species; Elephant, Asian

Mycobacterial Culture Result

No Isolation Made

Animal 10: Jenny eaSD #: TB12-10518 Sample: 12/19/2012 SpeCimen Type: Trunk Washings

SpecIes:

Elaphan~

As/an

Mycobacterial Culture Result

No Isolation Made

AnlmallD: Jenny Case tI: TB12-10517 Sample: 12/20/2012 Specimen Type: Trunk Washings
SpeclN: Elephant, Asian
Mycobacterial Culture Result

No IsolaOon Made

Page 2 cf4

Dale Generated:

3/612013

AW~S8lon :

i2'()50i13

Owner.

George carden

Referral Number.

AnlmallD: Judy Case tI: TB1210518 Sample: 1211812012 Specimen T~pe: Trunk Washings
Speole9: Erephant, AsIan

Mycobacterial Culture Result

AntmallD ~ Judy elise

Conlaminated

#: T812-10519 Sample: 12119/2012 Specimen Type: Trunk Washings

pecles; Elephant, Asian

Mycobacterial Culture Result

Acid

Isolate

Mycobacterium trlvlale

fast bacteria recovered

The Isola Ie was sequenced allha1E1S rRNA and the rpoB regIons 01 the genome. Results were blasted agalns\
RIDOM and GenBank. The Isolate ma!ohed Mycobacterium !r!viale al 99% sequence Blmllarlty based 011 rpoB
sequenolng. Mycobacterium trlvlale belongs to Ihe M, terrae compl6X.
nlmal ID: JUdy Case #: T812-10520 Sample: 1212012012 Specfmen Type: Trunk Washings
Spocles: Elephant, Asian

Mycobacterial Culture Resull

No Isolatlon Made

Animal ID: Liz Case #: T81210521 Sample: 12/18/2012 Specimen Type: Trunk Washings Speoles
: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnfmllllD : Uz Case #: TB12-10622 Sample: 12/19/2012 Specimen Type: TrunkWsshlnge Species

: Elephant, Asian

Mycobacterial Culture Result

No Iso lation Made

AnlmallD: Llz Cau8 #: TB1210623 Sample: 12120/2012 SpeCimen Type: Trunk Washings Species
: Elephant, Asian

Mycobacterial Culture Result

No Isolation Made

Animal ID; Tory Case #: T81210524 Sample: 12/1812012 Specimen Type: Trunk Washings
SpecIes: Eleph8n~ Asian

Mycobacterial CultUre Result

No Isolation Made

AnImal ID: Tory Case #: T81210525 Sample: 1211912012 SpecImen Type: Trunk Washings
Specl86: Elephant, Asian
Mycobacterial Culture Result
AnlmallP: Tory Case

No laolallon Made

#: T812-10528 Sample: 12120/2012 Speolmen Type: Trunk Washings

Speclu: Elephant, Asian

Mycobacterial Culture Result

No Isolation Made

Page 3 ot 4

Date Generated:

31512013

AccessIon :

12-050113

Ownec

George Carden

Ratelral Number:

AnlmallD: Vicky Case #: TB12-10527 Sample: 12/1812012 Specimen Type: Trunk Washings
Species ; Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnlmallD: Vicky Case #: T612-10528 Sample: 12119/2012 Specimen Type: Trunk Washings
Species! Elephant, Asian
Mycobaclarlal Culture Result

No Iso\atlon Made

Anima! lD : Vicky Case #: T812-10529 Sample: 12120/2012 Specimen Type: Trunk WashIngs
Species : Elephant, Asian

Mycobacterial Culture Result

Rssultll 8uttlorlzed by:

No Isolation Made

Dr. Suelee RobbeAusl9lmsn, Section Head, Mycobacteria and 8rucella Secllon

NVSL MB General Phone: 515337-7388

tllll!) U, Help You

(1111a new 'OOUDIl will b& updol4c1 p,,(lodlcally with Dp' lo!

.ubmi~e~)

To expedite the proCC3.'!lng of your sample(s), please Include Ihe word "sa m p!es" In tile addre~ on lite slllp

/0

labot.

This VllH !l3slsl dock receiving pel'sonnel in routing your 9~mple{g) ,

Page" of4

Dale Generaled:

3/612013

_V;

FINAL REPORT

National Veterinary Services Laboratories

V\'h~ )'il wry S<:l'vi('l'~ PO Box 844

.
....
Ames,lowe 60010

~: ,.;. ';- -~_:;V\~;, ' ;ir; I F'hona. 616-337-7514 Fox: 515337-7938

~!:,f.!1 ~l\"..:, ..'q,[,~"f. FEDERAL RELAY SERVICE (VoiceITfY/ASCIIISpenlsh) 1-!JOO-Bn-B339
The USDA Is 8n equal opportunity provIder end employer.
Laboratory T&et RlIport
...... Th lsla a conndenllal report Intended for official use ot'\ly .........

Owner

Accession Number:

George Carden Circus

Springneld, MO
Animal Location
Greene Counly MO

NFC Control Number:

Oats Collected:
Data Received:
Date Completed:

Submitter' 28999

Fair Grove Vel Service

Collected By:

Purpose:

13023392
19813040149

0710212013
09103/2013
01'. Michael D, Stafford
TB-10--4

PO Box 9

Fair Grove, MO 65646

FAX II: 417-7597803
Phone #: 417-759-7803

Referral/Relaln Tag Number:

This Is 9 bllhlble clIse.

NOTE : Condition of the asmple(s) WIle adequate unless othorwlu noted.

Animal 10: 80 Case #: TB13-05046 Sample: 6/25/2013 Specimen Type: Trunk Washings Specles~
Elephant, Asian
Mycobacterial Culture Result

Acid fast bacteria recovered

Isolate

Mycobacterium specles- not M. tuberculosis complex

AnlmallD: Bo Case #: T813-05047 Sample: 6/26/2013 Specimen Type : Trunk Washings Species:
Elephant. Asian
Mycobacterial Culture Result

No Isolation Made

An Imal 10: Bo Case #: TB13-05048 Sam pie: 6/27/2013 Specimen Type: Trunk Washings Species:
Elephant, Asian

Mycobacterial Culture Result

No Isolation Made

AnlmallD: Betty Cllse #: TB13-05049 Sample: 6/25/2013 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

Pags 1 of 2

Date Generated :

91312013

Accession:

13-023392

Owner:

George Carden Circus

Referral Number:

AnlmallD: Betty Case #: T813-05050 Sample: 6/26/2013 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

Animal 10: Betty Csse #: T813-05051 Sample: 6/27/2013 Specimen Type: Trunk Washings
Species; Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnimallD: Vicky Case #: T813-05052 Sample: 6/26/2013 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnlmallD: Vicky Case #: TB13-05053 Sample: 6/26/2013 Specimen Type: Trunk Washings
Species: Elephant, Asian
'
Mycobacterial Culture Result

No Isolation Made

Animal 10 : Vicky Case #: T813-05054 Sample: 6/27/2013 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

Animal 10: Janice Case #: T813-05055 Sample: 6/25/2013 Specimen Type: Trunk Washings
Species: Elephant, Asian

Mycobacterial Culture Result

No Isolation Made

Anima 110: Janice Case #: T913-05058 Sample: 6/26/2013 Specimen Type : Trunk Washings
Species : Elephant, Asian

Mycobacterial Culture Result

No Isolation Made

AnlmallD: Janice Case #: T813-05057 Sample: 6/27/2013 Specimen Type: Trunk Washings

Species: Elephant, Asian

Mycobacterial Culture Result

Results authorized by :

No Isolation Made

Dr, Suelsa RobbeAus(efman, Section Head, Mycobacteria end Brucella

NVSL MB General Phone : 515-337-7388

Sec~on

Halp Us Ha!pJ:.QJl
(1013 new .. ellen will be updsted ps(IOdlc.lly wlth UP$lor aubm!t!$I'(._)

Pleas6 remember to submit payment or your User Fee account number at the time of Bubmlsslon. If you
would like our billing department to contact you for a credit card number, please provide a valid contact
number,

Page 2 of 2

Dala Generated :

9/312013

N;ll j(, III \ I

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. - nil ", j" ... 1~I. llli' )"I\II.~ I (. 'f,/H I jl1\t~ lldf;, I

()IMllf;'(

for u(ilcial

I):.;" lH Ily

13044382

A c,,<'>sslOn Num ~()i'

(.;e()i'q( ~ (;11((\<-1'1

~~I'(il)yll"JII'I ;

MU

191j14(J10~39

Nr-C C(')I1(rol NUlllber:

Date! cullacto([:
DatI;) ROCGlvod :

Animal Locatloll
Gre[:ll\c Coul)ty MO

12/1 B12013
02/1 '112014

Data Compleled:
Cofleotacl By:

Dr, Michael

Fall' Grove Vel Selvice

PO Box 9

Purpose:

TB1D-4

I'al/ GroVI7. MO B564U

rAX ii. tI'17 ,759.. 7803
['I lone 11 ' 417 750 700::1

R()f"'rr~'motilln Ta~ Nlimber~

Submlttfll' 26999

"J.'l'"'n.<f

Thle Is

D, StaNord

hlllllbftl case.

NOTE : Condition of the 9!1mple(s) was adeqllllte- unless otherwl80 noleo.

Anlmal 10: Bo,

0365640f56 Case #: TB13 08H16 Sample: 12/11/1 J Specimen Type: Trunk

Washings Species:
E:lephal1'l, Asian
_ _ '_ ,_ _ , _ ,.. A". _
_

... '..- " __ ' "'-'-1"_' .... , . - , -

, .~-

, .~

... ,_,, ____

MycofJactedAI. Culture R0SLlH

'.,

_~~

..... _. , ___ .

_, _ , _ _ _

.___ u_.' " ; "'_."'_' , __

~ _.

_ _ _ _ _ _.. _ _ _ _

.. _ .

~_,_._J

No Isol~lI!)n Made

._'- ... , , ...... ,.. . . .-... . ... ..Cas2#:

,. -.. ,. . . ,. TBI308'18J
,. ._....-.... ---.Sl1lllple
, .,." . .:--.'121!2/13
--".-"_. , ,,---Specimen
"-"--"" . .Typo:
._-... _. TrllnJ<
--.,,--. --,,_. .- -...-... . .-.. J'
Aninl('lf JIJ: :3<),
(;
SpQ0lcs; EIElphCln(, Asian
03(-')!iGJW~iO

Wa~hin9::>
___ ., __ .... '-

_ ~

_.~.

' .... .. .. _ ... _ . . ......... ... _ U _ A _ _ _ _ _ _ _ _ . _ _ ..

Mycoh~lcler'iElJ

..;._~._,

Culture Result

. __ . _ _ _ , _

...'-"' _ _ _ _ _ _ _ _ _ _ ., _ _ _ . _ . _ _ _ _ _ _ _ _ _ _ .' __ _

No Iso/Eltion Made

:J

fAf~~-;j' lD:' Bo~03-6584056C;~e #0I3'13,:Oa-j88-sClmplp,: 12l13/13 Specimen Type: Trunk

~ashings Speclell: Elephant, Asian _

__._-_._._--_.--' __

Mycobacterl'll Culturo ResllH

_._--_.__.-_.

______ .. ____ .. __ ~ _ _ _ - _ - _ - - No Isolation Made

.._---,---_ .....__ .

..- ..
..
AnJmalID: B'3(ly, 036570847 CaBfI #: TB13 ..08189 SamplG: 121'111'1:1 Specimen Type: Trunk

!IN'1shil1as
-, -- ~ ~

, - - -

Sr~H:los :

- - _._- , , _ ... -

- - -- -

I--

,. -- .~ - ~'~. -'-

-,

#". - " -,~ - - - - - - _ . _ - - , . _ - - - - - - ....... ------~--~-------- ...... --~- ~"' - .... -

No Isol<ltion Made
-_'__ .. - .... _."--- ,_._-_._---_ .. ....------,, .- ._.. _...
Af)rm~IID : 801ly, 038;-i19847 C,\!?6 #; TEl'l:.1-08HJO Sample: 121'12/13 Spe<:lmen Type: Trunk
lNasrlings SpecJl.s : Eleph"ml, Asian
-_._---...'---,,- .,-,
.... . ,-----, . . - "-, ... -_.- - .. ,.- --, .......

... - ..... ,- _. ... ..

~-

E=.II-lph<mlc I\siClIl

.... , ~- ~ . ,- - .- . -~~, - , ._" _ "

Mycobr:tctedal elliturf! Result

-'"' '

---_...

_....

_ ._-"

_._-

Mycobacterial Cultlu e Result

_-_

''''' ' ' '

-- . ~-.~-

-,

---

_._-_._--_

__ _._]

.._...

----....------,- . - . ---- .~ ---- --

No Isolation Made

O~te G Clrum.rtecl:

2}'1'11201A

..

..--.". -

Accession:

1J04131l2

Owner:

George Carden

Referral Number:

Animal 10: Betty, 036579847 Case #: TB13Q8191 Sample: 12/13/13 Spec Imen Type: Trunk
Washings Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnlmallD: Cindy, 036549535 Case #: T81308192 Sample: 12111/13 Specimen Type: Trunk
Washings Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnlmallD: Cindy, 038549535 Case #: TB1~08193 Sample: 12/12/13 Speelman Type: Trunk
Washings Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

Animal 10: Cindy, 036649535 Case #: TB13-0S194 Sample: 12/13/13 Specimen Type: Trunk
Washings Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnlmallD: Janice, 4999761240 Case fl.: TB13-08195 Sample: 12/1'1/13 Specimen Type: Trunk
Washings Species: Elephant, Asian

Mycobacterial Culture Result

No Isolation Made

AnImal ID: Janice, 4999761240 Case #: TB13-08196 Sample: 12/12/13 Specimen Type: Trunk
Washings Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

An/mal 10: Janice, 4999761240 Case "Ii: T613.08197 Sample: 12/13/13 SpecImen Type: Trunk
Washings SpecIes: Elephant. Asian
Mycobacterial CultUre Result

No Isolation Made

Animal [D: Jenny, 036323638 Csse #: TB13-08198 Sample: 12/11/13 Specimen Type: Trunk
Washings Species: Elephant, Asian
Mycobaoterlal Culture Result

No Isolation Made

Animal 10: Jenny, 036323638 Case #: T813-08199 Sample: 12/12/13 Specimen Type: Trunk
Washings Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

An/maIID: Jenny, 038323638 Csse #: TB13-08200 Sample: 12/13/13 Specimen Type: Trunk
Washings Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

Page 2 of 4

Dale Generated:

2111/2014

Accession:

13044382

Owner:

Referral Number:

George Carden

Anima I 10: LIz Case #: T81308201 Sample : 12111/13 Specimen Type: Trunk Washings Species:
Elephant. As ian
Mycobacterial Culture Result

No Isolation Made

Anima! 10: Liz Case #: TB13-08202 Sample: 12/12/13 Specimen Type: Trunk Washings Species:
Elephant, Asian
Mycobacterial CultUre Result

No Isolation Made

A nlm al 10; Liz Case #: T813-08203 Sample: 12/13/13 Specimen Type: Trunk Washings Species:
Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

Animal ID: Tory, 4A18064623 Ca88 #: TB13-Q8204 Sample: 12/11/13 Specimen Type: Trunk
Washings Species: Elephant, Asian
Mycobacterial Culture Re!;lull

No Isolation Made

Animal 10: Tory, 4A18064623 Case #: TB13-08205 Sample: 12/12/13 Specimen Type: Trunk
Washings Species: Elephanl, Asian
Mycobacterial Culture Result

No Isolation Made

Animal 10: Tory, 4A18064623 Case #: T8130S206 Sample: 12/13/13 Specimen Type: Trunk
Washings Species: Elephant, Asian
MycobacterIal Culture Resu lt

No Isolation Made

Animal 10: Vicky , 036560042 Case #; TB13-08207 Sample: 12/11/13 Specimen Type: Trunk
Washings Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnlmallD: Vicky, 038580042 Case #: TB1308208 Sample: 12112113 SpecImen Type: Hunk
WashIngs Species: Elephant, AsIan
Mycobacterial Culture Result

No Isolation Made

Animal 10: VIcky, 036560042 Case#: T813-08209 Sample: 12/13/13 Sp6clmen Type: Trunk
Washings SpecIes: Elephant, Asian
Mycobacterial Culture Result

No Isolallon Made

Page 3 o( 4

Dale Generated:

2/1112014

Accasslon:

13-044382

Reeults .uthorlz&(i by:

Owner:

George Carden

Referral Number.

Dr, Suelee Robba-Austerman, Section Head, Mycobacteria and Brucella Section

NVSl MB General Phone: fi 15-337 -7'988

H&tp Us Help You

(ThIs new .. c~o n will be updstad pBnodloally wllh ~PI for :rubml!tllfli.)

Please remember to BI.lbmlt payment or your User Fee account number at the lime of submission. If you
would like our blUing department to contact you ror a credit card number, please provide 8 valid contact
number.

Page 4 of"

DElte Generated:

211 112014

_. __,_.~ug

27

2014

HP LASERJET FAX

lO:OBAH

p, 1

FINAl. REPORT

NIt/on.' Veterinary Servlcee t.abo/lltarln

PO

B~

844

Ama., 10wD 50010

PhOn.; 516-337,1614

Filii: 513-3J7.T938
, FEDERAL RELAY SERVICE (VolaefTTY/A8CIIIBp.~I.n) ~ . 60o-&17.8339
The U80A ~ .lin equal Opportl.llllly pro~i<Ior 'lind amploy.r.

Laboratory T.at Rlpo"

OW"lr
Cerdsri Clroua
Springl1C1ld, MO

Ma.nlon Humber:
NFO Conlnll Number:
Dati Col18ct8d:

1ge14034311

Greene county MO

D"t, A'.'v.d:
o.t. Ollmpl.l.d:

08(2D1'2.014
0811912014

liIubmll~r

CoI .. ct.d 9y1

Dr. Michael D. Stafford

rB1{)-4

Anlm.' L.ocatlO"

14..()20098

26999

Felr GrO~a Val SeM08

PO B<lx 9
FaIr Grove, MO 6~13
PAXN: 417-7597803
Phom, #: 4177597803

Purpo.. ;

/t11'errIIlR,t.ln Tag Humber:

TIlII , billab" eUI.

TB14-02481 8amplo: 6118/2014 SpecImen Type: Tf\.Ink Washings apecl .. :

Mycobacterial Culture Result

No IsolallOI1 Made

TB14-02462 Sampl., 6/17/2014 Ipaolman T\'pe: Trunk Washings Bpeol.a:

Myc:obllcterlal Culture Rosult

No laolatlon Made

Ima' ID! 90 Cae. ,: TB14.()2463 Sample: 6/18/2014 Speo lme" Type: Trunk Wuhlnga Speclea:
Ele han'. Asian

Mycobacterial Cullure ResLllt

No Isolation Made

nlmallD: Be/tty CU. #: TB14-D2464 Sample: 6/16/2014 Sp.clmen Type: Trunk WQshlng8
SpecIes: Elephant. Asian

Mycobacterfal Cullure Result

No laolst!on Made

," nl rna I to: BlJtffy C . . . II: T814..Q2486 Samp'e: 6/17/2014 _peclme" Type: Trunk Washings

Speol.lI; Elephant. Aslin

Mycobao1enPI Oulture Re8ult

No laolatlon Made

Page 1 or 2

08/21/2014 WHO 10.13

Dst" 06neral.,o:

(JOB NO.

e.t1912l1t4

6517)

IalOOl

Au~

27 2014 IOIOBAN

HP LRSERJET

FA~

Referrul Numbor:

nlmallO: 8eltty Can fr T814-0246El Simpla: 6/16/2014 Speolmen Type: TrunK Washings
Elephant, Asian

epec~.:

Mycooaotel1al Culture Re8ult

No laolatlon Made

Anlmell to: Vicky Cue #: TB1402467 Sample: 6/16/2014 Specimen Type: Trvnk Washings
Specl : Elephant, Asian
Mycobacterial Culture Reaull

No Isolatlon Made

"'maIIO: Vloky en.~: TB1402468 8emple: 6/1712014 9peolm.n Type: Trunk Washings
Specl. . : Elephant, Asian
MycobactarJal Culture RElsuH

No Isclatlon Made

nfmall0: Vicky Cu. AI: TB14024e9 Semple: 6/18/2014 SpecImen Type: Trunk Weshlnge
Speol : ElephBnt, Asian
Mycobaoterial Culture Result

No laolstlon Made

0(, Sualea Robt:l~u.tarmlln, 8ecllon H&ad, Mywbeoleria And BItJODlIa Seollon

NVSL MB Genel'9l Phone: 151&337-73118

Htlp U. Help Vaw

crnl I'>CIIir Hotlon ~Ibe updated p91iodb111y wflh tIPoI for wbmlt""")
Quality lIamples yiBld thllllTlost aoourate I'e!lulle. PleaSE: call If you htlve qU9!1tfo ns .

page 2 0(2

08/27/2014

Det, Gilnenallld:

no

10113

8/19/2014

[JOB 110, 6517]

""002

USDA

FINAL REPORf

Natlonlll Veterlnllry SGrvlces Laboratories

PO 9~x 8~~

Ames, Iowa 60010

Phon.: 615337-7614

Fax : 616 3377IlJ6

FEDERAL RELAY SERVICE (VolceITTYIASCilISp81)Ish) 1-00()'877-83:l9

111e USDA Is an eq(IBI opporlunl\y provider ~"d amployer.

Laboratory Teet Report

Sen$lllva Bul UnolailSln~dlSBnsltlve SeClJrily InformallOf\ DlSiemlnale on e Nead-T1).1(001'1 Basis Only

Owner

AC08Sslon Number.

15000343

Goorge Carden
Springneld, MO

Animal Looatlon
Greene Counly MO

Submitter. 26999
Fair Grove Vel Service
PO Box 9
Fair Grovel, MO 65648

Dale Colleoted:
Date Roo elved:
Date Comploted:
Colleoted By:
Purpoae:

01/06120,5
03/02/2016
Or, Michael D. Siafford
T8-10-4

Referrai/RetBln Tag Number:

FAX'll: 4177!!9-7803
Phone II; 417 -769-7803
This I, not a billable case,

NOTE: ConditIon of Ihe samplll(~1 was odequote unle!ls otherwlee noted.

AnlmallD: Bo Case #: TB15-00032 Sample: 1/02/2015 Specimen Type: Trunk Washings Species:
Elephanl. Asian

Mycobacterial Culture Result

No Isolallon Made

Animal ID: 80 Case #: T815-00033 Sample: 1/03/2015 Spaclmen Type: Trunk Washings Species:
Elephant. Asian

Mycobacterial Culture Result

No Isolation Made

AnlmallD: Bo Case #: TB15-00034 Sample: 1/0512015 Specimen Type: Trunk Washings Species:

Elephsnl, Asian

Mycobacterial Culture Result

No Isolation Made

Animal ID: Betty elise #: T815-00035 Sam pie: 1/0212016 Specimen Type: Trunk Washings
Species: Elephant, Asian

MycobactarlaJ Culture ResuU

No Isolation Made

AnImal ID: Betty Case #: TB 15-00038 Sample: 1/03/2015 Specimen Type: Trunk Washings
Spocles: Elephant, Asian

Mycobacterial Culture Resull

No Isolation Mede

Page 1 of 4

Dale Generated:

31212015

Accession:

16.000343

Owner:

Goerge Carden

Referral Number:

Animal 10: Betty Case #: T815-00037 Sample: 110512015 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Resull

No Isolation Made

Animal 10: Janice Case #: T815-00038 Sample: 1102/2015 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnlmallD: Janice Case #: T81500039 Sample: 1/03/2015 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture ResuU

No Isolation Made

Animal ID: Janice Case #: TB15-00040 Sample: 1/0412015 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnlmallD: Vicky Case #: T81500041 Sample! 1/02/2015 Specimen Type: Trunk Washings
Species: Elephanl, Asian
Mycobacteria! Cutture Result

No Isolation Made

Animal 10: Vicky ClIse #: T815-00042 Sample: 1/0312015 Specimen Type: Trunk Washings
Species: Elephanl, Asian
Mycobacterial Culture Result

No Isolallon Made

AnlmallD: Vicky Case #: T81500043 Sample: 1/04{2015 Specimen Type: Trunk Washings
SpecIes: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnlmallD; Cindy Caso #: T81500044 Sam pie: 1/02/2015 SpecImen Type: Trunk Washings
SpecIes: Elephant. Asian
Mycobacterial Culture Resull

No Isolation Made

llimallD: Cindy Case #: TB15-00045 Sample: 1/03/2015 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial CultUre Result

No Isolation Made

AnlmallD: Cindy Case #: TB16-00048 Sample: 1/05/2015 Specimen Type: Trunk Washings
Species: Elephant. Asian
Mycobacterial Cullure Result

No Isolation Made

Page 2 of 4

Date Generated:

31212016

Accession:

16000343

Owner:

Goerge Carden

Referlai Number:

AnlmallD: Tory Case #: T815-00047 Sample: 1/02/2015 Specimen TyPB : Trunk Washings Species
: Elephant, Asian
Mycobacterial Cullure Result

No Isolation Made

AnlmallD: Tory Case fl.: TB15-00048 Sample: 1/03/2015 Specimen Type: Trunk Washings Species

: Elephant, Asian
Mycobacterial Culture Result

No Isolallon Made

Animal 10: Tory Case #: TB15-00049 Sample: 1/05/2015 Specimen Type: Trunk Washings Species
: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnlmallD: Jenny Case fl.: TB1S-00050 Sample: 1/0212015 Specimen Type: Trunk Wa9hlngs
Species: Elephant, Asian
Mycobacterial Culture Result

No Isolalion Made

Animal fO; Jenny CU4;7 #: TB15-00061 Sample: 1/03/2015 Specimen Type: Trunk Washings
Species: Elephant, Asian

Mycobacterial CuNure Result

No lsolallon Made

Animal 10: Jenny Case #: TB 15"00052 Samplo: 1/04/2015 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterlel Culture Resull

No IsolaHon Made

AnlmallD: Liz Csse #: TB15-00053 Sample; 1/0212015 Specimen Type: Trunk Washings Species:
Elephant, Asian

Mycobacterial Culture Result

No Isolation Melde

AnlmallD: liz Cane #: T815-00054 Sample: 1103/2015 Specimen Type: Trunk Washings Species:
Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnlmallD: Llz Case #: TB15-00055 Sample: 1/05/2015 Specimen TYPB: Trunk Washings SpecIB&:
Elephant, Asian
Mycobacterial Culture Result

No lsolatlon Made

These samples ara rBlIubmls81on8 from Acoesslonli 14041275.

Page30r4

Dale Gef)eral6d:

31212015

Access ion:

1B-000343

Res ulls authorlzed by:

Owner:

Goerge Carden

RelerrBI Nt/mbet:

Dr, Suelee Robbe-Aulliermen, Section Head, Myoobaclerla and Brucella Sec((on

NVSL MB Gene(al Phone: 615-337-7388

HeiR Us HeiR YOII
(Thll n&w $$Clion wfll bi! ~pdQled pe~odi ca!l'V wllh lI~r for ovbmi\lurJ ,)

Quality samples yield the most accurate results. Please ca ll If you have questions.

Page 4 ot 4

Dale Generated:

31212015

USDA

National Veterinary Services Laboratories

FINAL REPORT

POBox 844
Ames, loWl! 60010
Phone: 615-3377514 Fox: 615337793B
FEDERAL RELAY SERV1CE (VolcaffTY/ASCIIISparJlah) 1-800...977-8339
Tho USDA Is an equal opportunity provider and employer.

Laboratory Test Report

Ssnsilive Bul Unclassified/Sensitive Serority In(orm8~on Olll8emlnatll on a Need-TI>-Know Bssls Only

Owner

Animal Location

Submltt&r - 26999
Fair Grove Vet Service

PO Box 9
Fair Grove, MO 65848
FAX # : 417-759-7803
Phone #: 417-759-7803

Accession Number.
NFC Control Number:
Date Collected:
Date Received :
Date Completed:
Collacted By;
Purpose:

15-019796
19815034330

OB119/2015
08/1812015

Dr, Amanda GuthrIe

TB-1D-4

Refllrral/Retaln Tag Number:

Thhs Iii

billable cue.

NOTE: Condition of the sample(s) was adequate unless otherwise noted.

AnlmallD: Betty Case#: TB15-02603 Sample: 6/12/2015 Specimen Type: TrunkWashings

Species: Elephant, Asian
Mycobacterial Culture Result

No Iso!ahon Made

nlmallD: Betty Case #: TB15-02604 Sample: 6/1412015 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result

No Isola lion Made

An ImalIC: Belty Case #: T815-02605 Sample : 6/15/2015 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnlmallD : Cindy Case #: TB15-02606 Sample: 6/1212015 Specimen Type: Trunk Washings
Species: Elephan~ Asian
Mycobacterial Culture Result

No Isolation Made

AnimallD: Cindy Case #: T815-02607 Sample: 6/14/2015 Specimen Type: Trunk Washings
SpeCies: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

Page 1 of2

Date Generaled:

8118/20i5

Accession:

15-019796

Owner:

Referral Number:

AnlmallD: Cindy Case #: TB15-02608 Sample: 6/15/2015 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result
AnlmallD: Bo Case #:
Elephant, Asian

T815~02609

Mycobacterial Culture Result

No Isolation Made
Sample: 6/12/2015 Specimen Type: Trunk Washings Species:

No Isolation Made

AnlmallD: Bo Case #: TB15-02610 Sample: 6/14/2015 Specimen Type: Trunk Washings Species:
Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

Animal ID: Bo Case #: TB15-02611 Sample: 6/15/2015 Specimen Type: Trunk Washings Species:
Elephant, Asian
Mycobacterial Culture Result

Results authorized by:

Help Us

No Isolation Made

Dr. Suelee Robbe-Austerman, Section Head, Mycobacteria and Brucella Section

NVSL M8 General Phone: 515-337-7388

Help You

(This new section will be updated periodically with (Ips fOT subml~8(&.)

Quality samples yield the mosl accurate results. Please call If you nave questlons,

Page 2 of 2

Date Generated'

8/18/2015

From: Ausherman, Sarah (mallto :sarah.ausherman@dellascltyhall,com]

Sent: Thursday, July 30, 2015 2:44 PM
To: Ben Johnson

Cc: Ruth line Kelly

Subject: RE: UnlverSoul Circus (letter to City of Dallas RE Elephants reactive to TB) - 2015 Dallas Animal Permits

Good afternoon,
Thank you for sending the additional documentation regarding Betty and Bo. The DCHHS has reviewed and notified us
that their position has not changed and they will not be able to be exhibited at Unlversoulln Dallas , Communication Is
attached.

That being said we can continue to process your event permit without Betty and 80 or If you would like to use
alternatlve animals. Please advise as soon as possible so we can coordinate efforts . As a reminder, If you plan to use
additional elephants/animals at the event the following documents will be required :

'.

Copies of the elephants' medical records;

Photographs of the elephants;
I nformation regarding any Identifying markings of the elephants; and
The name of the company's on-site veterinarian

please contact me directly should you

have any questions. Thank youl

Sarah Horn Ausherman I Special Event Manager

City of DalJas Office of Special Events
Sarah .ausherm an@dallasdtyhalJ,com
0214.939.27591 c 214.601 .5685 I f 214.939.2709

From: Ben Johnson [mallto:bJohnson@unlversouldrcus,com]

Sent: Tuesday, July 28, 2015 2:01 PM
To: Ausherman, Sarah
Cc: Ruthlfoe Kelly

---------------

EXHIBIT 3

Subject: Unlver$oul Circus (Letter to City of Dallas RE Elephants reactive to TB) - 2015 Dallas Animal Permits
Importance: High
Ms, Ausherman :
Last month while the circus was in Norfolk, Virginia, UnlverSoul Circus provided trunk wash test results to the Norfolk
Deputy City Attorney responsible for animal welfare, to counter claims made In June that Bo and Betty have been
positive for TB. Also Included in the documentation was a letter from Dr, Stafford, the elepha nts' veterinarian, and a
detailed explanation of the types of TB tests and USDA guidelines for TB for elephants. All of the documentation and full
statement are attached to this e-mail. In particular, I want to share with you the following excerpts from the June 15,
2015 correspondence from our Norfolk counsel to the Norfolk Deputy City Attorney, copied below:
I am sending YOll this in response to your expression of concern abollt whether two of the elephants currently traveling
with the UnlverSoul Circus (now In Norfolk) have tuberculosis. The two elephants at Issue are Betty and Bo. They
belong to larry Carden, who Is the trainer traveling with them. Your concern Is premised on your having been sent DPP
testing results for Betty and Bo (and other elephants owned by Mr. Carden) from December of last year. Those test
results show samples taken on December 9,2014 using a DPP test resulting In a "Reactive Ab Positive" finding In a report
dated December 23,2014. Since we spoke about this Friday afternoon, I have spoken with Mr, Carden, Dr. Michael
Stafford (the primary veterinarian for Bo and Betty, and the person who submItted the DPP samples at Issue In
December), and several officials at UnlverSoul Circus about the elephants' health, the December TB screen, and the
safety of their traveling and being exhibited here In Norfolk.
To be clear, I am assured by Dr. Stafford that neither Betty nor Bo have TB. He could not have been any more emphatic
about that Moreover, Dr. Stafford provided me the attached statement, dated Friday, June 12, that attests to Betty and
Bo meeting or exceeding the relevant USDA guidelines for the control of TS in elephants. A copy of his statement Is
attached ,
I am also attaching the USDA's own testing results of the more accurate trunk wash tests for TB done on both elephants,
both before and after the December DPP screening at Issue. The first of those was based on samples taken on June 16,
17, and 18, 2014. The more recent was based on samples taken on January 2,3, and 5, 2015. The reported finding of
"No Isolation Made" means that no positive TB cultures could be Isolated from the samples tested. It Is the way that a
negative trunk wash TB test result is reported by the USDA.
The complete correspondence from our lawyer to the City of Norfolk Is attached . It comprehensively (overs the TB Issue
as it relates to Betty and Bo, and should be sufficient evidence to allow Bo and Betty to perform In Dallas.
You can reach me at 404-588-1235 ext 8730 or Ruthline Kelly at 404-588-1235 ext 8734 with any questions. Also, Dr.
Michael Stafford has made himself available to discuss any concerns or questions as well.

Ben Johnson
Soul Circus, 'nCo

NEW ADDRESS
230 Peachtree Street, NW

Suite 2000 - 20 th Floor

Atlanta, GA 30303
404-787 -1821 mobile
bjohnson@unlversoulclrcus,com
www.facebook.com/univ~rsou\Circusandyvww.twltter.com/u niversoulclrc

From: Ruthllne Kelly

Sent: Tuesday, July 28, 2015 10:25 AM
To: Ben Johnson
Subject: F\tV: UnlverSoul Circus (Letter to City of Dallas RE Elephants reactive to TB)
Importance: High

From: Aushe rman, Sa ra h [rna lito: sarah,a ushermcl n@daliascltyhall.com]

Sent: Monday, July

27, 2015 5:36 PM

To: Ruthline Kelly; Danny Rodriguez

Cc: Ivery, Rhonda
Subject: UnlverSoul Circus (Letter to City of Dallas RE Elephants reactive to TB)

Importance: High
Hello,

I am reaching out to let you know we have denied the permission to exhibit Betty and Bo, your two elephants at the
UniverSoul event; as well as any other animal that has tested r2actlve to tuberculosis . Please see the attached
communication from Dallas County Health and Human Services.
Please let me know as 500n as possible If you plan to use any additional elephants at the event. The following
documents will be required asap If are planning replacements:

Copies of the elephants' medical records;

Photographs of the elephants;

Information regarding any Identifying markings of the elephants; and

The name of the company's on-site veterinarian

Please contact me directly should you have any questions. Thank youl

Sarah Horn Ausherman I Special Even! Manager

City of Dallas Office of Special Events
Sarah .ausherman@daUasci!yhall.com
0214.939.27591 c 214.601.5685 I f 214.939,2709

Zachary Thompson, MA
Director

Christopher Perkins, DO, MPH

Medical DirectorlHealth Authority

luly 30, 2015

Joey Zapata
Assistant City Manager, City of Dallas
Via email: ioey.zapata@dallascityhaILcom
Ron King
Executive Director of Convention and Event Services. City of Dallas
Via email: ron.king@dattascityhal1.com
Kris Sweckard
Director of Code Compliance, City of Dallas
Via email: luis.sweckard@daiiascityhal1.com
Re: Soul Circus Tnc. elephants

Dear Mr. Zapata, Mr. King, and Mr. Sweckard,

Thank you for sending us the additional information from Ben Johnson with Soul Circus, Inc.
regarding the company's desire to exhibit two elephants that have tested reactive for tuberculosis
at an event in Dallas next month.
Dallas County Medical DirectorlHealth Authority Dr. Christophel' Perkins who, by agreement,
serves as the Health Authority for the City of Dallas, Dallas County DiJ-ector of Tuberculosis
Control DI'. Garry Woo, and r have reviewed the recently submitted infonnatlon and our position
stands. While we recognize th6t the individuals charged with caring for these elephants have
differing viewpoints, our Investigation shows that the threat of potential transmission of
tuberculosis ITom elephants that carry the disease to arena and circus workers, members of the
pllblic, and other animals remains a serious concern, We believe no elephant that has tested
reactive for tuberculosis should be exhibited.

Sincerely.

jf1:m7~~
Director, Dallas County Health and Human Services
C: Dr. Christopher Perkins, Dallas County Medical DirectorlHealth Authority

2377 North Stemmons Freeway, Suite 600, Dallas, Te)(as 75207-2710

Office 214.819.2100' Fa)( 21<1.819 .6022

__ __ ___ _, ______ , ____ . '

_____ __
~

..

, _

_______ "

______

__

__

__ A

From: Ruthllne Kelly

Sent: Tuesday, July 28, 2015 10:25 AM
To; Ben Johnson
Subject: FW: UnlverSoul Circus (Letter to City of Dallas RE Elephants reactIve to T8)
Importance: High

Fro m: Ausherma n, Sarah [mailto: sa ra h,aus herma Q@dallascltyhall,com]

Sent: Monday, July 27, 2015 5:36 PM
To: Ruthllne Kelly; Danny Rodriguez
.Cc: Ivery, Rhonda
Subject: UnlverSoui arcus (Letter to City of Dallas RE Elephants reactive to TB)
Importance: High
Hello,

[ am reaching out to let you know we have denied the permission to exhibit Betty and Bo, your two elephants at the
UnlverSoul event; as welJ as any other animal that has tested reactive to tuberculosis. Please see the attached
communication from Dallas County Health and Human Services.

please let me know as soon as possible If you plan to I,Jse any'addltlonal elephants at the event. The follOWing
documents wHI be required asap If are planning replacements:
1

EXHIBIT 4

Caples of the elephants' medlca~ records;

Photographs of the elephants;

In formation regarding any Identifying markings

The name of the company's on-site veterinarian

Please

of the elephants; and

contact me directly should 'IOU have any questions. Thank youl

Sarah Horn Ausherman I Spedal Event Manager

City of Dallas Office of Special Events
Sara h. ausherm an@dallascltYhali,com
0214 .939,27591 c 214,601 .5685 I r 214,939,2709

DCHHS

Po.~ilies) nea.lth!j lives.

Dallas County Health and Human Services

Safe

Zachary Thompson, MA
Director

Christopher Perkins, DO, MPH

Medical DirectorlHealth Authority

July 27,2015
Joey Zapata
Assistant City Manager, City of Dallas
Via email: joey.zapata@dallascityhall.com
Ron King
Exocutive Director of Convcmlion and Event Services, City of Dallas
Via email: ron .king@dallascityhalJ.com
Kris Sweckard
Director of Code Compliance, City ofDailas
Via emall; kris .sweckard@dllllilScilYhal1.com
Dear Mr. Zapata, Mr. King, and Mr. Sweckard,
Dallas County recently received infOlmation indicating tbat two elephants scheduled to appear with Soul
Clrcus, Inc. (also known as UnlverSoul Circus) at the Soulhw6Sl Center Mall in Dallas next month have tested
reactive for tubercUlosis. Dallas County Immediately reached out to the Toxas Oeparunenl of State Health
Services and the United States Department of Agliculture to discuss the potential risks associated witb
bringing these elephants mto (he area.
Based on our communications with these agencies, Dallas County Medical Dil'ectorlHeelth Authority Dr.
Christopher Perkins who, by agreement, serves as the Health Authority for the City of Dallas, Dallas County
Director of Tuberculosis Control Dr. Garry Woo, Imd I respectfully request and recommend that tbe City of
Dallas deny Soul Circus, Inc. permission to exhibit these elephants, Betty and Bo, liS well as any other elephant
that has rested reactive fur tuberculosis. Given the potential transmission of tuberculosis from elephants that
CIIl'ly the disease to arena and circus workers, members of the public, and other animals, we believe that these
elephants should not be exhibited.
Should Soul CIrcus, Inc. seek to bring other elephants to the event scheduled in August, please request the
following infolmatlon and forward it to my office:
copies of the elephants' medical records;
photographs of the elephants;
infDlln8tion regarding any Identffying marldngs of the elephants; and
the name of the cDmpany's onsite veterinillian.
Please contact me If you have any questions.
Sincerely,

g~m~o~

;1~or, Dallas County Health and Human Services

c:

Dr. Christopher Perkins, Dallas County Medical Dil'ectorlHealLb Authority

2377 North Stemmons Freeway, Suite 600, Dallas, Texas 75207-2710
Office 214.819.2100' Fal< 214.819 .6022

From: "Ben Johnson" <biohnson@universoulclrcus.com>

To: "sarah.a usherman@dallascityhall.eom" <sarah.a usherman@dallascltyhall .com>
Cc; "Ruthllne Kelly" <rkelly@universouldrcus.com>

Subject: UnlverSoul Circus (Letter to City of Dallas RE Elephants reactive to TBl - 201S Dallas Animal
Permits
Ms . Ausherman:
Last month while the circus was In Norfolk, Virginia, Un IverSoul CIrcus provided trunk wash test results
to the Norfolk Deputy City Attorney responsible for animal welfare, to counter claims made In June that
Bo and Betty have been positive for TB. Also Included In the documentation was a letter from Dr.
Stafford, the elephants' veterinarian, and a detailed explanation of the types ofTS tests and USDA
guidelines for TB for elephants. All of the documentation and full statement are attached to this email. In particular, I want to share with you the folloWing excerpts from the June 15, 2015
correspondence from our Norfolk counsel to the Norfolk Deputy City Attorney, copied below:

I am sending you this In response to your expression of concern about whether two of the elephants
eu rrently travel ing with the UnlverSoul Circus (now In Norfolk) have tuberculosis. The two elephants at
Issue are Betty and Bo. They belong to Larry Carden, who Is the trainer traveling with them . Your
concern Is premised on your having been sent DPP testing results for Betty and Bo (and other elephants
owned by Mr. Carden) from December of last year. Those test results show samples taken on December
9, 2014 using a opP test resulting In a "Reactive Ab Positive" findl ng In a report dated December 23,
2014. Since we spoke about this Friday afternoon, I have spoken with Mr. Carden, Dr. Michael Stafford

EXHIBITS

(the primary veteri narlan for Bo and Betty, and the person who submitted the DPP samples at issue In
December), and several officials at UnlverSoul Circus about the elephants' healthJ the December TB
screen, and the safety of their traveling and being exhibited here In Norfolk.

To be clear, I am assured by Dr. Stafford that neither Betty nor Bo have TB. He could not have been any
more emphatic about that. Moreover, Dr, Stafford provided me the attached statement, dated FrldaYJ
June 12J that attests to Betty and Bo meeting or exceeding the relevant USDA guidelines fDr the control
of T8 In elephants. A copy of his statement Is attached.

am also attaching the USDA's own testing results of the more accurate trunk wash tests for TB done on
both elephants, both before a nd after the December DPP screening at issue. The first of those was
based on samples taken on June 16, 17, and 18,2014. The more recent was based on samples taken on
January 2, 3, and 5, 2015 , The reported finding of "No Isolation Made" means that no positive TB
cultures could be isolated from the samples tested. It Is the way that a negative trunk wash TB test
I

result Is reported by the USDA.

of Norfolk Is attached. It comprehensively

Bo, and should be sufficient evidence to allow Bo a nd Betty

The complete correspondence from our lawyer to

covers the TB issue as It relates to Betty and
to perform In Dallas.

the

City

You can reach me at 404-588-1235 ext 8730 or Ruthline I(el'y at 404-588-1235 ext 8734 with any
questions. Also, Dr. Michael Stafford has made himself available to discuss any concerns or questions as
well.

Ben Johnson
Soul Circus, Inc.

NEW ADDRESS
230 Peachtree Street, NW
Suite 2000 - 20 th Floor
Atlanta, GA 30303
404- 787 -1821 mobile
bj ohnson@universoulclrcus.com

wvvw. facebook.com/universoulclrcus and www.twitter.com/unlversoulclrc

From: Ruthljne Kelly

Sent: Tuesday, July 28, 2015 10:25 AM
To: Ben Johnson
Subject: F'II/; UniverSoul CIrcus (Letter to City of Dallas RE Elephants reactive to TB)
Importance: High

Fro m:

Ausherma n,

Sa rah [rna lito: sa ra h ,ausberma n@daliascltyhaI1,coml

Sent: Monday, July 27, 2015 5~36 PM

To: RuthlJne Kelly; Danny Rodriguez
Cc~

Ivery, Rhonda

Subject: UnlverSoul Circus (Letter to City of Dallas RE Elephants reactive to T6)

Importance:

High

Hello.
[ am reaching out to let you know we have denied the permission to exhibit Betty and Bo, your two
elephants at the UniverSoul event; as well as any other animal that has tested reactive to
tuberculosis. please see the attached communication from Dallas County Health and HUman Services.
Please let me know as soon as possible if you plan to use any additional elepha nts at the event. The
following documents will be required asap If are planning replacements:

Caples of the elephants' medical records;

Photographs of the elephants;
Information regarding any identifying markings of the elepha nts; and
The name of the company's on-site veterinarian

Please contClct me directly should you have any questions. Thank youl

Sarah Hom Ausherman I Special Event Manager

Cay of Dallas Office of Special Events
Sarah.ausherman@dallascltyhall.com
0214.939.27591 c 214.601.5685 I t 214.939.2709

DCHHS

Safe faW\i{;es) healthY (;ves.

Dallas County Health and Human Services

Zachary 'Thompson, MA
Direotor

Christopher Perkins, DO, MPH

Medical DixectorlHealth Authority

July 27, 2015

Joey Zapam
Assistant City Manager, City of Dallas
Via email: !oey.ZlIpBta@dalJascitvhall.com
Ron King
Executive Director of Convention And Event Services, City of Dallas
Via email: ron.king@dllilascityhall.com
Kris Sweckard
Director of Code Compliance, City ofDaUas
Via email: kris.sweckard@dallascityhall.com
Dear Mr. Zapata, Mr. King, and Ml. Sweckard,
Dallas County recently received infonnfltion indicating that two elephants scheduled to appeal" with Soul
Circus, mc, (also known as Un iV(lrSoul Circus) at the Southwest Center Mall in Dallas next month have tested
reactive for tuberculosis, Dallas County Immediately reached out to the Texas Department of State Health
Services and lhe United Stales DepBrtmMt of Agliculture to discuss the potential risks associated with
bringing these elephants Imo the !II'ea,
Based on our commun ications with these agencies, Dallas County Medical DircctorlHealth Authority Dr,
Chdstopher Perkins who, by agreement, serveg B.9 the Health Authority for the City of Dallas, Dallas County
Director of Tuherculosis Control Or. Garry Woo, and r respectfully request and recommend tbat the City of
Dallas deny Soul Circus, Inc, permission to exhibit these elephants, Betty and Bo, as well as flIIy other elephant
that has tested reactive for tuberculosis. Given the potential transmission of tuberculosis from elephants that
carry the disease (0 arena and circus workers, members of the public, and other aruma Is, we believe that tbege
elephants should not be exhibited.
Should Soul Circus, rnc, seek to bdng other elephants 10 the event scheduled in August, please request the
following information and forward it to my office :
copies of the elephants' medical records;
photogrnphs of the elephants;
infonnal1on regarding any Identifying markings of the elephants; and
the name offhe company's onslte veterinaJilm.
Please contact me if you have any questions.
Sincerely,

g,~m~C~<U

;1~or, Dallas County Health and Human Sel'vices

C: Dr. Christopher Perkins, Dallas COUDty MedIcal DirectorlHeahh Authority
2377 North Stem mons freeway, Suite 600, Dallas, Texas 75207-2710
Office 214,819 ,2100' Fal< 214.819.6022

From: Abel, ChrlslDpher A.

Sent; Monday, June 15, 20159:58 AM

To: cvothla.h!lll@oorfolk.goV

Subject: UnlverSoul arcus EIBphaots - December 2014 TB Screen

Importance: High
Cynthia - I am sending you this In response to your expression of concern about whether two of the elephants currently
traveling with the Ul)lverSoul Circus (now In Norfolk) have tuberculosis. The two elephants at Issue are Betty and
Bo. They belong to Larry Carden, who Is the trainer traveling with them. Your concern Is premised on your having been
sent DPP testing results for Betty and 80 (and other elephan ts owned by Mr. Carden) from December of last year. Those
test results show samples taken on December 9, 2014 using a oPP test resulting In a "Reactive Ab Positive" finding In a
report dated December 23,2014. Since we,spoke about this Friday afternoon, I have spoken with Mr. Carden, Dr.
Michael Stafford (the prima ry Veterinarian for Bo and Betty, and the person who submitted the DPP samples at Issue In
December), and several officials at UnlverSoul Circus about the elephants' health, the December TB screen, and the
safety of their traveling and being 8)(hlblted here In Norfolk.
To be clear, I am assured by Dr. Stafford that neither Betty nor Bo have TB. He could not have been any more emphatic
about that. Moreover, Dr. Stafford provided me the attached 'statement, dated Friday, June 12, that attests to Betty and
Bomeetlng or exceeding the relevant USDA guidelines for the control ofTB In elephants. A copy of his statement Is
attached.
I am also attaching the USDA's own testing results of the more accurate trunk wash tests for TB done on both elephants,
both before and after the December DPP screening at Issue. The first of those was based on samples tal<en on June 16,
17, and 18, 2014. The more recent was based on samples taken on January 2,3, and 5, 2015. The reported flndlng of
"No Isolation Made" means that no positive T8 cultures could be Isolated from the 'samples tested. It Is the way that a
negative trunk wash T6 test result Is reported by the USDA.
To put the whole elephantTB testing scheme Into perspective, there are three different types oftests for elephants In
order to determine whether an elephant has TB, First Is the TS STAT PAK, which Is a blood test that was used to detect
whether any TB antibodies exist. That test Is no longer avalla ble. The second Is the MAPIA DPP test. It Is a' quick blood
screen designed to detect TB antibodies. The report you showed me FrIday reflected a MAPIA DPP test that came back
posltrve for TB antibodies. This doe,S not mean that the elephant was positive for TB. The only way to determine
whether elephants truly have TB Is to take a culture and let It grow in the lab. This type of test-the third kind-Is called
a trunk wash test and Is required to be performed annually. As the name of the test Implies the elephant'S trunk Is
washed out by means of a saline solution being Introduced Into It and then draining the trunk Into a sample collection
container. The Intent Is to collect with the expelled saline solution any sputum or bqdy fluids that would be In the trunk
as WEll. The trunk wash protocol requires that a separate test be done 6n each of three successive daysto get a good
series of representative samples, Once collected, the sa mples are then cultured In the lab, to see whether any TB can be
Isolated from them. As you wrll see from the USDA trunk wash test results attached, Betty and Bo consistently came up
with no TB Isolation from their trunk washes six months before and two weeks after the December DPP test result VOll
were proVided. The trunk wash tests are considered good for a 'year, so the January 2015 test results attached are
current (and will be throughout Betty's and Bo's visit to Norfolk, and well beyond).
The USDA Issued guidelines In 2008 for controllIng TB In elephants , It then updated those with draftguld~lInes In
2010, Although the draft 2010 guidelines are the most recent, they have not been adopted, as I am Informed they are

being held up in litigation. To further c:ompllca te things, the 2008 gUidelines call for the use of the STAT PAK, but-as
noted above-It Is no longer available for use. So the current state of affairs Is that the 2008 guidelines are the effective
ones, but they have to be appl\ed without their STAT PAK requirement. The draft 2010 gUidelines call for use of the opp
test, but the DPP test has not yet been approved by the USDA. Mr. Carden stili uses It anyway, because some states
require It. The USDA gUidelines call for an annual trunk wash test, but Mr. Carden has that test done every six months as
a precaution that goes over and above what the law requires. I am told that the next trunk wash for the elephants with
the UnlverSoul Circus now In Norfolk is due to be conducted this weel<.
Under the USDA guidelines} an elephant should not travel whlle.l.t has TB or If It becomes "untested". An elephant
becomes I/untested" If It ~oes for more than 12 months without a negative trunk wash test being performed. Hence,
given all of that, there Is no reason why Betty and Bo cannot travel toes'e days.
Finally, you said you were concerned about the potential for an elephant with TB transmitting the disease to humans.
a m Informed by Dr. Stafford that no one really knows whether humans can contract TB from an ele'phant. To date, there
has been one suspicious case In the U.S., but no one has yet been able to prove that that person contracted T6 from an
elephant or, fra nkly, whether anyone could. Evidently, It Is much more likely that the transmission could go the other
way: that humans may be able to transmit T6 to elephants.
As I had indicated to you Friday, my understanding Is that this Is not the first time that PeTA has sought to use the TB
scare as a way to frustrate the Inclusion of elephants In traveling circuses.
Ho pefully. the Information provided above and In the documents attached wUI serve to dispel the notion that either
Betty or Bo have TB or pose any kind of public health rlsl<. Nevertheless, If you need any additional Information or
documentation In that regard, my client and I will be more than happy to provide It to you right away. - Chris

Christopher Abel
WILLCOX & SAVAGE, P.C,
440 Monticello Avenue, Suite 2200
Norfolk, VJrglnla 23510
Direct Dial: (757) 628-5547
Direct Fax: ("757) 333-3647
Cell: (757) 870-5344

. This message Is confidential, Intended only for the named reclplent(s) and may contain Information that Is privileged,
attorney work product or otherwise protected by applicable law. If you have received this message In error, please notify
the sender at 757-628-5500 and delete this message.

USDA

NaUonal Veterinary Services Laboratories

PO

Bo~

FINAL REPORT

844

Ames, 1CJ1,V9 60010

Phone: 615-337-7514 F~x: 515-337-7938
FEDERAL RELAY SERVICE (VolcetnY/ASClllSpsnlsh) 1800-877-8339
The USDA Is an equal opportunity provjder and IImployer.

Laboratory Teat Report

Sensitive Bul Unclassified/sensilive Secunty information - Disseminale on
Owner

II

Need-Te-Know Baals Only

Aceeulon Number:

14-020098

Carden CirCUli
Springfield. MO

Animal Location
Greene Counly MO
Submitter - 26999
Fair Grove Vel Service

PO Box 9
Fair GrovB, MO 65648
FAX #: 417-769-7803
Phone #: 417-769-7603

NFC Control Number;

Date Collected:
Date Received:
Date Completed:

19814034317
06125/2014

Collected By:

08119/2014
Dr. Michael D. stafford

Purpose:

T8-10-4

RaferrallRetaln Tag Number.

This 18

II

billable case.

NOTE: Condition of the samplo(s) wae adoquate unless otherwise noted.

Animal 10: Bo Case #: TB14-024B1 Sample: 6/16/2014 Specimen Type: Trunk Washings Species:
Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnimallD: 80 Caso #: TB14-02462 Sample: 6/17/2014 SpecImen Type: Trunk Washings Species:
Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

Animal 10: 80 Case #: T814-02463 Sample: 6/18/2014 Specimen Type: Trunk Washings Species:
Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

Animal 10: Bettty Case #: TB14-02464 Sample: 6/16/2014 Specimen Type: Trunk Washings
Species: Elephant. AsIan
Mycobacterial Culture Result

No Isolation Made

AnlmallD: Bettty Case #! TB14-02465 Sample: 6/17/2014 Specimen Type: Trunk Washings
SpecIes: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

Page 1 of 2

Dilta Generated:

8/1912014

Accession:

14-020098

Owner.

Cardan Circus

R~ferr81 Number.

AnlmallD: Betlty Case #: TB14-02486 Sample: 6/1812014 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnlmallD: Vicky Case #: TB14~02467 Sample: 6/16/2014 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnlmallD: Vicky Case #: TB14-02468 Sample: 6/17/2014 Sp8clm~n Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnlmallD: Vicky Case #: TB14-02469 Sample: 6J18/2014 Specimen Type: Trunk Washings
SpecIes: Elephant, Asian
Mycobacterial Culture Result

Results authorlzod by:

No Isolation Made

Dr. Suelee Robbe-Auslerman, Section Head, Mycobacteria and Brucella SecHon

NVSL MB General Phon6: 616-337-7388

Help Us Help You
(This now section will b~ updated periodically with Up. (or .... bmIUer .. )

QualHy samples yield the most accurate results. Please cal! If you have questions .

Page 2 of 2

Data Generated :

8/1912014

USDA

National Veterinary Services Laboratories

FINAL REPORT

PO Box 844
AmM, Iowa 50010
Phone: 615-337-7614 Fax: 515-337-7938
FEDERAL RElAY SERVICE (VolcafITY/ASCIl/Spanlsh) 1-80Q...S77-fl339
The USDA Is an equal opportunity provider and employer.

Laboratory Test Report

Sensitive But Unc1assltied/Sensltlve Security In(ormatlon - Disseminate on

Owner
Goorge Carden
Springfield, MO
Animal Location
Greene County MO
Submitter 26999

Fair GrovEl Vet Service

PO Box 9
Fair Grove, MO 65648
FAX #: 417-769-7803
Phone #: 417-759-7803

Nead-TcrKnow Basis Only

15-000343

Accesslorl Number:

Date Collected:
01/0612015
0310212016
Dr. Michael D. Stafford
TB-10-4

Date Received:
Date Completed:
ColIQcted By:
Purpoae:
RllfOrTilllRetalrl Tag Number:

This Is not a billable

CIISB.

NOTE: Condition 01 the sample(B) was adequate unleSB otherwise noted.

Animal 10: Bo Case #: TB15-00032 Sample: 1/02/2015 Specimen Type: Trunk Washings Species:
Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnlmallD: Bo Case #: T815-00033 Sample: 1/03/2015 Specimen Type! Trunk Washings Species:
Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

Animal 10: 80 CasEI #: TB15-00034 Sample: 1/05/2015 Specimen Type: Trunk Washings Species:
Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnlmallD: Betty Case #: TB15-00035 Sample: 1/02/2015 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnlmallD: Betty Case #; T81500036 Sample: 1103/2015 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

Page 1 of 4

Date Generated:

3/2/2010

Accession:

16000343

Owner.

Goerge Carden

Referral Number.

Animal ID: Betty Csse #: TB15-00037 Sample: 1/05/2015 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

Anlm81lD : Janice Case #: T815-00038 Sample: 1/02/2015 Specimen Type : Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

Anlm81lD: Janice Case #: T815-00039 Sample: 1/03/2015 Specimen Type : Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

Animal ID : Janice Case #: TB15-00040 Sample: 1/04/2015 Specimen Type : Trunk Washings
Species: Elephant. Asian
Mycobacterial Culture Result

No Isolation Made

AntmsllD: Vicky e8Se #: T815-00041 Sample : 1/0212015 Specimen Type: Trunk Washings
Speclee: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnlmallD: Vicky Case #: T815-00042 Sample: 1/03/2015 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnlmallD: Vicky Case #: TB15-00043 Sample: "110412015 Specimen Type: TrunkWashlngs

Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnlmallD: Cindy Case #; T615-00044 Sample: 1/02/2015 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

Animal 10: Cindy Case #: T815-00045 Sample: 1/03/2015 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

Animal 10: Cindy Case #: TB15-00046 Sample: 1/05/2015 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobaclerial Cu lture Result

No Isolation Made

Page 2 of 4

Date Generated :

3/212015

Accession:

16-000343

Owner:

Goerge Carden

Rarerral Number.

AnlmallD: Tory Case #: T815-00047 Sample: 1/02/2015 Specimen Type: Trunk Washings Species
: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

Animal ID: Tory Case #: T815-00048 Sample: 1/03/2015 Specimen Type: Trunk Washings Species
: Elephant, Asian
Mycobacterial Culture Result

No Isolatlon Made

AnlmallD: Tory Case #: T815-00049 SlImple: 1/05/2015 Specimen Type: Trunk Washings Species
: Elephant, Asian
Mycobacterial Culture Result

No Isolalion Made

AnlmallD: Jsnny Case #: TB15-00050 Sample: 1/02/2015 Specimen Type: Trunk Washings
Species: Elephanl, Asian
Mycobacterial Culture Result

No Isolation Made

AnlmallD: Jenny Case #: T815-00051 Sample: 1/03/2015 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result

No Isolatlon Made

AnlmallD: Jenny Case #: TB15-00052 Sample: 1/04/2015 Specimen Type: Trunk Washings
Species: Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AnimallD: Liz Case #: TB15-00053 Sample: 1/02/2015 Specimen Type: Trunk Washings Species:
Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

AntmallD: Liz Case #: T815-00054 Sample: 1/03/2015 Specimen Type: Trunk Washings Species:
Elephant, Asian
Mycobacterial Culture Result

No Isolation Made

Animal ID: Liz Case #: TB15-00055 Sample: 1/05/2015 SpecImen Type: Trunk Washings Species:
Elephant, AsIan
Mycobacterial Culture Result

No Isolation Made

These samples are resubmlsBlons from Acc8Sslon# 14-0"41276.

PBge 3 of 4

Date Generated:

31212015

Acceseion:

16-1] 00343

ReRu]tlt authorized by:

Owner:

Goerge Carden

Referral Number:

Dr. Suelee Robbe-Auslerman, Section Heed , Mycobacteria and BruceUa Section

NVSL MB Generill Phone: 616-33i-7388

Hilip

U8

Help You

[ThIs now section

wm be updated pe nodlcally wltl1Ups (or sUbmitters.)

Quality samples yield the most accurate results. Please eaUlt you have questions.

Page 4 of 4

Dale Generaied:

312(2015

DCHHS

~~ lives.
Dallas County Health and Human Services

Safe fa.Milies.,

Zachary Thompson, MA
Director

Christopher Perkins, DO, MPH

Medical DirectorlHealth Authority

July 30, 2015

Joey Zapata
Assistant City Manager, City of Dallas
Via email: joey.zapata@dallasci1yhall.com
Ron King
Executive Director of Convention and Event Services. City of Dallas
Via email: rotl .king@dallascityhall.com

Kris Sweckard
Director of Code Compliance, City of Dallas
Via email: kris.sweckard@dallascitvhall.com
Re: Soul Circus Inc. elephants
Dear Mr. Zapata, Mr. King, and Mr. Sweckard,

Thank you for sending us the additional information from Ben Johnson with Soul Circus, Inc.
regarding the company's desire to exhibit two elephants that have tested reactive for tuberculosis
at an event in Dallas next month.
Dallas County Medical DirectorlHealth Authority Dr. Christopher Perkins who, by agreement,
serves as the Health Authority for the City of Dallas, Dallas County Director of Tuberculosis
Control Dr. Garry Woo, and I have reviewed the recently submitted information and our position
stands . While we recognize that the individuals charged with caring for these elephants have
differing viewpoints, our investigation shows that the threat of potential transmission of
tuberculosis from elephants that carry the disease to arena and circus workers, members of the
public, and other animals remains a serious concern. We believe no elephant that has tested
reactive for tuberculosis should be exhibited.
Sincerely,

j~m&S~
Director, Dallas County Health and Human Services

C: Dr. Cluistopher Perkins, Dallas County Medical DirectorlHealth Authority

2377 North Stem mons Freeway, Suite 600, Dallas, Texas 75207-2710
Office 214.819.2100 Fax 214.819 .6022

EXHIBIT 6

8R ISKIN . C ROSS & S ANFORD , LLC

A TTO R NEYS A T LAW

1001 CAM BRIDGE S~UARE

TE~

S U ITED

FAX: 770.410.32.81

~PHAR~A.GA300D9

770A10.1555

WWW.BRISKINLAW.COM

August 17,2015
VIA E-MAlL TO
SARAH.AUSHERMAN@DALLASCTTVHALL.COM AND

REGULAR

U.S. MAIL

Ms. Sarah Hom Ausherman

Special Events Manager
City of Dallas Office of Special Events
650 S. Griffin Street
Dallas, TX 75202
Re:

Soul Ci.rcus, Inc . (the "Circus") Request for Permit for

Exhibition of Elephants

Dear Ms. Aushem1an:

This finn represents Soul Circus, Inc. d/b/a UniverSoul Circus (the "Circus").
The purpose of this correspondence is to clarify certain issues related to your email of
July 30, 2015, in which your office infonned the Circus that you will not permit the
exhibition of two elephants, Bo and Betty. during the Circus' upcoming tenure in Dallas.
We have reviewed the letter from Dallas County Health and Human Services
("DCHHS") provided with your email and it appears that the DCHHS analysis is based
on less than the best available veterinary science and evidence. Recognizing that
knowledge related to tuberculosis (''TB'') in elephants is limited to a small sector of the
veterinary medical practice, and even more limited in the non-veterinary medical
practice, we are providing you additional information and asking you to reconsider yoW'
denial. We would like to resolve this issue amicably and without the intervention of the
courts; however, as a traveling circus that has been providing wholesome entertainment
to families and children for decades--and for many years in Texas-the Circus must take
all steps necessary to .insure that we are abJe, once again this year, to provide the entire
circus entertainment experience to the general public in Dallas. including the
demonstration of the Circus' performing troop of elephants .

In short, the analysis provided by DCHHS is flawed and fails to recognize or give
proper scientific weight to the indisputable evidence that neither of the elephants, Bo nor
Betty, has TB. Further, the analysis fails to recognize that, as of the date of tlris letter,
there has never been a single reported case of transmission of TB from an eJephant 0 a
member of the general public arising from any public exhibition of elephants.

From the letter provided to us from the DCHHS, it appears that the sole basis for
the recommendation of the denial of the permit for exhibition of the elephants Bo and

9USINESS .

TECHNOLOGY.

COMMUNITY.

E)(HIBIT7

8RISK IN, CROSS

ATTO R N ~YS

&

SANFORD, LLC

AT LAVJ

L!;tter to Sarah Hom Ausherman

City of Dal las Office of Special Events
August 17, 201 5
Page 2 of 4

Betty is an unfounded fear that any elephant that has ever "tested reactive of
tuberculosis" poses a threat to the public. Such is simply not the case .
Enclosed with this letter, please find a copy of the supporting materials for the use
of the Cherubio Diagnostic Systems, Inc. DPP Vet TB Assay for Elephants. As the
literature clearly states, in the first paragraph, "The test is used as an aid in the diagnosis
of active tuberculosis (TB) in conjunction with other diagnostic tests." [11]
(Emphasis added.) The literature further states "A reactive result is suggesti~/e of active
TB" (2J (Emphasis added.) Of most importance is the following statement, found in the
third full paragraph : "The only USDA-recommended definitive diagnostic test to
detect TB in live elcpbantg is mycobacterial culture of trunk wash samples." [3] As
such, the DPP reactive immunochromatographic rapid test is merely a cbeap, easy, quick
screening tool, for which a follow up trunk wash is the only definitive test The DPP test
is subject to false positives, and the USDA guidelines, which are binding authority,
approve of trunk wash testing as the only defmitive test for active TB in elephants. As
such, any decision based solely on an old DPP false positive that was subsequently
proved to be a false posi tive by subsequent negative trunk washing tests ignores the best
scientific and medical evidence without any basis, and as such, is umeasonabJe, arbitrary,
and capricious. This is all the more so, when one considers that Betty and Bo meet all of
the requirements of both the USDA and the State of Texas [or exhibition in your state.

By means of further information, as you are aware, the USDA approved

guidelines for the control of tuberculosis in elephants, which were established by the
United States Animal Health Association ("USAHA") in 2008 (the "2008 Guidelines"),
and which defined the trunk wash test as the only definitive test for ncti ve TB in
elephants. The 2008 Guidelines were the onJy guidelines used for the control of TB in
elephants until December 2012. That is, in December 2012, USDA's Animal and Plant
Health Inspection Service ("APHIS") advised the public that it intends to use the 20 I 0
Guidelines for the Control of Tuberculosis in Elephants issued by USAHA. On
November 8, 20 10, USAHA issued a draft revision to the Guideline for the Control of
Tubercu10sis in Elephants (the "2010 Guideline"), which provided for more stringent
guidelines to exhibitioners.
Since there was confusion to the public concerning which guidelines to follow,

Soul Circus' elephant vendor, in an abundance of caution and in appreciation of the need
to fonow the best scientific evidence available, [ollowed the 20]0 Guidelines for the care,
testing, diagnosis, and treatment of Betty and Bo, the elephants in consideration for this
permit. This testing and care was provided and supervised by Michael D . Stafford,
D .V .M., who will testify t:hat he and tile owner of the elephants have fully complied with
the most restrictive protocols related to these animals, the 2010 USDA Guidelines.
I t is important to note that, under the 2010 Guidelines, elephants that test reacti ve
or positive to a DPP test, but also have a negative culture from a trunk wash and no

U $

I r..;

s _

TECH NOL OG Y .

BRIS KI N, CR O S S & SANF O R D , LLC

A T T O RN:;: ~

A T L. A W

Letter to Sarah Hom usherman

City of Dallas Office of SpcciaJ Events

August 17, 20) 5

Page 3 of 4

known exposure to TB fall under Group 3C . The 20 I 0 Guidelines prescribe how often
elephants that fall under Group 3C should be tested . The 20 I 0 Guidelines specifically
state that elephants should be "[mJonitor[ed] by culture (triple sample method) every 3
months for one year, then every 6 months fOT life. " The 2010 Guidelines further state
that elephants should undergo "repeat MAPIAlDPP testing every 3 ' months for the first
year, then every 6 months for an additional 2 years if elephant remains . .. reactive. If
all cultures remain negative after 3 years annual serological testing may resume as
described in these guidelines." The 2010 Guidelines then go further to state that " the
elephant may be able to travel and have public contact after 6 months of successful
documented USDA approved treatment."
The very first time that Bo tested reactive for TB was a Stat-Pak test in 2009, and
the very first time Betty tested reacti ve for TB was a State-Pak test in 2011. Immediately
after receiving these results) the staff and veterinarians in charge of the care of Betty and
Bo perfonned trunk wash tests every 3 months for one year (as prescribed by the 2008
and 2010 Guidelines), which all came back negative, and have been performing trunk
wash tests every 6 months thereafter (as prescribed by the 2010 Guidelines), which have
all come back negative for TB. Since all cultures remained negative for 3 years, annual
serological testing resumed. Currently, there are no restrictions on travel and exhibition
for Bo and Betty under the 20 10 Guidelines. As such, the Circus believes that it has fully
complied with the most restrictive suggested guidelines for treatment and testing.
Accordingly, the Circus suggests tbat the refusal of the City of Dallas Office of Special
Events to issue the requested permit is without scientific or legal basis,
As the materials provided to you previously have demonstrated, the trunk wash
tests performed on January 2,3, and 5, 2015, are all negative for Bo and for Betty. The
reported findings demonstrate that no positive TB culture could be isolated from the
series of samples . Under the '2010 Guidelines, these consistently negative tests mean
both elephants are under no restriction or special protocol of any kind and-in
accordance with even the most conservative interpretation of the applicable rules-may
be treated as if they never had a positive test. Consistent with those testing results, it
should be noted that the elephants' veterinarian has emphatically stated that neither one
has TB . The Circus previously has provided all of that evidence to your office.
While we certainly understand-and share-the City's concern over the
transmission of tubercuJosis, we also believe that the exclusion of Bo and Betty is
unreasonable, arbitrary, and capricious. It is clear from the correspondence from the
DCHHS that the 2008 Guidelines and more restrictive 2010 Guidelines were not
rev iewed or consulted in making tltis decision. As such, we believe that the decision is
based on less than a full Wlderstanding of the veterinary medical evidence, and, in fact,
ignores the clear and indisputable scientific evidence. Accordingly, we respectfully
hereby request that your office reverse its previous decision and grant the Circus the
requested perrni1s for the exhibition of both elephants. We will be happy to engage in

SU t ; I N E S $

TECH NOLOGY.

BRISKIN, C R O S S & S ANFORD, LLC

AT T O R N E Y S AT L AW

Lector to Sarah Hom Ausherman

Ollice of Sp<:cial E~-ents
August J 7, 2015

City of Dal1

Pnge 4 of4

any dialogue or to provide any other infonnation, including the names of recognized
experts in the field, should you so desire.
Given the reliance upon the exhibitor's compliance with the even more stringent
requirements of the 2010 Guidelines, the Circus is uncertain as to the legal or scientific
basis for the Department's denial. Please advise us of the specific statutory, regulatory,
or scientific basis for the deniaJ so that the Circus can evaluate an appropriate course of
action. The July 301h letter from DCHHS that was sent to the Circus merely states that
"our investigation shows that the threat of potential transmission of tuberculosis from
elephants that carry the disease to arena and circus workers, members of the public, and
other animals remains a serious concern." The letter from DCHHS does not state any
basis for their concern.
If you have any questions or would like to discuss this matter in more detail, feel
free to contact me at my office.
Sincerely yours,
BRISKIN, CROSS

& SANFORD, LLC

~~<jl?Z
Alan M. Briskin
Attorney at Law
Enclosure

celene:

Mr. Cedric Walker

President. Soul Circus, Inc .

Mr. Ben Johnson

Director of Operations, Soul Circus, {nco

Mr. John D. Fraser, Esq .

Ferguson, Braswell & Fraser PC

Mr. Christopher Abel , Esq.

Willcox & Savage, p.e
Mr. Joey Zapata
Assistant City Manager, City of Dallas

Mr. RanKing
Executive Director of Convention and Event Services, City of Dallas
Mr. Kris Sweckard
Director afCade Compliance, City of Dallas

TECHNOLOGY.

-C 0

!1 M U N I T Y

CATALOG II 65-9110-0

2 Tc.sl Kit

'HEMBIO
OIAGNOSTIC

SYSIEM5,

3661 Horseblock Road

Medford, New York 11763
U.S. Velerinary License No , 645

INC .

Mycobacterium bovis - Mycobacterium tuberculosis

Antibody Test Kit
DPp VetTB Assay for Elephants
A Rapid Immunochromatographic Test for the Detection of Antibodies to Mycobacrerium tuberClI/osls acd
Mycobacterillm bows in Elephant Serum, Plas!lUl or Whole Blood
FOR IN VITRO VETERINARY DIAGNOSTIC USE
READ rNSTRVCTIONS FOR USE CAREFULLY BEFORE PERFORMING TEST

PRINCIl'LE OF TEST

TNTEND ED USE

The DP? VorTB Assay is a slJ1gJe use Immunooh romalographic rap id teat for Ihe del~tion of andbodios to
M)lCobacler (~m I~berclrlo:rls and Mycobacleril/n! Mvls In
serum, plasma or whole blood from Ati1c~ elephants
(LoxodontrJ afrloana) and Asian elephants (Elephas
max/mil!). The test is usod iIB an aid in the dlagnosb of active
lubercuJ05il1 (TB) In conjunction with other dl!gnostlc
methods.

IT 8pe~iflo IlI)llbodiCll are pltSon( In the sample, the expected

fait l'eSUIt Is reac1lvo. A roa~tive result is suggestive of activo
Ta. In tho ab!lence of antibodlos, the cxp~t~d tc,l ~ult is

nOll/'\laetive.

SUMMARY AND EXPLANATION

Tuberculosis (113) in elephants Is a rc.-emel'glng zoonotic
disease caused pdmW'lly by Mycobaclerlll17l Nbercliions and,
In some ca~, by Mycobacltrrlum bOYi3 (I J. Tho ooly USDArecommended detlnitivo diagnostic te81 to detect TB ID live
elephants is mycob!oiGrlal culture of Irunk wash ~alllpies .
However, this metbod has poor diagnostic sensitivity, as h
c~n only Identity animals with elnonslve shedding of the
organhm tilal typlaally occurs late in the course of dis/llise
[2J. Rapid detection of lofeoted elepJuots I~ " crucial
prerequl~l1c fol' more effective control of Ta, IS carly
diagnosis allows Ilmely isolation and/ol' Initiation of
chemothel'llpy [1-3).

MATERIALS PROVIDED
Each kll contBins the follOWing lre~ :

2 DPP Ve(I'B tllSt devJC6:l

DPP Vt:ITS buffer viat (6mL)

Serological melhods 00fIS1itut6 an aiU'active altematlvo 8.1

they are relatively simple, inexpensive, non-Invasive, and
Uley do not de1Jend on detection ofmycobaoteria [3-4). None
of Ihe exlsling TB lesls alono iB suffielent co diagnose
dIBeQ~e . Therefore, new TB diagnostic algorithms ere bemg
developed, In which 90rologlcal assays may pta), ao important
r<>le [3-5] (see PERFORMANCE CHARACTERISTICS
below).

IO -61 9~O

Product Insert

Addldonal Material ReqOlred But Not Provided

Cloak, watch or othor tflnlng device
Disposable glovO!
Biohazard diBposal container
Collection dcvice<.I for specimen!
Pipettor capable of deliverbg 5ltL of sample

The use of Chembio D?P VetTB AMsy is an effective

approach for mpid animal-sido identification of ell>phants
infectod with M. mbBTolilosis or M. b(1'IU, liS this tesl is
highly accumte, user-fliendl)', safe, and easy to pel'form.

Rov 2

Nov nber 2012

The Chembio DPP VelTB Assay Is b(\$ed on immunochl'Olnt\tographlc technology. The ~I emplo)13 two
reC<lmbinanl antigens, an M 11iberClJlwl~ and an M. bovls
antigen, which are separately Immobilized on tho membrane
solid phase. It also utilizes recombinant Protein AlG corUugBtlld
to colloidal gold pel110les for antibody detection. Ths DPP
VetTB AJse.y \lIIe.!I !erurn, plasma or whole blood. The ample
is applied to the SAMPLE+BUF'PER well with the buffer.
After the sample and buffer have mJgrated onto the rut strip
additional buffer ~ ~dded to the BUFFER well. The buffer
feolUtalll.'l tho lawai flow of the rulcnsod produc!:l IUld
promote.! the binding of antibodies to the antigens. If present,
the antibodies bind to !he gold conjugated antibody binding
prott:ln. In a I'CIIctive sample, tha dye conJugated-lmmu~e
complex migrates on tbe nitrocellulose mtmlbrnne find is
CJIpTUred by the antigens immobilized In the TEST (\ 2) area
producing 8 plnklpwple line. In Iho absence of detectable
IDltibody, no speclfio Immune complex would be formed on the
test line. Rnd, therefore, no pinklpurplo line would appear In the
TIlST (J 2) I\relI. Unbound cOrU\lgBted gold pllrtlcles continue
to ro!grate illong the rnem bra no I!lld produc6 a pink/purple Hne
In the CONTROL (e) area. This procedural control serves to
dcmonltr8te tluu rhe reagents havo been properly IIppUed end
have migrated through ll1e dovico,

hS. I of4

STORAGE AND STABrUTY

The DPP VetTh Assay ihould be scored at 2 fO 30'C in the
or)glMl sea.led pouch. The dilu~nt should be stored in the
origInal viBI at 2 10 30'C. The klL Is slllble until Ihe date
printed on the box label and/or pouch.
NOTE: Do not lise ~)(p\red lest kits.
CAUTION: 00 not freeze testkics.

TEST PROCEDURE

2.

PRECAUTIONS
I.

2.

3.

4.

S.
6.
7.
8.

The telt is delligned FOR IN VITRO DlAGNOSl1C

USE only, Use the tesl on Iy in aCCI)nia.nce wIth
instructions supplied with the kit.
Handle aJl specimens as recommended fat' allY
potenrielly Infectious sO!"\lm or blood sp<:elmen In the
CDC.NTH manual, Blosafery In Microbiological (Jnd
Blomed/c(J1 LabnrotorlQ, 4'h ed., 1999.
US~ suitable protective clothing (glov~, lab coat, 8&fcty
glllSS6S) when hand ling WIIpl eo or (r.st dcviC6S afror
illmples have been applied. Avoid any conlBct berween
lIands, ~es, nose or mouth during specimen collection
lind testing.
Do not pipetle any material by mouth. Do not smoke, eat
or drink in areas whore sp.cimans or kit matorial are
kept.
All lestingshould be perfo, mad HI a lomperalure of 18 to
3 (}OC.
After the completion of the BIISSY. carefully dispose of
materials treating them as blohsZlIrdous weste.
Do not use CKJl!red lost kits. Do not freaze tllS! kits.
Do not mix reagents from different kit lots.

J,

If tes1 SAmples are refrlge\'ated, remove them flam the

refrigerator and allow Ihem to come to a temp~rature of 18
to 30'e befoto tostlng.
Remove the required number of DPP VetTB As~ay
dovlcea from their poucbes and place the devices on a nlll
sunsee area. It I. not neccsw)' ID remove the delioo~n(
from the package.
NOTE: If desiccant packet Is missing, DO NOT USE,
discard the test device and u now test device
should be u5td.
Label test units with sample namClS and/or identification
number!!, (see Figure I below)
Figura 1
T~ Window w~h

1\110

btu a line, and on e

a'Ben line

We!12

SAMPLE +SUFFER

Weill
U~\ng a 10)1.1

4.

dispoSllblo pipette (for whole blood) Or 6

laborB.lory pipertor (for S III of serum or plasma), relea!le
the specimen oarefully to the center 0 f the round
SAMPLH+BUFFBR Well 1. (Sec Figure 2 below)

5,

Onco (he specImen has blletl applied to the

SAMPLE+BUFFER Well 1, remove the cap, Invert the
buffer
boftlo,
hold
It
vertIcally
over
the
SAM.? LE+BUFPER Well 1, and add 1 drops (-6~ f1l) of
the buffer slowly 1010 SAMPLE+BUPPER weU. (See
Figure 3)

6.

W"it 5 Ullnut~, and then add 4 drops of the buffer tu the

squaro BUFFER Well 2, (See Figure 4 below.)
NOTE: The blue Bnd green colored Hiles should have
disappeared from 1he reotangular TEST &nd CONTROL
window. If not, dl!ICRrd tho test device and repoat the
procedure with B flew DPP test device.

SPECIMEN COLLECTION
The DPP VetTB AS.!iay can be performed on whole blood,
serum or plasm8.
Whole Blood: Collect whole blood into tubes containing

heparin or BDTA. Be sure to thoroughly mix whole blood by

Inverting capped tube several time.! JU!1 prior to resting.
Follow test procedure instructloD3.
Senlm: Serum is obtained from whole blood coli()Cted
aseptically by venipuncture Into a olean tube without
al\ticoagulant. AJlow tb.e bloed to clot at room temporature,
centrifuge a( 2000 rpm for 10 minutes at room temperature,
18 to )O"e, and separate the ~rum from tho clot wl1hln 24
hours of blood collection ..
PlAJm_: Collect whole blood with anliooagulant (heparin,
EDT A or sodl'Jm cll1llte), centrifuge lit 2000 '"pm for 10
minutes at room tomperatl.lle, 18 to 30C, and isolErte the
plasma supernatant.
Semplcii perfonn beost when tes1ed Immediately after
co lIectlon. Speolmens should be immediately refriget1lted at 2
[0 8C following collection and csn be u9Cd up 10 3 days, If
telling within 3 dHYs Is not pos~lbl6, 1ho speclmen9 should be
frozen at -20C or colder until use. Avoid repeated rroezi r.g
Hnd thewlng. DO NOT FREEZE WHOLE BLOOD.
NOTE: 1f speclmMs are to b~ stupp ed, they .hould be
pecked ill compliance with regulatiON covering the
mmspol1ation of etiologic agents. Venous whole
blood, serum and plasma specImens should be
shipped retTlgcratcd with cold paoks or wet iDe.

10-6294-0 Rov 1
N<rVembe: 2012

7.

8.

Ra&d the \esl r~sult 15 mlnlJtllS after the addition of th~

buffer into the BUFFER Well 2. In some ceses 8 test
line may appear in leu than IS minutes; however, IS
minutes are needed to l'Cport a non-reactlve l'eSult. Do
not raid results ~ftcr 2S mlnutos trom Addition of
SaruplH-Buffer to Wolll.

Nonreactive Res\llt
Only n pink/purple CONTROL (C) line is visIble. The sample
oonllllns no deteclalJle antibody to both TB lind
mycobacteriosis antlgons. A nonreactive resull doe.! nol
preclude the possibility ofTB infection.

After reading and rDeordlng test resultll. discard the used

tcst device5 and any olh~r t~sl luaterlais into Q biohaZllrd

waslo conl~lner.

QUALITY CONTROL
A pinldpurple color~ IIno should always appear in
CONTROL (C) eruu if the test has been performod correctly
and the devico Is worldng proporly, It sarve.9 as I1n interNlI
test proccdul-al control.

Invalid Result

INTERPRETATION OF RESULTS

A pink/purple line should always appear In the CONTROL (C)

area, whether or not a line appears In the TEST are~. Iflhol'e Is
no distinct pinldpurp!e line In Ihe CONTROL (C) area, the test
Is invalid IUId should be repeated using B new device.

Reactive ResD It
l. Three pink/purple lines. one line In the CONTROL area.
one line 10 the TBST (l) area and one line in the TEST (2)
area indicates a reactlvo resu It. This SUggOBI3 that the
slImple is raACtive for TB .

2. A plnklpurple TEST (2) line and II pln klpurpl~ CONTROL

line are visible. This S'Jgg~ts tlult the samplo u; rCllletlyo
for TB .
T6ST(2) Un.

LIMITATIONS OF THE PROCEDURE

1.

COl'ITROL (e) LIQ<

2.
3.
4.

5.
3. A plnklpurple TEST (I) line and II plnk/pul'ple CONTROL
line 81'e vl~lb Ie, This suggests that th~ sampla is CtIIIotiyO ror
TB or mycobActeriosis.
TBST (ljUn.

6.
7.
8.

COmROL (C) Lin,

9,

NOTEi Intensities of the TEST and CONTR.OL lines may

vary. Test IIn~ are conside red 1'e8ctlvtl regaJ'dles.l of
inrensity.
10 6'2.940 Rov '2.
November 2012

The Chomblo OPf> V~TB Assay 13 designed for detecting

antibodies against M. /uberCIIlosls and M. bovlN only fi'om
elephant pllIStna, serum or whole blood. Any other body
fluids or pooled samples or spcolmens from other thaD
elcpnont speci6.!l should not be u~cd.
Test rClllult'l mwt b~ read betw6cn 1520 minutes sfier the
addition oflne buffer 10 the square BUFPER Well 2.
Do not \lse hemolyzed blood samples.
Blood specimens must be lnoroughly milled Jtlll prior to
tesrlng.
Do not open the sealed roil pouch untll JUil prior to UOG.
Do not \lie kit contents beyond labeled expiration dale.
Read 1'OS\llts ie II weUlit aroa
A rOllctive r~Bult using tbe Cbetnbio DPf VolTB Assay
suggests the presence of antibodlos to M. /lIbcrcli/oJ/s and/or
M boy/s. Tho Cnemblo DPP V3I'Tl3 AS58), IS intondad liS an
aid In th~ diagnosis aDd rreatrceut ofTB in olepnants.
por ~ reaotivo result, (he Intensity of the test line doc:s 1101
necessarily cOITelate with the titer of antibody In the

speclll1on.
10. A nOIl-reaotlve result does not preclude the pos:libllity of
exposure to TB or Infection with TB. An antibody response
10 recent exposure may take several months to reaoh
detectablo levolg.
I!. In h~Bted elephants, Inle'lJl'et results with caution:
e, Treatment against T8 may reduce antibody responge.s.
thuB resulting ill non-reactive result:; in solllO c~ses.

Pes, J of4

b, A reactive result may persist in infected and tt'eBI~d

elephant! for months and yMI''l evon If en elephant Is
cons id ered cured .

II/barell/OS/S infccuon all10ng ~epliv~ A.ian elephants in B

SW5dish 1.00 . Vel. Rec. 156: J'/ I-I 75 .Lyashchenko, K.P.,
et aI., (2006) Tubercul05 is In elophants; antibody
response.. (0 deflned antigens of Mycobl1Clerfllm
h/bercu/OJls, potential for farly diagnosis. and monitoring
or t~atmenl. Clinical And Vaccine Immunology 13: 722-

I'ERFORMAi'lCE CHARACTERISTICS

Highly specl/lo and sensitive IlJ1tlbody binding antigens are

used in the DPP VetTB Assay. The diagnostic pllrfonmncB
WIU compared 10 the standerd USDA-~comDlendcd method,
trunk wash culture, and the DPP VetTB A.lSay diagnostic
pel'formllllce was found to be s\lperior [4-5).
further, it W8! shown that both Asian Bnd Aft'lean elephants
infected with M. II/bel'culas/! or M bovls oould be detected
by DPP VetTB Nsl\y up to sllveral years pr ior to finding
p08itlv\l culturo in tmnk washes r4-5] ,

732.
Greenwald, R. L)llI!Ihchenko, 0 ., fufundiari, J., Miller,
M., Mikota, S., Olsen, I.H., "Ball, R., Dumonc5Bux, G.,
Schmitt, D" Moller, T" Payeur, 1.B., Han'is, B" SofnHlko,
D., Wate~. W,R., Bnd L),a3hcbenko, K,P. (2009) Highly
accurate antibody assays fo!' easly Bnd rapid detection of
tubcl'Culosis in Aft'iean and ,A.,lan elephants. Clinical And
Vaccine immunology /6:605-612,
LYBShchenko K.P., Ore~nwBld R., Bsfilnd 1llTi J., Mlkola
S., Millor M.. Moller T., Vogc)nest L., Gtlrhe K.P ..
Rabbe-AustelmlUl S., Gal J., Dnd Walers W.R, (2012)
Field applJcBllon of serodlagnostlCB to idenllry elephl!1lts
who tubl:l'CLllosls prior to case confirmation by culture.
elin. Yacclne immltno/. [9(8):1269-75.

3.

4,

Sensitivity and Spedflclty

Sunsillvity of Ihe DPP VetTB Assay wes derenn ined by
iostlng 40 culture positive elephants. All 40 ~amples wore

FOR MORE INFORMATION, CONTACT:

reactive (Table I),

CHEMBIO DIAGNOSTIC SYSTEMS, INC.

The 8peclflclty of the DPP VetTB Assay wu determined by

testing sorum, pllClma, and/or whole blood samples collected
tram t47 trunk-wash culture negative elephants wilhou1

3661 HORSEBWCK ROAD

MEDFORD, NY 11763 [JSA
U.S. VeteriDary License No, 645
Tel: (631) 924-1135
Fay.: (63 I) 924-6033
Email: jnfo@chemhio.cQm
Web Sile: www.chembio.com

history ofTB , All 147 slI/llplcs were non-reactive (Table 2),

Table 1.
DI~l!no,llc

sens!tMI)' of DPP VetTH Assay

Elephllllt
species
Amcan
AfrlcBn

Mycooactel'ial

Asirul
Tobie 2.
Speclnci ~

Spe1::le5 Isolated

DPP VetTB
reactive

M tuberculMI9

616

M. boyls

1/1
33/3 3

M tuberculoJ/J

8rudl~

Elophant
ap5ci1;3

ORDER INC INFORMATION

of DPP VetTS Am,>,

DPP VerrB Assay

non-re~ctive

Atriono

79/79

A91an

68/68

Trunk wash
Mgative
79/79

cultur~

68168

REPRODUCIBILITY STUDIES
Reproduoibillty was evaluated at three independent
Illbol1ltories using two serials of DPP VeITB AasIl.Y. A
r8feren~ plDlel of 40 blindly-coded samples representing
negative, weakly reactive snd strongly reactIve Bera Wl."1'l!
lMled 3 tim68 on J different daYJ. The compliM re!ul\li from
J labol'elories demonstt'llted 98.6% acoUf8cy ,

REFERENCES
I.

2.

MikoUl, S.K., Peddle, L., Peddie, J., tsaZlt, R., Dunker,

F" West, 0., Lindsay, W., LIlrsen, R.S" Salman, M ,D"
Chatterjee, D" Payeur, I., Whlpplo, D.. Thoen, C.,
Davis, D. S., Sedgwick, C., Montall, R.J ., Ziccardi, M.,
Bnd Maslow, I. (2001) Bpldemlology and dlegno\s
MycobaCIBI'Ilim Il/bercII/Mls in ~apt!vo Asian elephants
(E/ephaJ maxinlUJ). J. Zoo Wild', Med. 321 1-16.
Lewerln, 8.g., Olsson, S-L., Eld, K., R1ikcn, B~
GllebrclJlichael, S., Koivula, T., Kd(leolus, 0 " Bnd
B61ske, G. (2005) Outbreak of Mycobacrerlum

I D.4I294 0 R"" 2

NOV'embcr 20\2

Pag.~.fd

Cet#

Product

65-9[ [0-0
65-91 [1-0
65-9112-0

DPpe VetTB 2 Test Kit

DPp4P VetT8 5 Te.llt Kit
DPP~ VolTB 20 Test Kit

II

Antigens licon~cd from

Slalens S&I'Um I MTlwt
wwyt,s3i.dk

Elephant TB Guidelines 1

GUIDELINES FOR THE CONTROL OF TUBERCULOSIS IN

ELEPHANTS 2010
UNITED STATES ANIM:AL HEALTH ASSOCIATION (USAHA)
ELEPHANT TUBERCULOSIS SUBCOMMITTEE
8 November 2010 DRAFT REVISION

TABLE OF CONTENTS (topics are bookmarked)

1. Introduction
2. Definitions
3. Aruma] Testing
4. Culture Collection Procedure
5. ElephantTB STAT-PAJ( and MAPIATM Collection Procedure
6. Ancillary Diagnostic Tests
7. IB Management Groups
8. Principles of Anti-tuberculosis 111erapy
9. Anti-tuberculosis Drugs
10. Dosages and Routes of Administration
11. Blood Levels
12. Postmortem Examination
13. Employee Safety and Health
14. Reportin g
15. Appendices
Appendix 1. References
Appendix 2. Acknowledgments
Appendix 3. A Trunk Wash Technique for the Diagnosis of TB in Elepharlts
Appendix 4. Testing Lab oratori es
Appendix 5. USDA Standard Operating Procedure for Processing Elephant Trunk
Washes for the Isolation of Mycobacteria
Appendix 6. Contacts for Questions
Appendix 7. Sources for Anti-tuberculosis Drugs
Appendix 8. SSP Serum Banking Fonn
Appendix 9. TB Management Groups - Flowcharts
These guidelines are available on the Internet at the following sites:
1. http://www.aphis.usda.gov/animal welfare/index.shtml (available to the public)
2. www.aazv.org (available to AAZV members by password)
3. www.elephantcare.org (available to the public)
4. www.elephanttag.org (available to the public)

EXHIBIT 8

Elephant TB Guidelines 2

1. INTRODUCTION
Tuberculosis (TB) is caused by bacteria in the genus Mycobacterium. Over 100 species
comprise this genus. Mycobacteria infect a broad range of species including humans, nonhuman primates, carnivores; marine mammals, psittacine birds, reptiles, fish, artiociactylids,
pachyderms, and domestic and non-domestic ungulates. Species susceptibility to specific
mycobacteria varies (Montali 2001).
In mammals, the term ''tuberculosis'' is used to define disease caused by Mycobacterium
tuberculosis (M tb) complex organisms. The M tb complex includes M. tuberculosis, M. bovis,
M. africanum, M microti, M . canetti, M. caprae, and M. pinnipedii. A vaccine strain derived
from M. bovis (M bovis BCG) is sometimes included as a separate member of this complex.
The term "mycobacteriosis" refers to infection with any mycobacteria but is generally used to
defme disease caused by non-tuberculous mycobacteria (NTM). "Atypical mycobacteria" or
"mycobacteria other than TB" (MOTT) are other tenns used to describe this group. Most NTM
are saprophytes found in soil or water but they may occasionally cause disease in humans and
animals, including elephants .

Mycobacterium tuberculosis is the predominant disease-causing agent in elepbants although

cases caused by M bovis have occurred. Mycobacterium szulgai, an uncommon NTM species,
was associated with fatal disease in two African elephants (Lacasse 2007) and Mycobacterium
elephantis, a rapidly growing mycobacterium, was isolated from a lung abscess of an elephant
that died of chronic respiratory disease (Shojaei 2000). Mycobacterium avium is commonly
isolated from elephants (payeur 2002), but to date has not been associated with clinical disease.
The National Tuberculosis Working Group for Zoo and Wildlife Species has been monitoring
TB in elephants since 1996. The original Guidelines for the Control of Tuberculosis in
Elephants were released in 1997 and modified in 2000, 2003, and 2008. The Guidelines include
recommendations for the testing, treatment, and surveillance ofTB in elephants and are revised
as new information becomes available. The 2010 guidelines include updated information on
diagnostic tests and add further clarification to TB management groups.

2. DEFINITIONS
Ancillary diagnostic test: A subordinate OT auxiliary test to be used in support of a primary test
to diagnose disease.
Airborne transmission. Airborne transmission occurs by dissemination of either airborne
droplet nuclei or small particles in the respirable size range containing infectious agents that
remain infective over time and distance (e.g., spores of Aspergillus spp, Mycobacterium
tuberculosis bacilli). Microorganisms carried in this manner may be dispersed over long
distances by air currents and may be inhaled by susceptible individuals who have not had faceto-face contact with (or been in close proximity to) the infectious anima.l or person (Siegel 2007).

Elephant TB Guidelines 3

Attending veterinarian: a person who has graduated from a veterinary school accredited by the
American Veterinary Medical Association's Council on Education, or has a certificate issued by
the American Veterinary Medical Association's Council on Education Commission faT Foreign
Veterinary Graduates; has received training and/or experience in the care and management of the
species being attended; and who has direct or delegated authority for activities involving animals
at a facility subject to the jurisdiction of the Secretary (i.e. a USDA licensed facility).
Atypical mycobacteria: see non-tuberculous mycobacteria
Contact transmission:
Direct contact transmission may occur during activities such as touching or riding an
elephant, being touched by an elephant, examining, medicating, bathing, and handling
Indirect contact transmission involves contact with a contaminated intermediate object, such
as occurs during cleaning cages and equipment and handling soiled laundry. Injuries from
contaminated sharps, such as scalpel blades, needles, and necropsy knives, may result in
exposure to pathogens . (NASPHV 2006)

Culture positive for M.th complex: Isolation and identification of M. tuberculosis complex
organisms from any site using standard mycobacterial methods.
Culture positive (M.th complex) elephant: An elephant from which a M. tuberculosis complex
organism has been isolated from any body specimen. A culture positive elephant is considered
positive until it has met the treatment requirements as outlined in the CWTent Guidelines.
Dual Path Platform (DPP) VetTB Assay: A new generation screening kit for the rapid
detection of IgG antibodies to M tuberculosis or M. bovis in elephant serum, plasma, or whole
blood. The DPP has shown 100% correlation with MAPIA TM (Greenwald et a1. 2009).
ElephantTB STAT- PA.l( Assay: A qualitative screening kit for the detection of antibodies to
M tuberculosis and M bovis in elephant sera, plasma, or whole blood (Lyashchenko 2005, 2006,
Greenwald 2009).
ELISA: Enzyme-linked irnmunosorbent assay; a test used to detect and measure either antigen
or antibody.
Exposure: Risk of transfer of an infectious agent from 8 TB infected elephant(s) or
contaminated environment through contact (direct, indirect) or airborne modes of transmission.
Fondte: An inanimate object or material on which disease-producing agents may be conveyed.
Gamma-interferon test: A whole blood in vitro assay that can be used as an ancillary
diagnostic test for TB (not currently available for use in elephants).
Genotyping assay: A technique for the identification and analysis of polymorphism in certain

Elephant TB Guidelines 4

types of repeat units in DNA. Restriction fragment length polymorphism (RFLP) and variable
number tandem repeat (VNTR) are examples of genotyping techniques.
Herd: A group or groups of elephants, maintai.ned on common ground. Alternatively, two or
more groups of animals under common ownership or supervision that are geographically
separated, but that may have an interchange or movement of animals or personnel without regard
to health status.
Incidence: The rate at which a certain event occurs, for example, the number of new cases of a
specific disease occurring during a certain period.
Index animal: The animal in which a disease is first diagnosed.
Infected elepbant: an elephant from which Mycobacterium tuherculosis complex has been
identified through culture, PCR or other molecular techniques or that is reactive on the
ElephantTB STAT -P AK@ Assay and the MAPIA TM.
Intradermal tuberculin test (skin test): The injection of purified protein derivative (PPD)
tuberculin into the skin for the purpose of detecting exposure to tuberculosis. In cattle, the test
site is either the caudal fold (CFT) or cervical region (e.g. comparative cervical test, CCT) and
the test is read by observation and palpation at n hours (plus or minus 6 hours) following
injection. In hwnans, the test site is the foreann and the test is read at 48-72 hours . The
intradermal tuberculin test is not a reliable test in elephants (Mikota 2001, Lewerin 2005) .
Licensed veterinarian: a person who has graduated from an accredited school of veterinary
medicine and who has a valid license to practice veterinary medicine in the U.S.
MultiAotigen Print ImmunoAssay (MAPIA TM): A confIrmatory test to the ElephantTB
STAT -P AK Assay for detection of antibodies to M tuberculosis and M bovis in elephant sera
or plasma (Lyasbchenko 2000, 2006, Greenwald 2009).
Mycobacteria otber tban TB (MOTT): See non-tuberculous mycobacteria.
Mycobacteriosis: A disease caused by non-tuberculous mycobacteria (NTM).

Mycobacterium: A genus in the family Mycobacteriaceae.

Mycobacterium avium (M. avium): A non-tuberculous mycobacteria that is the primary
causative agent of tuberculosis in birds . M avium may be isolated from non-clinically affected
elephants and is usually considered an environmental contaminant.

Mycobacterium bovis (M. bovis); The primary causative agent of tuberculosis in cattle, bison,
and cervids; may also affect a variety of mammals including pigs, humans, primates, and nondomestic ungulates.

Mycobacterium tuberculosis (M.th): The primary causative agent of tuberculosis in humans;

Elephant TB Guidelines 5

may also affect a variety of animals, including primates, pigs, cattle, dogs, parrots, elephants, and
rhinos.

Mycobacterium tuberculosis complex (M.th complex): A group of mycobacteria which

includes M. tuberculosis, M bovis, M. ajricG.J1um, M microti, M canetti, M. caprae, and lvI.
pinnipedii. A vaccine strain derived from M. bovis (M bovis BCG) is sometimes listed as a
separate member of this complex.
Mycobacterium Tuberculosis Direct Test (MTD): A nucleic acid amplification test used in the
diagnosis ofTB. The MTD utilizes a technique that replicates RNA from bacteria of the M
tuberculosis complex.
No isolation: Absence of growth of M th complex organisms from trunk wash, feces, tissue or
other samples using standard mycobacterial culture methods. Failure to isolate organisms may be
due to the following reasons:
1. The animal is not infected
2. The animal was not shedding at the time of sample collection
3. Sampling error (culture overgrowth by contaminating organisms, inadequate sample, or
laboratory error)
4. Improperly handled or shipped sample
Non-reactive: Absence of response; in the context of serological testing for TB in elephants, a
non-reactive result indicates that an antigen-antibody reaction has not occurred in the presence of
an appropriate positive control response.
Non-tuberculous mycobacteria (NTM): Mycobacteria that generally do not cause the
fonnation of granulomas. Most NTM are saprophytes found in soil or water. They are typically
non-pathogenic but may occasionally cause disease in humans and animals, including elephants.
Also referred to as "atypical" mycobacteria or "Mycobacteria Other Than T8" (MOTT).
Nucleic acid amplification test: A technique that amplifies entities such as DNA or RNA.
peR (polymerase-chain reaction): A nucleic acid amplification technique in which specific
sequences of nucleic acid (DNA or RNA) are replicated, allowing for detection of target
sequences.

Premises: A parcel of land containing elephants, administered by a person, government entity

(city, county, state, region) or organization (zoological society, corporation).
Prevalence: The total number of cases of a specific disease in a given population at a given time.
Rapid Test: see ElephantTB STAT -P AK Assay
Reactive: Presence of response; in the context of serological testing for TB in elephants, a
reactive result indicates that an antigen-antibody reaction has occurred.

Elephant TB Guidelines 6

Report date: The date the laboratory reports the results.

Spoligotyplog: A genotyping assay
Variable number tandem repeat (VNTR); A genotyping assay
Submission date: The date the sample is received at the laboratory.
Test date: The date the sample is collected.
Tested elephant: An elephant that has been tested for tuberculosis according to the protocol
established in these guidelines.
Triple sample method: A method of culture collection whereby 3 samples are obtained on
separate days.
Trunk wash: A procedure used in elephants to obtain a sputum sample using one of the
approved methods outlined in Section 4 - Culture Collection Procedure.
Sensitivity: A measure of the ability of a test to identify infected animals. Sensitivity is the
frequency of a positive or abnonnal test result (e.g. a test that is outside of the reference interval)
when a disease is present (i.e. the percentage of true positive results). Sensitivity = [Tp..;.. (TP +
FN)] X 100 where TP = true positive; FN = false-negative).
Specificity: A measure of the ability of a test to identify non-infected animals. Specificity is the
frequency of a negative or "normal" test result when a disease is absent (i.e. the percentage of
true-negative (TN) test results. Specificity = [TN..;.. (TN + FP)] X 100.
Untested eJephant: An elephant is considered "untested" if it has not had three trunk washes
obtained by the method outlined in this protocol within a 12 month period or if fewer than three
valid culture results are obtained or if it has not been tested with the ElephantTB STAT-P AK
Assay performed by a USDA veterinarian trained and certified to perform the test.

3. ANNUAL TESTING
To adequately address the concerns ofTB in the general elephant population, all captive
elephants must be tested annually by culture and with the ElepbantTB STAT-P AK Assay (a
blood test). Samples for cultures and blood must be collected by or under the supervision of a
licensed veterinarian according to current USDA requirements. Blood collection for the
Guideline-required ElepbantTB STAT -P AK Assay must be witnessed by a federal or state
veterinarian and performed as licensed by the USDA Center for Veterinary Biologics. See
further information below under ElephantTB STAT -P AK Assay. It is required that elephants
with a reactive ElephantTB STAT-PAK Assay result be tested using the confirmatory
MultiAntigen Print ImmunoAssay (MAPIATM). See item 5 below.

Elephant TB Guidelines 7

Elephants should be tested within 30 days of the established annual test date. Blood for
ElephantTB STAT -P AK Assay and culture should be collected within a 2 week period. AU
elephants must be tested every calendar year. Note that the date the sample is collected is the
"test date," the date the sample is received at the laboratory is the "submission date," and the
date the laboratory reports the results is the "report date."
Record keeping of TB testing and treatment by the attending veterinarian is of upmost
importance. It is recommended that attending veterinarians maintain open communication with
the United States Department of Agriculture (USDA) and State Veterinarian, particularly
concerning elephants under treatment for TB or in cases of exposure to TB positive elephants . It
is recommended that at least a 1 ml aliquot of sera collected at the time ofTB testing be sent to
the elephant serum bank (See appendix 8).

4. CULTURE COLLECTION PROCEDURE (also see Appendix 3)

Samples for culture must be collected by or under the supervision of a licensed veterinarian
using the "triple sample method ." This method consists of obtaining three samples from the
trunk on separate days. Ifpossible, collect samples within a seven-day period. Do not pool
samples. Samples should be taken after water has been withheld for at least two hours to reduce
sample dilution and contamination. Light exercise prior to collection may facilitate obtaining
secretions from lower in the respiratory tract, which is desirable. Of the foUowing methods, the
trunk wash with bag seems to provide the most effective way to collect samples at this time.
Samples collected by swab are Dot acceptable. As there is a risk of human exposure to sputum
produced during this procedure, personal protective measures are recommended for personnel
during sample collection. These should include gloves and HEPA-filt.er masks certified by the
National Institute for Occupational Safety and Health CNIOSH) to protect against TB (see
Employee Health and Safety) .
A. Trunk wash with bag (or other suitable container) - Using a catheter tip syringe, instill 60
ml sterile saline into the trunk. Raise the trunk as high as possible to distribute the fluid deeper
into the trunk. Lower the trunk and place a clean, one-gallon plastic bag over the end of the trunk
and hold in place until the elephant exhales into the bag. Transfer at least 20 ml of the sample to
a sterile leak proof, screw-top container. Sterile 50-ml conical screw-top plastic centrifuge tubes
are preferred and are available free of charge from the National Veterinary Services Laboratories
(NVSL) - call 515-337-7388 .
B. Trunk wash - Using a 14 French feeding tube, introduce 60 rol of sterile saline into the trunk
then aspirate. Transfer at least 20 rol of the sample into sterile leak proof, screw-top container.
Methods A and C are preferable to this method.

C. Forcible exhalation - Mucous collected without instilling saline into the trunk is acceptable
if elephants are trained to forcibly exhale into a clean plastic collection bag and the volume
collected is at least 20 ml. This may allow sampling of secretions from other areas of the
respiratory tract and may be a preferable sample. Transfer the sample into sterile, leak proof,
plastic screw-top container.

Elephant TB Guidelines 8

Storage
Do not expose samples to sunlight or heat. Consult receiving laboratory to determine whether
samples should be refrigerated or frozen prior to shipment. For those laboratories that
recommend freezing (i.e. NVSL) freeze samples as soon as possible after collection and keep
frozen until shipment. Freeze at -20C (conventional freezer). As standard frost-free freezers
undergo cyclic freeze-thaws to limit frost, freezers that do not have this feature are preferred.
Freezing at -80 cC (ultra-low temperature freezer) is also acceptable. Frozen samples must be
shipped within 2 weeks of sample collection to the testing lab.

Packaging and Shipping

All three refrigerated or frozen samples may be submitted together. Label containers with the
animal ID and date of collection and put the same information on the submisslon form. Place
screw-top containers in double zip-lock bags. Do not send samples in glass containers or
packaged only in plastic bags. Sterile 50-ml conical plastic centrifuge tubes with lids sealed
with parafilm or electrical tape are preferred.
Place samples on ice packs or dry ice and ship overnight via Federal Express, Airborne, or other
overnight carrier. Do not ship by U.S. mail as samples may be irradiated which will render
them unacceptable. Packaging and shipping should be in accordance with the International Civil
A viation Organization Technical Instructions for the Safe Transport of Dangerous Goods by Air
2009-2010 (http://www.icao.int/icaonetldcs/9284.html). Also helpful is the 2007 WHO
document "Guidance on Regulations for the Transport oflnfectious Substances
O1ttp:l/www.who. inUcsr/rcsourcesJpu b! icationslbio safety/\\' H 0 CDS EP R 2007 2ee .pd 0
Packaging and shipping samples and cultures should be in accordance with Department of
Transportation regulations - 49 CFR Parts 171, 172, 173 and 175- Hazardous Materials :
Infectious Substances; Harmonization with the United Nations Recorrunendations; Final Rule,
published June 2,2006 in the Fedcrnl Register.
http://i su 1. indstate. cdu/tercli n fecti ousm aterial/ pd flHaza rdou s%2 0Materi a!s In fectious%20su bsta
nccs%20hannonizatlon%20with%20the% 20UN%20final%20rule.pdf
hltp:l/ec fr .gpoaccess. gOY /cgi/t/tex tltextidx.?c=ecfr&rgn=div5&view=text&node=49:2.l.l.3.8&idno=49
Send samples to NVSL or other laboratory facility offering comparable procedures for
identification of mycobacteria species. When submitting samples to NVSL, use VS Fonn 10-4,
Specimen Submission Form. This form is available online in Word or pdf fonnat:
http://www .aphis. usda. gOY /ao i rna I health/lab info serv iceslfonns pub Iicatioos.shtm I.

Request mycobacterial culture with speeies differentiation.

Positive cultures from laboratories that do not have the capability to differentiate M . tuberculosis
complex organisms mU'lt be forwarded to NVSL or other qualified laboratories for speciation.
Culture of mycobacteria requires a minimum of eight weeks . Laboratory reporn that do not
provide a definitive result due to contamination/overgrowth or other causes are considered
invalid. Additional samples should be collected and resubmitted to replace those reported as
contaminated.

Elephant TB Guidelines 9

Note: Other mycobacteria species such as M . avium, M. kansQsii, M. elephantis, and M.

fortuitum have been isolated from elephants. At rms time, there is no substantive evidence that
these organisms are pathogenic for elephants. However, Mycobacterium szu/gai, an unusual
non-tuberculous mycobacterium, has been associated with pathology in elephants (Lacasse
2007).

5. ELEPHANTTB
PROCEDURE

STAT~PAK~

ASSAY SAMPLE COLLECTION

Blood collection for the Guideline-required ElephantTB STAT -PAK Assay must be witnessed
by a federal or state veterinarian and performed as licensed. It is advisable to also bank a serum
sample. Blood from elephants with reactive ElephantTB STAT -P AKoo Assay results must be
submitted for MAPIA TM /DPP testing to:
Cherubio Diagnostic Systems, Inc.
3661 Horseblock Road
Medford, NY 11763
Tel: 631-924-1135
Fax: 631-924-6033
Email: customerservice@chembio.com
Contact Chembio for shipping instructions.
The USDA veterinarian is responsible for shipping the sample but the owner must pay for
shipping and must contact Chembio to arrange payment for the MAPIATM or DPP@ test.

6. ANCILLARY SCREENING / DIAGNOSTIC TESTS

A number of other ante mortem tests have been under investigation to diagnose TB in elephants.
Following is a summary of those tests and current recommendations for their use.

Intradermal Tuberculin Test

A correlation between the intradennal tuberculin test (skin test) and culture results has not been
established (Mikota 200 1, Lewerin 2005). Therefore, intradeImal tuberculin testing cannot be
deemed reliable for screening or diagnosis and is not recommended.

Enzyme Linked Immunosorbent )-ssay (ELISA)

A multiple antigen ELISA was developed at the Animal Population Health Institute at Colorado
State Uruversity (Larsen 2000). This test was used for detecting dIe presence of elephant serum
antibodies to mycobacteria and investigations showed high sensitivity and specificity for
detecting infected elephants and monitoring elephants over time. However, ELISA testing is not
currently available.

Acid Fast Smears

Acid fast stains of trunk wash smears or other tissue are not reliable indicators of tuberculosis
when used as a sale diagnostic test.

Elephant TB Guidelines 10

7. TB MANAGEMENT GROUPS (1-4)

All elephants will fall into one of four management groups (1-4) based on test results or will be
untested (group 5). A culture positive elephant is defIDed as an elephant from wruch
Mycobactel'ium tuberculosis or Mycobacterium bo vis has been isolated from any body site or
specimen. A culture positive elephant is considered positive until it has met the treatment
requirements as outlined for Group 4. Exposure history bas been incorporated into the
Guidelines as ongoing data collection has indicated that it is an important risk factor. FLow charts
are included in Appendix 9 to iLLustrate the management groups.

GROUP 1: Culture negative; ELephantTB STAT-P~non-reactivej no exposure to

culture positive elephant in past 12 months.
Monitor annually by culture (triple sample method) and ElephantTB STAT-P AKI!i (single serum
sample collected concurrently) .
No treatment or travel restrictions .
No elephant should move into a facility where there is an untested elephant.
If an elephant has had exposure to other untested elephants in the previous 3 months, then a

STAT-PAK test should be repeated in 3 months time to confirm. If the ElephantTB STATP AK@ remains non-reactive, the elephant continues in Group 1.

GROUP 2: Culture negative; ElephaoffB STAT-PAJClI non-reactive; exposure to culture

positive animal within the last 12 months.
Monitor by culture (triple sample method) and ElephantTB STAT -PAK every 3 months for one
year post-exposure, then every 6 months for 2 years, then annually thereafter if all cultures
remain negative and ElephantTB STAT -PAK remains non-reactive.
No travel or public contact until 2 additional non-reactive ElephantTB STA T-P A.K tests

are performed at 3 and 6 months post-exposure (6 month restriction) .

o If non-reactive at 6 months, travel/public contact restrictions removed as long as
additional testing can be performed as outlined above .
If the results during any of the follow-up testing change, the individual elephant will
change group. No elephant should move into a facility where there is an untested
elephant.

Note : The exact time to sem-conversion is unknown.

GROUP 3: Culturenegatwe; ElephantTB STAT-P~ reactive

It is required that blood from elephants with reactive ElephantTB STAT -P AK results be
submitted for MAFIA / DPP testing (see item 5 aboveJ. Based on MAPIA TM /DPP results
and exposure history, the elephant will fall into one of the following subgroups :
A. Culture negativej STAT-PAK reactive, MAPIATM/DPP@ non-reactive, no

known exposure
Monitor by culture (triple sample method) every 3 months for the ftrst year after
becoming ElephantTB STAT-PAK reactive, then every 6 months for the next 2
years. Repeat MAP LA TM /DPP@ every 6 months for the first year if elephant

Elephant TB Guidelines 11

remains STAT-PAK reactive. If all cultures and MAPtAfMfDPP remain

negative/non-reactive during this period, annual testing may resume.
No treatment or travel restrictions.
If the cultuTe becomes positive or MAPIA TM/DPP becomes reactive during any of
the foUow-up testing the individual elephant will change category.
No elephant should move into a facility where there is an untested elephant.

B. Culture negative; STAT -P AK reactive, MAPIA IDPP non-reactive. known

exposure to TB culture positive elephant (00 time limit on exposure history)
Monitor by culture (triple sample method) every 3 months for one year postexposure, then every 6 months for two years then annually thereafter if aU cultures
remain negative. Repeat MAPIA TM/DPP every 6 months for the first 3 years if
elephant remains STAT-PAK reactive. IfaU cultures and MAPIA TM/DPP remain
negative/non-reactive during this period, annual testing may resume after 3 years.
No travel or public contact for fIrst year; if results are unchanged at the first year,
restrictions are removed.
If the culture or MAPTA TM/DPP results change during any of the follow-up testing
and become positive, the individual elephant will change group.

Culture positive elephants that have completed a course of anti-tuberculosis therapy

may remain ElephantTB STAT-P AK reactive and fall into this group . If appropriate
treatment has been documented and approved by USDA, these animals will not have
traveVpublic contact restrictions unless there is a change to positive culture and/or
reactive MAPIA TM/DPP@ results during foHow-up testing.
C. Culture negative; STAT-PAK reactive. MAPIATM/DPP reactive, no known
exposure
Monitor by culture (triple sample method) every 3 months for one year, then every 6
months for life. Repeat MAPIA TM/DPP every 3 months for the first year, then
every 6 months for an additjonal 2 years if elephant remains STAT -P AK reactive.
If all cultures remain negative after 3 years annual serological testing may resume as
described in these guidelines.
No travel or public contact until the first year of testing has been completed.
Treatment should be considered. If serological conversions are demonstrated to be
recent (within me past 12 months then prophylactic treatment can be used. If
serological conversions are longer standing or unknown, then full treatment may be
advisable. Individual cases should be evaluated in conjunction with USDA. If
treatment is perfonned, the elephant may be able to travel and have public contact
after 6 months of successful documented USDA approved treatment.
If the culture or MAPJATM/DPP@ results change during any of the follow-up testing
the individual elephant v,ri1l change group.
Note: The STAT-PAK and MAPIATM IDPP tests have been shaYI'll to be early
indicators of TB infection. Retrospective studies have shown elephants may be
serologically reactive months to years in advance of detection by culture [Greenwald
2009).
D. Culture negative; STAT -PAK reactive, MAP LATM/ DPP reactive, known

Elephant TB Guidelines 12

exposure to TB culture positive elephant (no time limit on exposure history)

Monitor by culture (triple sample method) every 3 months for one year postexposure, then every 6 months for life. Repeat MAPIA TM/DPP every 3 months for
the first year, then every 6 months for an additional 2 years if elephant remains
STAT-PAK reactive. If aU cultures remain negative after 3 years, annual
serological testing may resume as described in these Guidelines.
No travel or public contact until the first year of testing has been completed.
Treatment should be considered. If serological conversions are demonstrated to be
recent (within the past 12 months) then prophylactic treatment can be used. If
serological conversions are longer standing or unknown then full treatment may be
advisable. Individual cases should be evaluated in conjunction with USDA. If
treatment is perfonned, the elephant may be able to travel and have public contact
after 6 months of successful documented USDA approved treatment.
If the culture or MAPIA TM/DPP@ results change during any of the follow-up testing
the individual elephant will change group.
Culture positive elephants that have completed a course of anti-tuberculosis therapy
may remain ElephantTB STAT -PAK reactive and fall into this category. If
appropriate treatment has been documented and approved by USDA, these animals
will not have travel/public contact restrictions unless there is a change in their results
during follow-up testing. It has been shown that the MAPIA TM/DPP@ will decline
and may indicate a response to treatment so on-going annual monitoring with
MAPIA TM/DPP is required for life as changes in MAPIA TM may detect relapse.

Considerations for ElephantTB STAT-PAK reactive elephants.

Elephants may develop antibodies to mycobacterial antigens months to years prior to detection
by culture, however, the time intervals between exposure, seroconversion, and shedding are not
precisely known. Numerous variables such as age, genetics, immune status, nutritional
condition, other concurrent health problems, and other factors influence the development of
disease in an individual animal following exposure to a pathogenic agent. Results of
MAPIA TM/DPP tesring are useful in helping determine potential risk categories as defined
above and determine which animals require more frequent surveillance or should undergo
prophylactic treatment (Greenwald 2009).
There may be a possible association with chronic inflammatory conditions, such as arthritis, in
elephants that are ElephantTB STAT-P AK reactive, but non-reactive on MAFIA TM/DPP and
with no known TB exposure based on a small number of cases. Review history for possible
exposure to a culture positive animal or previous treatment for TB since this may also affect
results. Nonetheless, it is important to monitor these elephants for possible development of
infection and disease. Retrospective analyses of banked serum samples are strongly encouraged
to provide a more complete serological history.
Elephant.<; that are culture negative, ElephantTB STAT-PAK reactive and MAPIATM/DPP@
reactive are at increased risk of either latent or active TB. Factors to consider in the decision to
administer treatment vs. increased monitoring include exposure history, age, whether the
elephant travels, potential exposure of personnel or public, side effects of treatment, concurrent
health problems, etc. Increased monitoring and travel/pUblic contact restrictions is required based

Elephant TB Guidelines 13

on risk. If culture results during any of the follow-up testing become positive, the individual
elephant wj]l move to Category 4.
Consideration should be given to minimizing or eliminating contact with the public tha.t would
result in exposure by contact or aerosol transmission and to providing personal protective
equipment such as a NIOSH certified N95 respirator 1N95 face mask for staff when working in
close proximity to elephants that are under enhanced surveillance. Employees must be respirator
fit tested before they use the N95 respirator.
Based on a history of exposure to a culture positive animal, or other considerations, the attending
veterinarian may elect to administer prophylactic or full treatment after consultation with USDA.
Effective prophylactic therapy is defined as the administration of a specific number of doses of
two anti-TB drugs within a specified time. It must be demonstrated that adequate anti-TB drug
levels are achieved in the blood of the elephant under treatment. Acceptable anti-tuberculosis
drugs include isoniazid (INH), rifanlpin (RIP), pyrazinamide (PZA), ethambutol (ETH), or a
fluoroquinolone such as levofloxacin, moxofloxacin, ciprofloxacin, or enrofloxacin. Isoniazid is
recommended as one of the two drugs if a known exposure case isolate is INH sensitive. PZA
should not be given if M. bovis infection is suspected since this organism is inherently resistant
to PZA.

Prophylactic therapy is for 9 months can be administered using either of the following
schedules:
Prophylactic Treatment Schedule 1 (preferred):
Administer two anti-TB drugs daily for 9 months (270 total doses). The ftrst 60 doses should be
administered within a period of90 days (i.e. no more than 30 days of "refused medication"
should occur. It must be docwnented that the elephant received 270 total doses at a dosage level
sufficient to achieve adequate drug serum levels.
Prophylactic Treatment Schedule 2:
Administer the two anti-TB drugs daily for two months (as above, the first 60 doses should be
administered within a period of 90 days) . Adequate levels of both drugs must be demonstrated
in two serum samples collected approximately two weeks apart. Serum samples should be
collected as soon as the elephant is accepting medication reliably. If acceptable levels (see
below) are not achieved, the dosage should be adjusted and serum levels tested again (two
samples collected approximately two weeks apart) . It must be documented that the elephant
received the first 60 doses at a dosage level sufficient to achieve adequate drug serum. levels.
Once this has been demonstrated, administer the two drugs every other day but at twice the
previous dosage level for an additional 9 months (105 total doses of every other day dosing plus
the initial 60 doses for a total of 165 doses). It is not necessary to repeat serum drug levels when
changing to the every other day schedule.
Note: Pyridoxine 50 mg is administered to humans receiving INH for treatment of active or
latent tuberculosis to prevent the development of peripheral neuropathy. Although this side
effect has not been reported in elephants, it may be possible. At the discretion of the attending
veterinarian, Vitamin B6 (pyridoxine) can be given prophylactically at a dose of 0.8-1 mglkg

Elephant TB Guidelines 14

daily.
Concomitant use ofINH, rifampin, and PZA with other hepatotoxic drugs should be done with
caution.
Refer to TB Drugs section for starting dosages, routes of administration, side effects, blood
levels, and other information.
Monitoring of Prophylactically Treated Elephants
During the 9 months of treatment, elephants should be closely observed for changes in appetite,
behavior, and any other signs that may be attributable to adverse drug effects . Monthly blood
tests (CBC and serum chemistry profile) are recommended to monitor general bealth and
possible drug effects on the liver. Liver tests (AST, ALT, LDH, bile acids, and bilirubin) should
be included in the serum chemistry panel. Isoniazid may cause hepatitis and anemia. In
addition, leukopenia has occurred in at least one elephant apparently due to JNH toxicity).
GROUP 4: M. tuberculosis complex positive culture
Animals that have had Mycobacterium tuberculosis complex isolated from any sample (sputum,
stool, tissue, etc .) are considered culture positive for TB. A culture positive elephant is defmed
as an elephant from which Mycobacterium tuberculosis complex organism has been isolated
from any body site or specimen..
The ElephantTB STAT~PAK and MAPLA TM/DPP tests must be performed on blood from
culture positive elephants. Serum for MAPIA TM/DPP testing must be submitted regardless of
ElephantTB STAT -P AK resu Its .
Positive cultures must be submitted to NVSL for genotyping.
A culture positive elephant is considered positive until it has met the treatment
requirements as outlined below. These elephants must be separated from the public for the
duration of the treatment period. Separation from previously non-exposed elephants is also
recommended_until treatment is completed. Precautions to safeguard personnel health and safety
should be instituted immediately (see Employee Safety and Health section) . Elephants with
cultures that yield non-tuberculous strains of mycobacteria are not considered infected and are
not a risk to other animals or humans. Options for Category 4 elephants include:
Options:
A. Treatment: This is the preferred option for culture positive elephants whenever possible.
1. If the organism was isolated at a l.aboratory other than NVSL and they do not perform
mycobacterial species differentiation and DNA fingerprinting, the owner must request that the
laboratory submit the isolate to NVSL or other qualified laboratory for mycobacterial species
differentiation and DNA fingerprinting.
2. Antimicrobial sensitivity testing should be perfonned on all positive isolates. Sensitivities
should be requested for the following drugs: isoniazid, rifampin, pyrazinamide, ethambutol,
ciprofloxacin (or other fluoroquinolone), and amikacin. (Antimicrobial susceptibility testing for

Elephant TB Guidelines 15

M tuberculosis complex organisms is now available at NVSL).

3. Perfonn ElephantTB STAT-PAK and MAPIA TM every 3 months during treatment then every
6 months for 2 years then according to the schedule in the group that the elephant falls into posttreatment. Serological monitoring of treated elephants with MAPIA has shown changes that
may indicate successful treatment or recrudescence of infection (Lyashchenko 2006).
4. Beginning with the onset of treatment, cultures should be collected by the triple sample
method every 2 months for the first 6 months of treatment, then every 6 months for the
remainder of the elephant's life. This intensive screening by culture ensures adequate therapy
during the treatment period and after trea1ment has ended to ensure that the anima] does not
revert to a positive culture, which would again pose a risk to animals or humans.
5. Pending antimicrobial susceptibility results, initiate empiric therapy with 3 or 4 of the
following drugs: isoniazid, rifampin, pyrazinamide, and ethambutol or a fluoroquinolone
(moxifloxacin is preferred). Following the human model, initiating empiric treatment with four
drugs is considered "ideal." However, the difficulties associated with training an elephant to
accept medications are aclrnowledged. After determining sensitivities, continue treatment using
one of the following schedules:
Schedule 1 (preferred): Administer 3 drugs to which the isolates are susceptible daily for 2
months. The first 60 doses should be administered within a period of 90 days (i.e. no more than
30 days of "refused medication" should occur). Adequate blood levels of aU 3 drugs must be
demonstrated in two samples collected approximately two weeks apart. Serum samples should
be collected as soon as the elephant is accepting medication reliably. If acceptable levels (see
below) are not achieved, the dosage should be adjusted and serum levels tested again (two
samples collected approximately two weeks apart). It must be demonstrated that the elephant
received the fIrst 60 doses at a dosage level sufficient to achieve adequate drug serum levels.
Trealment is then continued daily for an additional 10 months with 2 drugs to which the isolate is
susceptible for a total nwnber of doses (with two drugs) of 300. As above, the inclusion ofINH
is recommended. The total number of doses for the entire treatment is 360. The entire treatment
should be completed within 15 months (this allows for "refused medicine" days and periods of
interruption that may be needed if side effects are noted).
Schedule 2: Administer 3 drugs to which the isolate is susceptible for 2 months. The first 60
doses should be administered within a period of 90 days (i.e. no more than 30 days of "refused
medication" should occur). Adequate levels of all drugs must be demonstrated in two samples
collected approximately 2 weeks apart. Serum samples should be collected as soon as the
elephant is accepting medication reliably. If acceptable levels (see below) are not achieved. the
dosage should be adjusted and serum levels tested again (two samples collected approximately
two weeks apart). It must be demonstrated that the elephant received the first 60 doses at a
dosage level sufficient to achieve adequate drug serum levels. Continue treatment with two
drugs at twice the dosage used in the initial period every other day for 10 months (150 doses). It
is not necessary to repeat serum drug levels. The total number of doses is 210. The entire
treatment should be completed within 15 months (this allows for "refused medicine" days and
periods of interruption that may be needed if side effects are noted). Animals that have not

Elephant TB Guidelines 16

completed treatment are considered as non-treated.

Note: Peripheral neuropathy can sometimes occur in humans receiving INH. Although this side
effect has not been reported in elephants, it may be possible. At the discretion of the attending
veterinarian, Vitamin B6 (pyridoxine) can be given prophylactically at a dose of 1 rog/kg daily.

Travel: Elephants in Group 4 should not travel or have public contact (direct or indirect) until
treatment is completed according ta the guidelines.

Additional Monitoring of Treated Elephants

Elephants should be closely observed for changes in appetite, behavior, and any other signs that
may be attributable to adverse drug effects. Monthly blood tests (CBC and serum chemistry
profile) are recommended to monitor general heaJth and possible drug effects on the liver. Liver
tests (AST, ALT, LDH, bile acids, and bilirubin) should be included in the serum chemistry
panel. Isoniazid may cause liver damage and anemia. In addition , leukopenta has occurred in at
least one elephant apparently due to INH toxicity).
B. Quarantine without treatment: This option may be considered especially for animals that
are already housed alone and not considered a good candidate for treatment (ex. bull elephant).
Additional precautions must be taken for human safety (such as the use ofN-95 masks, gloves,
etc). Quarantined elephants should be kept out of range from non-infected animals and should
be monitored fOT signs of TB disease.
No travel is permitted.
No public contact that would result in exposure by contact or aerosol transmission is
permitted.
No exposure to other elephants is permitted.
Additional testing (trunk wash culture, ElepbantTB STAT-PAK@ft.AAPIATM/DPP@),
ancillary tests and nucleic acid amplification are recommended for data collection.
C. Euthanasia: Tbis option may be considered for those animals that are showing clinical signs
considered to be poor candidates for treatment, or for other factors based on the clinician's
discretion. A thorough postmortem examination must be performed (see section II).

Group 5: Untested If an elephant cannot complete procedures as outlined far official annual
testing, it should not be permitted to have public contact that would result in exposure by contact
or aerosol transmission, or contact with other tested elephants (or their enclosures or equipment).
Untested elephants should not be moved from their home facilities. A tested elephant should not
move into a facility housing an untested elephant unless it can be demonstrated that there will be
no direct contact with the untested elephant or with its enclosure or equipment. If a tested
elephant(s) is in contact or housed with an untested elephant, the tested elephant carolOt travel
nor have public contact until the Wltested. elephant is tested unless approved by USDA.

8. PRlNCIPLES OF ANTI-TUBERCULOSlS THERAPY

The American Thoracic Society has published guidelines for the treatment of tuberculosis in
humans (see references). In brief, it is necessary to treat active TB with multiple drugs to

Elephant TB Guidelines 17

prevent the emergence ofresistant strains of bacteria. For individuals exposed to TB (positive
skin test), but no signs of active disease (negative chest radiograph, negative sputum cultures),
treatment is typically with a single drug (INH).
The guidelines for the treatment ofTB in elephants are based on the assumption that animals
with known active disease are treated similarly to humans. However, for elephants, the
treatment period has been extended. For a category 3 elephant with negative cultures and
presumed exposure based on positive serologic response, i.e., positive ElephantTB STAT-PAK
(and MAPIA TM), treatment is a "modified" regime - with two drugs for 9 months. Skin testing
is not reliable in elephants . Acid-fast smears are not reliable on elephant trunk washes.
For humans, treatment of primary tuberculosis is to empirically administer 4 first line drugs
while waiting for antimicrobial sensitivity testing. This assures that initial treatment includes at
least 2 drugs to which the organism is susceptible . And, the additional number of antibiotics
results in more rapid clearance of bacteria from the sputum thereby decreasing the public health
risk.
Once susceptihility tests are received, and the sputum has reverted to being smear negative, the
number of drugs is decreased to two first line drugs for the remainder of treatment. When the
index case is known, and the index isolate is known to be susceptible to all anti~mycobacterial
drugs, then initial treatment may be limited to three drugs. However, in the va.st majority of
ca~es the index case is not lrnown with certainty and four drugs are given. Moreover, in regions
or situations when the frequency of resistance exceeds 10%, empiric initial therapy for humans
cons ists 0 f fi ve drugs .
The length of therapy for humans is currently 6 months for active tuberculosis. This includes the
initial period of 3-5 drugs as above and 2-drugs for the remainder of treatment. For individuals
with resistance to a single antibiotic, treatment is extended to 12 months with 2 drugs to which
the organism is susceptible. For individuals infected with multi-drug resistant tuberculosis
(MDR-TB), treatment is for at least 12 months with 2-4 drugs based on the susceptibility pattern
(lower numbers of agents are employed if the isolate is susceptible to INH or rifampin). Because
the long term outcome and efficacy of treatment for TB of non-human species is currently
unknown, treatment of elephants iE s1ructured for a 12-month course.

9. ANTI-TUBERCULOSIS DRUGS
Antituberculous agents are divided into first and second line agents. Fjrst line agents include
isoniazid, rifampin, pyrazinamide, ethambutol, and streptomycin. These are agents with the
greatest activity and the best side effect profiles . Second line agents include those with less
activity and/or greater side effects. Second line agents include capreomycin, ethionamide,
cycloserine, and thiacetazone. The fluoroquinolones (FQ; moxifloxacin, ciprofloxacin,
levofloxacin, and enrofloxacin) wlllie not considered as l't line agents have significant
bactericidal activity against M. tuberculosis. Moreover, published studies report the equivalency
of FQ substitution for ethambutol in the treatment of TB in humans and studies are underway to
investigate FQ use for the treatment of latent TB infection. Linezolid, a drug active against Gram
positive bacteria such as Staphylococcus aureus, MRSA, enterococcus, and VRE has also been

Elephant TB Guidelines 18

shown to have significant activity against M. tuberculosis and has been used successfully in
salvage regimens. Amikacin, an amino glycoside (as is streptomycin), is a mainstay in the
trea1ment of non-tuberculous mycobacterial infection and has been used in salvage regimens
against MDR-TB. Phannacoldnetic studies ofINH, RIP, EMB , and PZA in elephants have been
published (Maslow et aJ. 2005a, Maslow et a1. 2005 b, Zhu et a1. 2005, and Peloquin et al .
2006) .

FIRST LINE AGENTS

Isonicotinicacid hydrazide (Isoniazid, INH)

Mechanism of action: INH acts to inhibit cell wall synthesis through blockage in the mycolic
acid pathway. The specific target enzymes are unknown; however, evidence supports a role for
the catalase enzyme, katG, as modifying INH to an active form . Postulated targets of the
activated form of Thl'H include ketoacyl synthetase and inhA.
Metabolism and excretion: INH is acetylated in the liver through the action of N-acetyltransferase. The acetylated product is then excreted in the urine. Some ethnic groups (Native
Americans, Eskimos, and Orientals) as well as others carry a recessive allele encoding for rapid
acetylation ofINH those results in more rapid clearance and lower bioavailability. 1t is not
known whether elephants are polymorphic in this enzyme and differ in the speed of acetylation.
Toxicity: The major adverse effects documented in humans are hepatitis (principally
hepatocellular inflammation with a transaminitis) and peripheral neuropathy. Uncommon
adverse reactions include headaches, optic neuritis, seizures, psychosis, encephalopathy,
twitching, rashes, and gastrointestinal upset. A histamine like reaction can be observed when
products with tyramine (red wine, cheese) are ingested. Risk factors for hepatic toxicity in
humans include age greater than 35 yr, concomitant viral hepatitis (Hepatitis B or C), and other
hepatic toxins (drugs, alcohol). Vitamin B6 (pyridoxine) is given at a dose of 50 mg daily (-1
mglkg) to prevent the development of peripheral neuropathy.
Toxicity in elephants: Observed toxicities ofINH have included inanition., transarninitis, and
anemia. Fermented products (mash or other feeds) should likely be avoided to minimize
potential histamine reactions. Liver values (SOOT, SGPT, and bilirubin) should be monitored
monthly for 2 months and then bimonthly ifno liver toxicity is observed. INH has caused
irreversible leukopenia in camels; reversible leukopenia has been observed in one elephant that
was considered as possibly / probably related to INH
Route of administration: In humans INH is administered orally. In elephants, lNH is
preferentially administered as an oral bolus. However, rectal absorption is efficient, yielding
levels similar to oral bolus dosing. In bongo antelope, INH has also been successfully
administered via intramuscular injection.

Rifampin (RIF)
Mechanism of action: Rifarnpin is a semi synthetic derivative of rifamycin, an antibiotic
derived from the fungus Streptomyces mediterranei. Rifampin acts to inhibit the DNAdependent. RNA-polymerase thus blocking formation of messenger RNA (the first step in

Elephant TB Guidelines 19

protein synthesis).
Metabolism and excretion: Rifampin is acetylated in the liver. Both the unaltered and
acetylated drug is excreted into the bile. Rifampin is then reabsorbed whereas the acetylated
fonn is not.

Toxicity: The major toxicity of rifampin is hepatitis . Other side effects include gastrointestinal
upset, renal failure, hemolysis, acute renal failure, and thrombocytopenia. It is avoided in
pregnancy during the fIrst trimester because of possible teratogenicity.
Rifampin is also a strong inducer of the cytochrome P450 hepatic enzymes that may increase the
metabolism of concurrently administered drugs. A prime example is exogenously administered
steroids used for in vitro fertilization. For animals being treated for other conditions, potential
drug-drug interactions should be ruled out.

Toxicity in elephants: The toxicity in elephants is unknown. Similar adverse reactions to

humans should be expected. Therefore it is recommended that in addition to liver tests, serum
creatinine, electrolytes and CBC be monitored per the schedule listed for INH.
Route of administration: Rifampin is administered to humans orally although intravenous
administration is used in patients unable to tolerate oral dosing. In elephants rifampin appears to
be absorbed well as an oral bolns although acceptance is low because of the drug's bitterness.
Rifarnpin is not absorbed rectally; there is no known experience with parenteral administration in
elephants or other animals. Urine and feces may become orange colored while on this drug.

Pyrazinamide (PZA)
Mechanism of action: Pyrazinamide is a synthetic antibiotic derived from nicotinic acid. Its
mechanism of action is unknown; however the presence of an intact pyrazinamiciase is required.
Since Mycobacterium bovis lacks this enzyme, it is resistant to PZA.
Toxicity: Toxicities observed in humans include arthralgias and arthritis, hyperuricemia,
hepatitis, gastrointestinal upset, and photosensitivity (skin rashes).
Toxicity in elephants: The toxicity for elephants i~ unknown, however hepatitis may have been
observed. Similar adverse effects as documented for humans should be expected.
Route of administration: In humans, pyrazinamide is administered orally. rn elephants both
oral and rectal dosing have yielded acceptable blood levels. Pyrazinamide has been successfully
administered to bongo antelope via subcutaneous injection.
PZA is should not be given if M bovis infection is suspected since this organism is inherently
resistant to PZA."

Ethambutol (EMB)
Mechanism of action: Ethambutol is a specific inhibitor of the arabinosyl transferase thereby
inhibiting fonnation of arabinogalactose and lipoarabinomannan, which are the dominant lipids

Elephant TB Guidelines 20

in the M tuberculosis cell wall.

Toxicity: The major toxicity of ethambutol is optic neuritis, which may result in decreased
visual acuity, a central scotoma, and loss of red-green discrimination. Ethambutol may also
cause peripheral neuropathy, headache, rashes, art.hralgias, hyperuricemia, and rarely
anaphylaxis.
Toxicity in elephants: The toxicity for elephants is currently unknown.

Route of administration: Ethambutol is administered orally to humans and elephants. Rectal

administration is irritating and poorly tolerated resulting in expUlsion of the drug. Subcutaneous
administration has been given successfully to bongo antelope.

Streptomycin
Mechanism of action: Streptomycin is an amino glycoside antibiotic derived from the fungus

Streptomyces griseus that acts on the 30S ribosome to inhibit protein synthesis.

Toxicity: Similar to other aminoglycosides, streptomycin admirUstration may result in auditoryvestibular and renal toxicity. Specific symptoms include ataxia, vertigo, nerve deafness, and
renal failure. Most symptoms are reversible if the drug is discontinued immediately after their
occurrence.
Toxicity in elephants: The toxicity for elephants is currently unknown but is likely the same as
for humans .

Route of administration: Streptomycin is administered via intramuscular injection to humans.

There is no experience in administering streptomycin to elephants.
SECOND LINE AGENTS

Fluoroquino]ones: Moxifloxacin, Ciprofloxacin, Levofloxacin, Enrofloxacin

Mechanism of action: Fluoroquinolone antibiotics act to inhibit the topoisomerases DNA
gyrase and topoisomerase IV. Both of these enzymes are needed during DNA replication to fir:st
unwind supercoiled DNA and then to again achieve a supercoiled structure of DNA. Of the
commercially available fluoroquinolones, moxifloxacin has the greatest in vitro activity and in
vivo activity in a mouse model of infection followed by ciprofloxacin and levofloxacin
(Neurmberger EL et ai, Moxifloxain-containing regimens of reduced duration produce a stable
cure in murine tuberculosis, Am J Respir Crit Care Med 2004, 170: 1)31-4 ). The antituberculous activity of enrofloxacin, a derivative of ciprofloxacln is unknown. Gatifloxacin also
has excellent in vitro activity against strains of TB, although the drug was recently withdrawn
due to reports of antibiotic associated diarrhea and QT-proJongation. Studies are underway
examimng the role of Moxifloxacin in standard treatment and prophylaxis regimens (BllfTllan et
al. Moxifloxacin versus ethambutol in the fIrst 2 months of treatment for pulmonary tuberculosis.
Am J Respir Crit Care Med 2006, 174: 331-8; Pletz MW et a1. Early bactericidal activity of
rnoxifloxacin in treatment of pulmonary tuberculosis: a prospective, randomized study,

Elephant TB Guidelines 21

Aotimicrob Agents Chemother 2004, 48: 780-2).

Toxicity: The quinolone antibiotics may result in arthropathy, cartilage defects in adolescent
animals, photosensitivity, antibiotic related diarrhea, and electrocardiographic prolongation of
the QT interval.
Toxicity in elephants: The toxicity for elephants is unknown.
Route of administration: These agents are administered either orally or intravenously
(levofloxacin only). Orallevofloxacin has been administered to bongo antelope, although poor
serum levels were observed. Oral levofloxacin has been used to successfully treat a Klebsiella
spp. infection of the hock in a horse. (J Maslow, personal communication). Enrofloxacin has
been used to treat one elephant with disseminated multi-drug resistant TB as part of a multi-drug
regimen. The animal developed photo-induced blepharitis, although this adverse effect had been
episodic during infection and was initially detected prior to the institution of enrofloxacin. Thus,
the causal association to enrofloxacin is unknown.

Amikacin
Mechanism of action: Amikacin is an amiooglycoside antibiotic that acts on the 30S ribosome
to inhibit protein synthesis. Isolates that are resistant to streptomycin may be susceptible to
amikacin.
Toxicity: Similar to other arninoglycosides amikacin administration may result in auditoryvestibular and renal toxicity. Specific symptoms include ataxia, vertigo, nerve deafness, and
renal failure. Most symptoms are reversible if the drug is discontinued immediately after their
occurrence.
Toxicity in elephants: The toxicity for elephants is currently unknown but is likely the same as
for humans.
Route of administration: Amikacin is administered via intravenous injection to humans.
Amikacin has been administered via intramuscular injection to bongo antelope yielding
acceptable serum levels (unpUblished). A pharmacokinetic study of amikacin in African
elephants has been conducted (Lodwick, LJ., Dubach., 1.M. and Phillips, L.G., 1994.
Pharmacokinetics of amikacin in African elephants. J Zoo Anim. Med 25: 367-375). There is
no published infoITJlatlon regarding amikacln in Asian elephants. Amikacin in one Asian
elephant given 1M 3 times a week at 14 mg/kg yielded good blood levels (acceptable levels in
elephants unknown) aod was eliminated ahnost completely from serum within 72 hours .
However, significant toxicity occurred with prolonged use of this drug at this dose (personal
communication, Dr. G Dumonceaux).
Other second line agents have not been used for mycobacterial infections in elephanis.
Clinicians contemplating the use of agents other than those listed should consult with the USDA
on an individual basis .
The fOUI first-line drugs used to treat tuberculosis in humans are isoniazid (INH), rifampin (RlF),

E1ephant TB Guidelines 22

pyrazinamide (PZA) and ethambutol (ErH). Second-line drugs used in cases of drug intolerance
or multi-drug resistant organisms include amikacin and a fluoroquinolone. Both
fluoroquinolones and linezolid have been used in cases of multidrug resistance in humans
(Veziris, N . et al. Fluoroquinolone-containing third-line regimen against Mycobacterium
tuberculosis in vivo. Antimicrob Agents Chemother 2003, 47: 3117 -22) .

10. DOSAGES AND ROUTES OF ADMINISTRATION

Anti TB drugs must be directly administered. Placing drugs over food does not produce reliable
blood levels and this is not an acceptable method of treatment. Drugs vary in palatability and
acceptance so some experimentation may be required to determine a workable regimen for each
individual elephant.
Isoniazid and PZA can be given either orally or rectally. Rifampin and ethambutol should only
be administered orally (effective blood levels of rifampin cannot be achieved with rectal
administration and ethambutol is quickly expelled when given rectally). Below are suggested
starting doses, but actual doses may need to be adjusted in order to achieve adequate blood levels
and I or reduce effects of toxicity.
Drug

Isoniazid

Rifampio
Pyrazinamide
Ethambutol

Dosage
(mglkg)

Route

5
4
4
10
30
30

Oral
Oral
Rectal
Oral only
Oral or rectal
Oral only

Formulation

premixed suspension
Powder
premixed suspension
Powder
Powder
Powder

Target
cone
(I-!g/ml)
3-5
3-5
3-5
8-24
20-60
2-5

Cmax
(hr)

1-2
0.5-1
0.25-0.5
2-4

l-2
1-2

The dosages quoted above are based primarily on the pharmacokinetic studies of drug
administration to the first herds of treated elephants as reported (Maslow et al 2005a., Maslow et
a12005b, Zhu et a12005, Peloquin et aI2006). Recent studies have demonstrated that lNH
achieves Cmax much more quickly than previously thought when administered rectally Dosages
are considered as estimates with the goal of achieving target serum concentrations as listed in
#10 below without causing significant side effects that interrupt treatment. Serum drug levels or
drug side effects may dictate that dosages be adjusted up or down accordingly. Sequential
MAPlA TM tests may also be used to monitor response to treatment (Lyashchenko 2006) . Second
line agents should only be considered and administered following consultation with the facility
USDA inspector.

11. BLOOD LEVELS

Target blood levels for elephants treated with each of the anti-tuberculosis drugs are based on the
experience in humans. Target serum concentrations are listed in the table above. Blood levels
approximating those found in humans have been reported for elephants with each of the four 1st

Elephant TB Guidelines 23

line agents INH, RIP, PZA, and EMB (Maslow et aJ 2005a, Maslow et al 200Sb, Zhu et al 2005,
Peloquin et al 2006).
Blood levels should be determined to measure the maximal concentration of drug (Cmax). While
INR, PZA, and EMB are rapidly absorbed with a Cmax occuning between 1-2 hrs, drug
absorption may vary between elephants and may also vary drug to drug. Recent studies have
demonstrated that lNH achieves Cmax much more quickly than previously thought when
administered rectally. Importantly, the time to Cmax (Tmax) may vary over the course of
treatment due to multiple factors such as food intake, drug acceptance, etc. Thus, at the start of
treatment and periodically through the course of therapy it is important to measure drug levels at
multiple time points until Cmax for each drug and animal is determined.
For INH, PZA, and EMB it is recommended that drug levels be determined at lhr, 1.5hr, and 2
hr and for RIP at 2hr, 3hr, and 4hr except ifINH is administered rectally and then 15 min and)O
min blood levels are recommended to accurately measure the Cmax. Tfthe first measured time
point represents the greatest level for any drug, then Tmax may have already passed and earlier
time points should be assessed. Conversely, if the last measured time point represents the
greatest concentration for any drug, then Tmax may occur later than the range chosen and later
time points mould be assessed. During the initial phase of treatment, time ranges should always
be assessed to detennine the true Tmax .
NOTE: Target blood levels for anti-TB drugs in elephants have not been rigorously established.
Until further studies can be conducted, target blood levels of 80ti-TB drugs for elephants must
necessarily be based on human data. Although achieving blood levels comparable to humans is
the ideal goal, the attending veterinarian should be aware that there is unpublished evidence that
some elephants cannot tolerate anti-TB drugs at the doses required to achieve the above levels .
Isoniazid, in particular, has caused side effects. It may be necessary to reduce the dose of an
anti-TB drug to eliminate side effects, which may result in lower blood levels. The attending
veterinarian should carefully document observed side effects, dosage changes and associated
anti-TB drug levels in these cases. Variations to these Guidelines require consultation with the
facility USDA inspector.

12. POSTI\10RTEM EXAMINATION

It is essential that a post-mortem examination be performed on a1l elephants that die. The
examination must include a thorough search for lesions of tuberculosis regardless of
exposure status. A comprehensive elephant necropsy protocol has been prepared by the
Elephant SSP and is available at these websites:

www.elephanttag.ol1!;
www.eJephantcare.or g
Prior to any planned euthanasia of an elephant, trunk washes, blood for serology and any other
ancillary tests should be performed regardless of whether or not TB is suspected. In this way,
valuable data can be gathered to evaluate the efficacy of the current testing protocol. In the event
of a sudden death, collect post-mortem blood and separate serum for other tests.

Elephant TB Guidelines 24

It is recommended that a trained veterinary pathologist direct the necropsy if possible. 10 the
event of an elephant necropsy (elective or otherwise), contact Dr. Scott Terrell (Elephant SSP
Pathology Advisor) for further instructions and possible participation:

Scott P. Terrell, DVM, Diplomate ACVP, SSP Pathology Advisor, Disney's Animal Kingdom,
1200 N Savannah Circle, Bay Lake, FL 32830, W (407) 938-2746; H (407) 251-0545; Cell
(321 )229-9363; email Scott. P.Terrell@disney.com
The following infonnation is excerpted from the SSP Elephant Necropsy Protocol:

Prntective equipment for tuberculosis cases - Mandatory

Respiratory protective equipment should be available during any elephant necropsy procedure
regardless of the historical TB testing status oftbe animal. In animals with an unknown, suspect,
or positive TB test history, respiratory protection should be considered mandatory. OSHA
standards (29CFR 19] 0.134) require that "workers present during the performance of high hazard
procedures on individuals (humans) with suspicious or confirmed TB" be given access to
protective respirators (at least N-95 level masks).
Similar precautions should be taken during an elephant necropsy. According to the draft CDC
guidelines for the prevention of transmission of tuberculosis in health care settings, respiratory
protective devices used for protection against M. tuberculosis should meet the following criteria:
1. Particulate filter respirators approved include (N-, R~, or P-95, 99, or 100) disposable
respirators or posltive air pressure respirators (p APRs) with high efficiency filters)
2. Ability to adequately fit wearers who are included in a formal respiratory protection
program with well-fitting respirators such as those with a fit factor of greater than or
equal to 100 for disposable or other balf-mask respirators
3. Ability to fit the different face sizes and characteristics of wearers. This can usually be
met by supplying respirators in at least 3 sizes. P APRs may work better than half-masks
for those persons with facial hair.

Consult these websites for OSHA and CDC guidelines:

1. OSHA TB standards and rules: http://www.osha.goy/SLTC/tuberculosi s/standards.html

2. Guidelines for Preventing the Transmission of Mycobacterium tuberculosis in

Health-Care Settings, 2005:
http://www .cdc .gov/nchstp/tblF ederal_RegisterlNew _ Guidelines/TBl CGuidelines.pdf
Necropsy procedures
All elephants undergoing necropsies should have a careful examination of the tonsillar regions
and submandibular lymph nodes for tuberculous appearing lesions. These lymph nodes may be
more easily visualized following removal of the tongue and laryngeal structures during the
dissection. All lymph nodes should be carefully evaluated for lesions since other sites may also
be infected (ex. reproductive or gastrointestinal tract). Collect any nodes that appear caseous or
granulomatous for mycobacterial and standard bacterial culture (freeze or ultrafreeze), and

Elephant TB Guidelines 25

fixation (in buffered 10% formalin). In addition, search thoracic organs carefully for early stages
ofTB as follows: after removal of the lungs and trachea, locate the bronchial nodes at the
junction of the bronchi from the trachea. Use clean or sterile instruments to section the nodes.
Freeze half of the lymph node and submit for TB culture to NVSL or a laboratory experienced in
mycobacterial culture and identification (even if no lesions are evident). Submit sections in
formalin for histopathology. Carefully palpate the lobes of both lungs from the apices to the
caudal borders to detect any:firm B-B shot to nodular size lesions. Take NUMEROUS (5 or
more) sections of any suspicious lesions . Open the trachea and look for nodules or plaques and
process as above. Regional thoracic and tracheal lymph nodes should also be examined and
processed accordingly. Split the trunk from the tip to its insertion and take samples of any
plaques, nodules or suspicious areas for TB diagnosis as above. Look for and collect possible
extra-thoracic TB lesions, particularly if there is evidence of advanced pulmonary TB.

13. EMPLOYEE HEALTH AND SAFETY

All employees that are in direct contact with eJephants should be tested for TB annually
following established human testing guidelines. New employees should be tested prior to
contact with elephants.

Any employee with a positive intradennal test (i.e. a positive intradermal reaction to purified
protein derivative (PPD) of M tuberculosis) should be evaluated for the possibility of active TB.
It is recommended that health care providers who manifest a positive PPD receive INH
prophylaxis unless there is a contraindication to treatment. Conversely, those declining
treatment are followed yearly with a chest radiograph and clinical evaluation to determine
whether they have developed active disease.
A positive skin test may result from either exposure to M tuberculosis, M. bovis, BeG injection,
or exposure to non-tuberculous strains of mycobacteria. The American Thoracic Society has
published guidelines for the interpretation of intradermal testing . If inoculation with BCG
occurred more than 10 years ago, a positive PPD test should not be considered a reaction due to
BeG, but should instead be considered as positive for exposure to TD.
Employees with acid-fast positive sputum smears should be removed from animal contact until it
is determined whether this represents infection with an organism of the M tu.berculosis camp lex
(M tuberculosis or M. bovis). Treatment guidelines and recommendations for contact with
animals and humans are available through state public health departments . At the present time
there is no known tramfer of non-tuberculous strains of mycobacteria between humans and
animals (or human to human) via aerosolization or any other route and thus, there are no
restrictions placed on animals or humans known to be colonized or infected such organisms.
Any facility housing a known culture-positive (M. tuberculosis complex) animal should develop
a program to protect employees from TB exposure, to include the use of appropriate face masks
(N95 HEPA filtered masks, certified by the National Institute for Occupational Safety and Health
to protect against IB), disinfection procedures, and the use of separate implements for infected
animals. The local public health department should be contacted for further guidelines.

Elephant TB Guidelines 26

Measures to protect staff from infected animals should include the use of respiratory (N95)
HEP A filtered masks during all direct or indirect contact with infected animals, such as cage
cleaning, medication administration, feeding, watering, etc. The facility should contact local
health agencies and should provide additional other protective gear such as gowns, gloves, etc.
No specific precautions are necessary for animals that are culture positive for mycobacteria other
than M. tuberculosis and M. bovis.
Best practices for the safe conduct of work in biomedical and clinical laboratories and animal
facilities in regards to Mycobacterium tuberculosis are listed in the 5th Edition of Biosafety in
Microbiological and Biomedical Laboratories published by the U.S. Department of Health and
Human Services in 2007. http://www .cdc.gov/od/ohsibiosfiv/bmbI5/BMBL 5th Edition .pdf

14. REPORTING
Tuberculosis is a reportable disease. Positive culture results must be reported to the State
Veterinarian and appropriate public health agencies.

15. APPENDICES
APPENDIX 1. REFERENCES CITED AND ADDITIONAL READING
Anon. 2003. Treatment of Tuberculosis. ATS, CDC and Infectious Diseases Society of America,
MMWR 52: No.RR-11 (June 20, 2003) 1-88 .
Auclair, B., Mikota, S.K., Peloquin, C.A., Aguilar, R., Maslow, J.N. 2002. Population
phannacokinetics of antituberculous drugs and treatment of Mycobacterium bovis infection in
bongo antelope (Tragelaphus eurycerus isaaci). J Zoo Wildl Med. Sep; 33(3): 193-203 .
Ball, R.L., Dumonceaux, G., Olsen, J.H., Burton, M.S., Lyashcbenko, K. Comparison oftnmk
wash results matched to mlJltiantigen print immunoassay (MAPlA TM) in a group of captive
Asian elephants (Elephas maximus). 2006. Proceedings American Association of Zoo
Veterinarians. 303 -304.
Centers for Disease Control and Prevention and National Institutes of Health. 2007. Biosafety
in Microbiological and Biomedical Laboratories, 5 th ed. u.s . Goverrunent Printing Office,
Washington, D.C. 143- t47~ http://www.cdc.gov/odJohs/biosfty/bmb15/BMBL 5th Ed ition.pdf
Davis, M. 2001. Mycobacten'um tuberculosis risk for elephant handlers and veterinarians. Appl
Occup Environ Hyg. 16: 350-353.
Greenwald, R., Lyashchenko, 0., Esfandiari, J., Miller, M., Mikota, S., Olsen, J.R., Balt, R.,
Dumonceaux, G., Schmitt, D., Moller, T., Payeur, J.B., Harris, B., Sofranko, D., and Waters,
W.R., Lyashchenko, K. 2009. Highly accurate antibody assays for early and rapid detection of
tuberculosis in African and Asian elephants. Clinical and Vaccine Immunology t6(5): 605-612.

Elephant TB Guidelines 27

Isaac, R. The elephant trunk wash - An update. Elephant Managers Association Annual
Conference. Orlando, Florida. November 9-11, 2001.
Isaza, R, and Ketz, C. A trunk wash technique for the diagnosis of tuberculosis in elephants.
Proceedings ofthe 39th international symposium of the European zoo and wildlife medicine.
Vienna, Austria . May 12-16, 1999. 121-124.
Lacasse, c., Terio, K., Kinsel, MJ., Farina, L.L., Travis, D .A., Greenwald, R., Lyashchenko,
K.P., Miller, M., and Gamble, K. 2007. Two cases of atypical mycobacteriosis caused by
Mycobacterium szulgai associated with mortality in captive African elephants (Loxodonta
a./ricana). J. Zoo Wildl. Med. 38 (1): 101107.
Landolfi, lA., Mikota, S.K., Chosy, 1., Lyashchenko, K.P., Giri, K., Gairhe, K., Teno, K.A.
2010 . Comparison of systemic cytokine levels in Mycobacterium spp. seropositive and
seronegative Asian elephants (Elephas maximus). J Zoo Wildl Med. 41(3): 445-455 .
Landolfi, LA., Schultz, S.A., Mikota, S.K., Teno, K.A. 2009. Development and validation of
cytokine quantitative, real-time RT-PCR assays for characterization of Asian elephant immune
responses. Vet Immunol Immunopathol. Sep 15; 131(1-2): 73-78.
Larsen, R.S., Salman, M.D., Mikota, S.K., Isaza, R., Montali, RJ. and Triantis, J. 2000 .
Evaluation of a multiple-antigen enzyme~linked immunosorbent assay (ELISA) for detection of
Mycobacterium tubercu.losis in captive elephants. J Zoo Wildl Med 31: 291-302.
Lewerin, S.S., Olsson, S.L., Eld, K., Roken, B., Ghebremichael, S., Koivula, T., Kallenius, G.,
and Bolske, G. 2005. Outbreak of Mycobacterium tuberculosis infection among captive Asian
elephants in a Swedish zoo . Vet Rec. 156(6): 171-175.
Lyashchenko, K., Singh, M., Colangeli, R.., and Gennaro, M.L. 2000. A multi-antigen print
Lrnmunoassay for the development of serological diagnosis of infectious disease. J ouma! of
Immunological Methods 242: 91-100.
Lyashchenko, K., Miller, M., and Waters, W.R. 2005. Application ofMAPJATM (Multiple
Antigen Print Immunoassay) and rapid lateral flow technology for tuberculosis testing of
elephants. Proe American Association of Zoo Veterinarians, 64-65.
Lyashchenko, KP., Greenwald, R., Esfandiari, J., Olsen, J.H., Ball, R., Dumonceaux, G.,
Dunker, F., Buckley, c., Richard, M., Murray, S., Payeur, J.B., Andersen, P., Pollock, J.M.,
Mikota, S., Miller, M., Sofranko, D., and Waters, W.R. 2006. Tuberculosis in elephants:
antibody responses to defined antigens of Mycobacterium tuberculosis, potential for early
diagnosis, and monitoring of treatment. Clin Vaccine Immunol 13(7): 722-732.
Maslow, J.N., Mikota, S.K., Zhu, M., Isaza, R., Peddie, L.R. , Dunker, F., Peddie, 1., Riddle, H,
and Peloquin, C .A. 2005. Population pharmacokinetics of isoruazid in the treatment of
Mycobacterium tuberculosis among Asian and African elephants (Elephas maximus and
Loxodonta africana). J Vet Pharmacal Ther. 28(1): 21-27.

Elephant TB Guidelines 28

Maslow, 1.N., Mikota, S.K., Zhu, M., Riddle, H., and Peloquin, C.A. 2005. Pharmacokinetics of
ethambutol (EMB) in elephants. J Vet Pharmacol Ther. 28: 321-323 .
Maslow, 1. Tuberculosis and other mycobacteria as zoonoses. 1997. Proceedings American
Association of Zoo Veterinarians. 110-115.
Micha.lak, K., Austin, c., Diesel, S., Bacon, J. M., Zimmennan, P., and Maslow, J. N. 1998.
Mycohacterium tuberculosis infection as a zoonotic disease: Transmission between humans and
elephants. Emerging Infect. Dis. 4: 283-287.
Mikota,S.K., Dumonceaux,G., Miller,M., Gairhe,K., Giri,K., Cheeran,J.V., Abraham,D.,
Lyashchenko,K., Larsen,S., Payeur,]., Waters,R., Kaufman,G. 2006. Tuberculosis in elephants:
An update on diagnosis and treatment; implications for control in range countries. Proceedings
International Elephant Conservation and Research Symposium, 109-118.
Mikota, S.K., Mi11er, M., Dumonceaux, G., Giri, K., Gairhe, K., Hamilton, K., Paudel, S.,
Vincent, B. Elephant tuberculosis diagnosis: implications for elephant management in Asian
range countries. 2006. Proceedings American Association of Zoo Veterinarians. 142-143.
Mikota, S.K., Peddie, L., Peddie, J., Isaza, R., Dunker, F., West, G., Lindsay, W., Larsen, RS.,
Salman, M.D., ChatteIjee, D., Payeur, J., Whipple, D., Thoen, c., Davis, S., Sedgwick, c.,
Montali, R.J., Ziccardi, M., and Maslow, 1. 2001. Epidemiology and diagnosis of
Mycobacterium tuberculosis in captive Asian elephants (Elephas maximus). J. Zoo Wildl. Med.
32: 1-16.
Mikota, S.K., Larsen, R.S., and Montali, R.I. 2000. Tuberculosis in elephants in North
American. Zoo Biology 19: 393 -403.
MoHer, 1., Roken, B.O., Lewerin, S.S., Lyashchenko, K., 2006 . The elephant Rapid Test (RT)
the future diagnostic test for TB (M. tuberculosis) in elephants? Call for a validation study in
Europe. Proceedings International Elephant Conservation and Research Symposium 119-124.
Moller,T., Roken,B., Petersson,L., Vitaud.,C., Lyashchenko,K.. 2005. Preliminary results of a
new serological test for detection ofTB-infection (Mycobacterium tuberculoSis) in elephants
(Elephas maximus and Loxodonta africanum) - Swedish Case studtes. Verh.ber.Erkrg .Zootiere.
42,173-181.
Montali, R.J., Mikota, S.K., and Cheng, LJ. 2001. Mycobacterium tuberculosis in zoo and
wild1ife species. Revue Scientifique et Technique Office International des Epizooties 20(1):
291-303.
Montaii, R.J., Spelman, L.H., Cambre, RC., Chatterjee, D. and Mikota, S.K. Factors influencing
interpretation of indirect testing methods for tubercuJosis in elephants . 1999. Proc . Amer.
Assoc . Zoo Vet. 109-112.
Murphree, R., Dunn , J .R, Warkentin, I.V., SChaffner, W., and Jones, 1.F. 2010 . Mycobacterium
tuberculosis infection among employees of an elephant refuge. 2010 National Tuberculosis

Elephant TB Guidelines 29

Conference, 06/22-6/24, 2010>Atlanta, GA.

National Association of State Public Health Veter:irurr.ians (NASPHV), Veterinary Infection
Control Committee. 2006. Compendium of veterinary standard precautions : Zoonotic disease
prevention in veterinary personnel.
http://www .nasphv .org/Docliments/VeterinaryPrecautions.pdf
Oh, P., Granich, R, Scott, J., Sun, B ., Josepb, M., Stringfield, C , Thisdell, S., Staley, J.,
Workman-Malcolm, D., Borenstein, L., Lehnkering, E., Ryan, P., Soukup, J., Nitta, A., Flood, J.,
2002. HUman exposure following Mycobacterium tuberculosis infection of multiple animal
species in a Metropolitan Zoo . Emerg Infect Dis 8: 1290-1293.
Payeur, J.B., Jarnagin, J.L., Marquardt, lG., and Whipple, D.L. 2002 . Mycobacterial isolations
in captive elephants in the United States. Ann NY Acad Sci 969: 256-258 .
Peloquin, CA., Maslow, J.N., Mikota, S.K. , Forrest, A., Dunker, F., Isaza, R. , Peddie, L.R.,
Peddie, J., and Zhu, M. 2006 . Dose selection and pharmacokinetics ofrifampin in elephants for
the treatment of tuberculosis. J Vet Pharmacol Ther. 29: 581-586.
Peloquin, CA. 2003. Clinical pharmacology of the anti-tuberculosis drugs. rn Davies, P.D .O.
(Editor) . Clinical Tuberculosis. London. England. Arnold Publishers, 171-190.
Peloquin, c.A. 2002. Therapeutic drug monitoring in the treatment of tuberculosis. Drugs
62(15): 2169-2183 .
Peloquin, C.A. 1997. Using therapeutic drug monitoring to dose the antimycobacteriai drugs.
Clinics in Chest Medicine 18: 79-97.
Ryan, C.P. 1997. Tuberculosis in circus elephants . Pulse Southern California Veterinary Medical
Assoc. 8.
S iegel JD, Rhinehart E, Jackson M, Chiarello L, and the Hea1thcare Infection Control Practices
Advisory Committee, 2007 Guideline for Isolation Precautions: Preventing Transmission of
Infectious Agents in Healthcare Settings, June 2007.
http://wwv/.cdc.gov/ncidoct/dhgp/pdf/iso\ation2007.pdf
Zhu, M., Maslow, J.N., Mikota, S.K" Isaza., R., Dunker, F., and Peloquin, c.A. 2005 . Population
phannacokinetics of pyrazinamide in elephants. J oumal of Veterinary Pharmacology and
Therapeutics 28: 403-409.

Elephant TB Guidelines 30

APPENDIX 2. ACKNOWLEDGMENTS
The following individuals have contributed to the historical development of these
GuideUnes:
Dr. Wilbur Amand, Director Emeritus American Association of Zoo Veterinarians
Dr. Miava Binkley, USDA, Animal Care
Dr. Genevieve Dumonceaux, Florida Aquarium
Dr. Freeland Dunker, Steinhart Aquarium
Dr. Murray Fowler, University of California., Davis
Dr. Werner Heuschele, San Diego Zoo (in memoriuro)
Dr. R.amiro Isaza, University of Florida - Gainesville
Dr. Barbara Kahn, USDA, APHIS, Animal Care
Dr. Scott Larsen, University of California, Davis
Dr. William A. Lindsey, Feld Inc.
Dr. Konstantin Lyashchenko, Chembio Diagnostic Systems, Inc.
Dr. Joel Maslow, University of Pennsylvania
Dr. Bob Meyer, USDA, APHIS, Veterinary Services
Dr. Susan K. Mikota, Elephant Care International
Dr. Richard Montali,
Dr. C. Douglas Page, Jacksonville Zoo
Dr. Linda Peddie and Dr . James Peddie, America's Teaching Zoo, Moorpark College
Dr. Mo Salman, Colorado State University
Dr. Dennis Schmitt., Feld Inc.
Dr. Scott Terrell. Disney' s Animal Programs
Dr. Dominic Travis, Lincoln Park Zoo
Dr. Charles Thoen, Iowa State University
Dr. Gary West, San Antonio Zoo
Ms. Diana Whipple, USDA, ARS, National Animal Disease Center
Dr. Michael Ziccardi, University of California, Davis

The following individuals are members of the U.S .Animal Health Association TB Scientific
Su bcommittee:
Dr. Chuck Massengill, Missouri Department of Agriculture
Dr. Susan K. Mikota, Elephant Care International
Dr. Michele Miller, Palm Beach Zoo
Dr. Kathy Orloski, USDA, APillS, Veterinary Services
Dr. Janet B . Payeur, USDA, APHIS, National Veterinary Services Laboratories
Dr. W. Ray Waters, USDA, ARS, National Animal Disease Center

The following individuals have contributed to the 2019 Guidelines:

Dr. Joel Maslow, University of Pennsylvania
Dr. Denise Sofranko , USDA (regulatory advisor only)

Elephant TB Guidelines 31

APPENDIX 3. A TRUNK WASH TECHNIQUE FOR THE DIAGNOSIS OF

TUBERCULOSIS IN ELEPHANTS
Ramiro Isaza, DVM, MS and Cornelia Ketz, DVM

Summary
A trunk wash is a practical method of collecting a sample from an elephant's distal respiratory

tract for Mycobacterium culture and is the technique recommended in the "Guidelines for the
Control of Tuberculosis in Elephants" by the National Tuberculosis Working Group for Zoo and
Wildlife Species. The procedure, however, is potentially dangerous to the handlers and requires
cooperation of the elephant. Because of the limitations of using culture results as a screening test,
the trunk wash results should be interpreted with care. A positive culture result identifies an
elephant that is shedding tuberculosis organisms whereas a negative result is non-diagnostic.

Introduction
Tuberculosis in Asian elephants (Elephas maximus) has been sporadically reported in the literature
for many years (1, 2). The isolation of Mycobacterium tuberculosis from elephants in the United
States has resulted in the development of the "Guidelines for the Control of Tuberculosis in
Elephants" by the National Tuberculosis Working Group for Zoo and Wildlife Species
(http://www .aphis. usda. govfacIE I ephTBGuidelines2000. htrnl). Compliance wi th this po Iicy
requires that all elephants have annual mycobacterial cultures. In these guidelines, the trunk wash
is recommended as the most practical method of obtaining a culture sample from an elephant. This
paper describes the trunk wash technique as the authors are currently using it.

Materials and methods

The trunk wash technique requires that the elephant allow the handlers to restrain and manipulate
the tip of trunk. This is difficult in an untrained elephant in that most elephants resent this
manipulation, and the trunk is many times stronger than the combined force of several handlers. It
is therefore important that the animals be trained to present the trunk, aUow gentle manual restraint,
and manipulation of the trunk tip during the collection of the sample. The training period varies
with the individual elephant, the prior behavioral conditioning of the animal, and the skill of the
handlers. In our experience, most animals can be adequately trained for the procedure in 2-4 weeks.
The materials needed for a trunk wash include: Sterile 0.9% saline solution, sterile 60 ml syringe,
I gallon plastic zip lock type bags (heavy duty), and sterile, 50 ml, screw top, plastic jar or
centrifuge tube. As long as attention is given to collecting a clean sample from the distal nasal
passages, the materials and techniques for the sample collection can be modified. For example,
some clinicians prefer to use a 14-gauge red rubber tube feeding tube inserted into the trunk tip
instead of simply flushing the sterile saline into the trunk tip. Another common variation is to use
a sterile plastic container to catch the trunk wash fluid instead of a plastic bag.

Procedure
A routine screening of an elephant should consist of a series of three trunk wash samples collected
on separate days withjn a one-week period. Trunk washings should be collected in the morning
and prior to water being offered to the animal. These recommendations are made in an attempt to
obtain a representative sample of the nasal flora from the previous night, and to avoid the dilution
effect caused by elephants drinking water with their trunks.

Elephant TB Guidelines 32

The elephant's trunk is manually restrained by the handlers so that the tip is held up. The 60 ml
syringe filled with sterile saline is then inserted into one of the nostrils and the saline quickly
flushed into the trunk. The handler then lifts the trunk tip as high as possible to help the fluid flow
as far into the trunk as possible. The I gallon plastic bag is then slipped over the trunk tip and the
tip of the trunk is lowered to allow the fluid to drain. If possible, the elephant is anowed to exhale
into the bag during this collection phase of the procedure. A good sample should retrieve a
significant portion of the saline that was placed into the trunk (about 40 rnl). The sample should
contain visible mucus from the inside of the trunk and often contains dirt and food particles that
are nonnally found inside the trunk . The collection of moderate amounts of foreign material does
not invalidate the sample. If, however, the collector feels the contamination is excessive, a second
flush may be attempted.
Once the sample is collected in the plastic bag, it is carefully transferred into a labeled container.
Ideally, the sample is refrigerated and sent directly to a laboratory for processing and
mycobacterial culture. If the sample cannot be sent directly for culturing, it may be frozen in a
regular freezer (-20 to -10 0c) until it can be sent to the laboratory. Often the recommended three
daily cultures samples are collected and frozen until all samples are collected and the batch of
samples can be sent to the laboratory together.

Discussion
Identification of a M. tuberculosis infected animal has significant management implications to
both the animal and the collection. Management of the infected animal may require isolation of
the exposed herd, potential removal of the animal from exhibit or shows, and if elected, treatment
of the animals and exposed herd which can be very expensive. In the worst case, a positive
diagnosis may lead to euthanasia of the infected animals. For these reasons , the screening test
selected needs to be definitive and have as few false positives as possible. A positive culture of M.
tuberculosis is, therefore, the only diagnostic test result used as a basis for making decis ions in the
guidelines.
The trunk wash as a method of collecting a culture sample from elephants was selected by the
National Tuberculosis Working Group for Zoo and Wildlife Species because it is a practical
method of obtaining a culture sample from a large proportion of the elephant population. The
procedure requires no sedation or undue stress to the animal. Additionally, the procedure requires
no specialized or expensive equipment.
An important consideration of this procedure is that it can potentially be very dangerous to the
handlers. This is particularly true when attempted on an uncooperative elephant, because any
attempts to manually restrain the trunk in an uncooperative elephant can lead to injury. The time
spent training the elephant to accept this method will greatly increase the efficiency and safety of
the procedure. In some cases, with potentially dangerous or unpredictable animals, an increased
level of handler safety can be obtained by having the animal lie in sternal or lateral recumbency
prior to sample collection. This technique does not guarantee safety or successful sample
collection, as it still requires cooperation of the animal and does not replace adequate training. In
the case of elephants managed under protective contact, the anima l's trunk can be handled though
a set of bars. This method still requires that the animal is fully cooperative and, therefore, usually

Elephant TB Guidelines 33

requires extensive training prior to the collection.

A second safety issue is the potential for zoonotic infection. Recently there has been
documentation of a zoonotic transmission of tuberculosis between humans and elephants (3).
During the collection of the trunk wash sample, there is exposure to aerosolized mucus from the
elephant's respiratory tract. The authors, therefore, suggest that the collectors and handlers wear
protective gear during the collection process. Minimal precautions would include a well fitted
respirator or face mask capable of filtering 0.3 micron particles, disposable gloves, and working
in a well-ventilated, sunlit, area.
Mycobacterial culture as the primary method of detecting infected animals has several limitations
that are best illustrated by examination of the underlying biological assumptions. The ftrst
assumption is that most infected elephants have respiratory infections. Although the literature
suggests that most infected elephants have respiratory infection, there have been no comprehensive
necropsy studies to conflrm these observations. The second assumption is that most infected
animals shed mycobacterial organisms into the respiratory tract. There is little data that determines
if and when an infected animal will begin shedding organisms . It is unknown what proportion of
elephants can carry latent or "walled off" infections that would be missed with culturing
techniques . A third assumption is that animals that are shedding will pass mycobacteria organisms
at least once in the three-day testing period. Currently it is unknown if shedding animals pass
organisms periodically or continuously. Finally, the samples collected from the distal trunk are
often contaminated with normal bacterial flora and foreign material. It is assumed that these
contaminants do oat routinely overgrow or mask the growth ofpathogeruc mycobacteria., although
00 studies have tested this assumption. The interpretations of the culture results should, therefore,
be limited. A positive culture is strong evidence that the animal is shedding mycobacteria and is
infected; negative culture results provide little information as to whether the elephant is infected
or not.
Culturing the distal trunks of all the animals in a population will only detect animals shedding
tuberculosis through the trunk, and not detect all animals that are infected. However, with time
and repeated cultures of all animals in the population, it may be possible to detect and treat most
of the elephants shedding infectious organisms. If these animals are then treated properly and
shedding of organisms stops, the spread of tuberculosis from elephant to elephant should decrease
in the population.

References
1. Mikota, S., Sargent, E.L., Ranglack., G.S. Medical Management of Elephants. West
Bloomfield, MI, Indira Publishing House. 1994.
2 . Mikota, S.K., Peddie., L., Peddie, J., Isaza, R., Dunker, F., West, G ., Lindsay, W., Larsen,
R.S., Salman, M.D., ChatteIjee, D., Payeur, J., Whipple, D ., Thoen, c., Davis, D.S ., Sedgwick,
c., Montali, R.J., Ziccardi, M., and Maslow, I. Epidemiology and diagnosis of Mycobacterium
tuberculosis in captive Asian elephants (Elephas maximus). J Zoo Wild) Med 2001 Mar. 32 (1):
1-16.
3. Michalak, K., Austin, C., Diesel, S., Bacon, I .M., Zimmerman, P., and Maslow, J.N.
Mycobacterium tuberculosis infection as a zoonotic disease: Transmission between humans and
elephants. Emerg Infect Dis. 1998. Apr-Jun 4(2): 283-287.

Elephant TB Guidelines 34

APPENDIX 4. TESTING LABORATORIES

CULTURES, ANTIMICROBIAL SENSITIVITY, GENOTYPING
USDA APHIS VS
National Veterinary Services Laboratories (NVSL)
1920 Dayton Avenue
Ames, LA 50010
Lab web site: hnp:/lwww.aphis.usda.gov/animal health/lab info serviccsldiagnos lests .shtmJ

Dr. ,Tanet Payeur

Scientific Outreach Coordinator
(515) 337-7003 Fax: (515) 337-7397
Email: .Jan~t.B.Payeur@aphis.usda.gov

Dr. Beth Harris

Head, Mycobacteria and Brucella Section
(515) 337-7362 Fax: (515) 337-7315
Email: Beth.N. Harris@laphis.usda.gov

Dr. Suelee Robbe-Austerman

Veterinarian, Mycobacteria and BruceUa Section
(515) 337-7837 Fax: (515) 337-7315
Email: Suelee.Robbe-Austerrnan(m,aphis.usda.gov
*

Send trunk washes to NVSL either frozen or on icepacks by overnight express (Federal
E;q>ress handles diagnostic samples). Containers should be leak proof and double-bagged (50 ml
conical screw-top centrifuge tubes are preferred) and are available free of charge from NVSL.
If lesions are submit1ed for culture, tissues should be frozen and sent on ice packs
-overnight. Lesioned tissues should be split and Yl should be sent to the histopathology lab so
peR can be run to see if the tissne is compatible for tuberculosis. There is no charge fOT
histopathology on lesioned tissue.

Use the VS Form 10-4 for submission, not the VS 6-35 form found in the TB kit. If the
forma1ized tissue is sent separately from the frozen tissue, please indicate on the submission
forms that there are 2 separate packages coming from the same animal so that the reports
can be combined and accession numbers coordinated when they reach NVSL. It is also
helpful to call or email NVSL contacts when sending TB suspects to schedule testing and relay
any relevant history of the case.
NVSL Trunk wash cost: $98 per sample for processing which includes a Oen Probe DNA
probe on any isolate. If the sample is positive for mycobacteria and speciation is requested, the
charge is $122.00 per sample which includes biochemical analysis, 165 rDNA sequencing
analysis, spoliotyping and VNTR genotyping. DNA fingerprinting of M tuberculosis or M bovis
isolates is also available. Antimicrobial susceptibility testing is available for M. tuberculosis

Elephant TB Guidelines 35

complex organisms for $112.00 per isolate. Please contact NVSL at (SI5) 337-7388 for test
schedule.
To establish an account at NVSL for billing, contact Connie Osmundson (515) 337-7571
Email: Coonie.J.Osmundson@aphis.usda.gov .

Of

(User fees as of October 1, 2010). Call lab before shipping samples for current prices and
schedule of testing or check prices at the NVSL web site:
http://www.aghis.usda.goY/animal health/lab info serv ices/diagnos tests.sht1l11
Mycobacteriology Lahoratory at National Jewish Medical aod Research Center
National Jewish Medical and Research Center
Director: Leonid H eifets. M.D.
1400 Jackson St.
Denver, CO 80206
(303) 398-1384
E-mail: heifets l@nic.org
For price list, shipping instructions, and requisition form:
hnp: //www.national jewish.org/research/clinical-labs/aboutlleam/mvcobac/index.aspx
Serum sample submission: it is important to protect the samples from light by wrapping the tubes
in tinfoil and to separate the serum and freeze it without delay, transferring the serum to a tube or
cryovial that is also wrapped in tin foil. Samples should be sent on dry ice as well.

mSTOPATHOLOGY
Scott P. Terrell, DVM, Diplomate ACVP
SSP Pathology Advisor
Disney's Animal Kingdom
1200 N Savannah Circle
Bay Lake, FL 32830
W (407) 938-2746;
H (407) 251-0545;
Cell (321) 229-9363;
Email Scott.P.TerreJI@disney.CDm
Send sections in formalin of any gross lesion and complete set of tissues including lung, liver,
spleen, mesenteric lymph nodes, bronchial lymph nodes and other major organs. Use leak proof
container.

USDA APIDS NVSL Pathobiology Laboratory

1920 Dayton Avenue
Ames, JA 50010
(515) 337-7521
Fax (515) 337-7527
Lab web site: http://www.aphis.usda.goY/animal health/lab info services/dia..,anos tesls. shtml

Elephant TB Guidelines 36

Dr. Art Davis

Director ofPathobiology Laboratory
(515) 337~7526
Email: Arthur.J .Davis@aphis.usda.gov

Dr. Mark HaU

Head Pathological Investigations
(515) 337-7927
Email : Mark.Hall@aphis.usda.gov
Send fonnalin sections of any gross lesion and target tissues (lung, liver, mesenteric and
bronchial lymph nodes). Use leak proof container. Please indicate on submission form if a
sample was submitted for culture so that tbe testing can be coordinated and results
combined on one form.

ANTI TB DRUG LEVELS

Infectious Diseases Pbarmacokinetics Laboratory (IDPL)
National Jewish Medical and Research Center
1400 Jackson St.
Denver, CO 80206
Web site: http://vvww .nationaljewish .org/research/~ ~J in ical ~labs/about/learn/infectiolLs/i dpl.a~;px
Refer to the above website for specimen handling instructions and to downJoad
Requisition forms.
Infectious Disease Pharmacokinetics Lab, College of Pharmacy, and Emerging Pathogens
Institute
Cbarles Peloquin, Pharm.D.
Professor and Director
University of Florida
1600 SW Archer Rd., Rm P4~33
PO Box 100486
Gainesville, FL 3261 O~0486
Tel: 352-273~6266
Fax: 352~273~6804
Qe log uin@cop. ufl.edu
Call or email for infonnation on sample submission .
The National Veterinary Services Laboratories
USDA APHIS NVSL
1920 Dayton Avenue
Ames, IA 5001 0
Web site: http://www.aphis.usda.gov/animai health/ lab info services/diagnos tests.shtml

Elephant TB Guidelines 37

Dr. David Kinker

Head, Serology Section
515-337-7950

Email: David. R.K inker@aphis. usda.gov

Call before shipping samples for current prices.

Chembio Diagnostic Systems, [nco

3661 Horseblock Road
Medford, NY 11763
Tel: 631-924-1135
Fax: 631-924-6033

Erna il: custom erservice(aJ,chem bio. com

Call Chembio before shipping samples for current prices on veterinary products such as
EJephantTB STAT-P AK, MAPIA TM or DPP.

Elephant TB Guidelines 38

APPENDIX 5. USDA Standard Operating Procedure for Processing Elephant

Trunk Washes for the Isolation of Mycobacteria
United States Department of Agriculture
National Veterinary Services Laboratories
Standard Operating Procedure
Processing Elephant Trunk Washes for the Isolation of Myc<lbacteria
Mention of trademark or proprietary product does not constitute a guarantee or warranty of the
product by USDA and does not imply its approval to the exclusion of other products that may be
suitable.

1.

Purpose

The purpose of this Standard Operating Procedure (SOP) is to describe the procedure for
processing elephant trunk washes for the isolation of Mycobacterium tuberculosis used in the
Mycobacteria and Brucella (ME) section.

Warning: Mycobacterium "ovis, M. tuberculosis and M. avium are pathogenic to humans

and they are a Class In pathogen. All procedures must be performed in a Class II or Class
III biological safety cabinet.
2.

Materials
2.1
2.2

2.3
2.4
2.5

2.6
2.7

50 mJ sterile conical centrifuge tubes

15 ml sterile conical centrifuge tubes
SorvaU RC 3BP refrigerated centrifuge
N-AcetylL cysteine (NALC); Sigma catalog number A-7250
NaOR-Sodium citrate solution; (NVSL #10687)
2.5.1.1 Dissolve 29 gm of sodium citrate dehydrate in 1000 m1 of Super Q
fuO. Dissolve 40 gm of sodium hydroxide pellets in 1000 m1 of
Super Q H20. Combine the 2 solutions and dispense as requested.
Autoclave for 15 minutes at 121C.
Sterile distilled water
lohne's antibiotic mixture (contains vancomycin, amphotercin B, and nalidixic
acid); NVSL #20215
2.7.1 Brain Heart Infusion Broth (NVSL #10009)
18.5 g
2.7.1.1 Combine 37 gm of Difco BRI w/out dextrose (BBL # 250220)
with 1000 rol Super Q H20. Bring to a rolling boil. Dispense as
requested. Autoclave 20 min. at 121C for flasks, 15 min . for
tubes.
1000 m1
2.7.2 Super QH20
2.7.3 Nalidixic Acid (NVSL #40153)
10 ml
2.7.3.1 Mix 10 g of nalidixic acid with 500 ml of distilled H20 . Add lON
NaOH drop by drop until solution clears and QS to 1000 ml. Filter

Elephant TB Guidelines 39

2.8
2.9

2.10
2.11
2.12
2.13
2.14

3.

sterilize solution thru a O.22)lm filter into sterile jug with bell end.
Dispense (wearing gloves), 20 mJ into a 50 ml sterile conical tube.
Caution - chemical is carcinogenic.
10 ml
2 .7.4 Vancomycin (NVSL #40151)
2.7 .4.1 Combine 9.346 gm with 1000 ml of distilled Super Q H20 and mix
weI\. Filter sterilize. Dispense into 50 rol sterile tubes in 20 .5 ml
amounts.
5 m1
2.7.5 Amphotericin B (NVSL #40154)
2.7.5.1 Add 10 ml wann sterile distilled H20 to a 100 mg Amphotericin B
(Fungizooe) . Shake gently until dissolved and dispense as
requested.
2.7.6 Combine BHl broth and Super QfuO . Autoclave for 20 min at 121C.
Cool to 50C. Remove 25 rol of cooled broth and discard. Add Nalidixic
Acid, Vancomycin, and Amphotericin B. Mix wel1. D ispense in tubes
and cover with foil became the Amphotericin B is light sensitive . Store in
-20C freezer. Media is good for 3 months.
3 7C CO~ incubator preferred
Media set-up (one tube of each per sample):
2.9 .1 Middlebrook 7HI0 w/glycerol; NVSL #10941 or BBL Middlebrook and
Cohn 7HIO Agar tubes (BBL #220959).
2.9.2 Middlebrook mIl w/glycerol; NVSL #10942 or BBL Seven H11 Agar
tubes (BBL #221392) or BBL Selective Seven H11 Agar tube (BBL
#297639).
2.9.3 Stonebrinks; NVSL #10451
2.9.4 Mycobactosel L-J medium (BBL #221414)
2.9.5 Bactec l2B medium vial with Panta and Erythromycin (32 ,ug/ml)
I ml tuberculin syringes
5 ml syringes
Slant trays, media tube baskets
Vortex
Sterile swabs

Procedures
3.1

Carefully pour 10 - 12 ml of the trunk wash into a 50 ml conical centrifuge tube.

If there is less than 10 ml, use the entire sample .
3.1.1 At this time, also pour 10 - 12 mJ of sterile distilled water into a 50 mJ
conical centrifuge tube; this sample wiJl be labeled "negative control" and
will be processed the same as the rest of the samples.

3.2

Pour 10 to 12 ml (or whatever is left if < 10 ml) of the remaining trunk wash into
a 15 m1 conical centrifuge tube for storage.
3.2.1 These 15 ml centrifuge tubes are stored at -20 2 0 C for a minimum of 8
weeks or until the bacterial isolation procedure is completed.
3.2.2 Samples from cases in which no isolation was made are retained in a
-20 2 C freezer for at least 6 months .

Elephant TB Guidelines 40

3.3

3.4
3.5

3.6

3.7

3.8

3.2.2.1 Samples that have no isolation and are older than 6 months can be
discarded by the procedure in the current version ofMBSOP0008 .
3.2.3 Samples from cases in which mycobacteria have been isolated wiU be
retained for one year and stored in a -20 0 C freezer.
Allow the 10 - 12 ml of the trunk wash in the 50 ml conical tube to stand
Or
undisturbed for 15 - 20 minutes to allow sediment to settle to the bottom.
Alternately:
Pulse spin the 10 - 12 ml of trunk wash in the 50 ml centrifuge to spin down
excess sediment. This can be accomplished by centrifuging for 1 minute and 40
seconds at 3000RCF, lOoe, using the Sorvall RC 3BP centrifuge.
Slowly pour the supernate, trying not to disturb the sediment, into a sterile 50 rnl
conical centrifuge tube.
Prepare the N-Acetyl-L-cysteine (NALC)lNaOH- sodium citrate solution
according to the following proportions:
Volume (ml)
NaOH- sodium citrate a (mn
NALC (g)
50
0.25
50
100
0.50
100
150
0.75
150
200
200
1.00
300
300
1.50
a. 1: 1 mixture of 4% NaOH to 2.9% sodium citrate
b. Allow NALC to dissolve in solution before use
c. Discard this solution after 24 hours
Add an equal amount of the NALC solution to the trunk wash supernate up to a
maximum of 10 ml using a sterile pipette .
3.6.1 Be careful to avoid cross contamination of samples when adding the
NALC solution.
Vortex or vigorously hand shake for 20 5 seconds
Routinely allow the trunk wash to remain in contact with the NALC for 15 5
minutes.
3.8.1 If the sample is extremely cloudy or appears to be contaminated, the
NALC solution may need to remain in contact with the sample for up to
20 5 minutes.
NALC is a mucolytic agent and this procedure reduces or eliminates
contaminating bacteria while releasing Mycobacteria which may be
trapped in mucin and cells, allowing them to grow. Observe the tube for
clearing before proceeding to the next step.
Add enough sterile distilled water to the NALC/wash solution to fill the
centrifuge tube
Centrifuge the waterlNALC/wash solution for 20 minutes at 6000g and lOoe.
Carefully pour off and discard the supernatant.
Re-suspend the sediment with 2 mls of sterile distilled water and 1 ml of the
Johne 's antibiotic mixture.
3.8.2

3.9
3.10
3.11

3. 12

Elephant TB Guidelines 41

3.13
3.14
3.15

3.16

3.17

3.18

3.19
3.20

3.12.1 Vortex the re-suspended mixture for 20 5 seconds.

Incubate overnight at 37 2C.
Vortex or vigorously hand shake the incubated sample for 20 5 seconds .
Using a sterile swab per specimen, swab the sample over the entire surface of
each of the solid media tubes listed in section 2 of this SOP.
3.15.1 The four solid media tubeR are Middlebrooks 7HIO w/glycerol,
Middlebrooks 7H11 wI glycerol, Mycobactosd LJ, and Stonebrinks.
3.15.2 If one of these media is not available, contact the section head or their
designate for which media to be substituted.
Prepare the BACTEC 12B bottles.
3.16.1 A 5 m.l syringe is u"ed to add 2 ml of Erythromycin to the reconstituting
fluid supplied for the PANT A solution for a final concentration of
321-1g1ml.
3.16. 2 Using a new 5 ml syringe, add 5 ml of the reconstituting fluid to the vial
of PANT A.
3.16.3 Using a 1 ml tuberculin syringe, each vial of the BACTEC media is
inoculated with 0.2ml of the reconstituted supplement.
Inoculate the BACTEC media with your sample.
3.17 .1 Using a 1 ml tuberculin syringe, inoculate the BACTEC media with
:s 0.5 ml of the sample
Place the inoculated solid media on a slant rack and incubate overnight at
37 2C in 10% C02, if available .
3 .18.1 Incubation in an atmosphere of C02 will encourage earlier growth.
3.18.2 After being incubated overnight on a slant rack, the media tubes can be
stored in the 37C incubator in an upright position.
Place the inoculated BACTEC bottles in a locked 37C incubator.
Read the media tubes as outlined below and record the results on the appropriate
worksheets ;
3.20.1 SolidMedia
3.20.1.1
3.20.l.2

3.20.1.3

Read tubes weekly for weeks 1~ 7 after inoculation.

For tubes in week 8, read and record the results and discard
the tubes if there is no suspicious growth for Mycobacteria
noted in the tubes.
For tubes that contain contamination, discard the media
tubes that are overgrown. If the entire set of media is
overgrown, the case may need to be retested.

3.20.2 BACTEC Bottles

3.20.2.1
BACTEC bottles are read twice weekly for the first 3
weeks and then once a week until week 6.
3.20.2.2
Results of the growth indicator values are recorded on the
BACTEC worksheet assigned to the case on the bottle each
time the BACTEC bottle is read by the BACTEC 460
machine.

Elephant TB Guidelines 42

3.20.2.3

4.

BACTEC bottles that have a growth indicator value of 300

or rugher are examined by acid fast staining. See the
current version ofMBSOP2210 for these procedures.
BACTEC bottles that are to be discarded are stored in a
locked cabinet in the biological safety level 3 laboratories
until they are removed by the personnel from
Environmental Health and Safety. See the current version
ofMBSOP2007 and MBSOP2008 for these procedures.

References
4 .1
Della-Latta, P. and 1. Weitzman. 1998. Mycobacteriology. p. 169-204 In H.D.
Isenberg (ed .), Essential Procedures/or Clinical Microbiology. American
Society for Microbiology, Washington, D .C.
4.2

Zimbro, MJ . and O.A. Power. 2003. Difco & BBL Manual- Manual of
Microbiological Culture Media. Becton, Dickinson and Company, Sparks, 1vID.

Elephant TB Guidelines 43

APPENDIX 6. CONTACTS FOR QUESTIONS

DrAGNOSIS AND TREATMENT
Michele A. Miller, DVM, MS, PhD
Chief Veterinary Officer and Director of Conservation Medicine
Palm Beach Zoo
1301 Summit Blvd.
West Palm Beach, FL 33405
Phone: 561-833-7130 ext 224
Cell: 561-727-9630
Email : mmiller@.palmbeachzoo .org

Dr. Genevieve DumoDceaux

The FIOlida Aquarium
701 Channelside Drive
Tampa, Florida 33602
Cell : 813-465-9234
Work: 813-367-4055
Email : gd um on ceauz(dl flaquarium .org

Dr. Susan K. Mikota

Director of Veterinary Programs and Research
Elephant Care International
166 Limo View Lane
Hohenwald, TN 38462
Tel : 931 -796-7102
Cell : 931-628-5962
Email : srnikota@el ephantcare.org
Webs ite: www.eJephantcare .org

ELEPHANT TD STAT~PAJ( ASSAY AND MAPIA

Konstantin Lyashchenko, Ph.D.
Research Director, Mycobacterial Immunology
Chembio Diagnostic Systems, Inc.
3661 Horseblock Road
Medford, NY 11763
Tel: 631-924-1135, exUl1
Fax: 631-924-6033
Email: klvashchenkofa>.chembio.com
REGULATORY

Dr. Denise Sofranko

USDA-APHIS-Animal Care
Field SpeciaJist for Elephants
Voice Mail: 240-461-9142

Elephant TB Guidelines 44

Email: Denise.M. Sofranko@aphis.usda .gov

2150 Centre Avenue
BLDG B Mail Stop #3-Wl1
Ft. Collins, CO 80562-8117

HUMAN HEALTIl, ELEPHANT THERAPY AND TREATMENT

Joel Maslow, MD, PhD
Division of Infectious Diseases
Philadelphia Veterans Affairs Medical Center and the
University of Pennsylvania School of Medicine
University & Woodland Avenues
Philadelphia, PA 19104
Tel: 215-823-4021
Fax : 215-823-5171
Email: joel . ma~lowlal med.va . gov
NECROPSY, PATHOLOGY
Scott p, Terrell, DVM, Diplomate ACVP
SSP Pathology Advisor
Disney's Animal Kingdom
1200 N Savannah Circle
Bay Lake, FL 32830
Tel : 407-938-2746
Home: 407-251-0545
Cell: 32 1-229-9363
Email : Scott.P.T errell@disncy.com

INTERNET
These guidelines are available on the Internet at the following sites:

1. http://v,ww.aphis.usda.gov/animal welfare/index.shtml (available to the public)

2. www.aazv .org (available to AAZV members by password)
3. www.elephantcare.org (available to the public)
4. www .ele12hanttag.org (available to the public)

APPENDIX 7. SOURCES FOR ANTI-TUBERCULOSIS DRUGS

There are various veterinary compounding phannacies that have experience with formulations
for elephants. Please contact one of the consultants in Appendix 6 for information. Select
veterinary compounding phannacies are also listed on w w.elephantcare.org.

Elephant TB Guidelines 45

APPENDIX 8. Elephant Serum Bank S bmission Form

Institution/owner:

-------------------------------------

Submitter:
Address:

-----------------------------------------------

Tel:

_ _ _ _ _Fax: _ _ _ _ _ _ Email: ________________

Animal Information

AsiAn [)

African [) ISIS#

Name
Sex: [ J male

Studbook # _--,------:-_ _
Age: _______ [ 1actual [ J estimate

r J female

SAMPLE COLLECTION INFORMATION

Date of sample collection:
TIme lIf collection: _______
Site of sample collection: [ J ear vein [ ] leg vein [ J other: _________
Health status of animal: ! J nonnal [ 1abnormal
Fasted: [ J no [ ] yes - how long ___________
Weight ____________ [ 1actual [ ) estimated
Type of res1raint: ( ] manual [1 anesthetized/sedated [ ) behavioral control
Temperament of animal: [ 1calm [ 1active [ ] excited
Type of blood collection tube:
[ ] no anticoagulant (red-top)
[ ) EDT A (purple)
[ J heparin (green)
[ ) other: ---:-::-:-_______ ___
Sample handling;
1separation of plasmalserum by centrifugation
(Check all that apply)
[ 1stored as whole blood
[ 1 /Tozen plasma
[ 1 other - describe __________________
TO EXPOSURE ST ATUS
[ ] Known infected animal
( ] Known exposure to culture positive source within the past 12 month~
r J Known exposure to a culture positive source within the past \-5 years
[ 1No know exposure to 8 culture posirive source in the last 5 years
TREATMENT INFORM ATlON
Is elephant currently receiving any medication or under treatment? [ J yes [ ] no
If yes, please list drugs and doses: _ _ _ _ _ _ _ _ _ _ _ _ _ __

Time between blood collection and last treatment: ____________

Ship samples overnigbt frozen witb sbipping box marked "PLACE 1N FREEZER UPON ARRIVAL"

Send completed form with samples to:

Michele A. Miller, DVM, MS, PhD
Chief Veterinary Officer and Director of Conservation Medicine
Palm Beach Zoo, 1301 Swnmit Blvd., West Palm Beach, FL 33405
Phone: 56\-833-7130 ext 224 ; Cell: 561-727-9630;
Email : mm illertm.pa lmbeac hzo o.org

Elephant TB Guidelines 46

Consent Form for Use of Serum by Elephant SSP

I give consent for the serum submitted to the Elephant Species Survival Plan (SSP) serum bank
to be used for research on any elephant related issues based on recommendations by the
veterinary advisor and/or steering committee.
The results could be reviewed and used by the SSP veterinary advisor in providing health-related
recommendations and publications.

1ll1derstand that all results and recorrunendations regarding the individual elephant will be kept
confidential .
_ _ Yes,
results.
_ _

No,

r agree to allow the SSP to use our sample for designated research and testing

r do Dot consent to the use of our sample and test results unless specified.

Signature, title

Printed name

Institution

Date

Phone number

Email address

Address

Conunents: ______________________________________________________

Elephant TB Guidelines 47

Appendix 9. TB Management Groups

TB Management Group 1

Group 1: Culture
nega tive; S AT-PAK
DOD-reactivc:; 80 exp08Bre
past lllDOBtbJ

No treatment or travel
restr'ctions

I
If exposed to untested
elephant in previous 3
months repeal STAT-PAK

in 3 months

TB Management Group 2

G oup 2: Culture
aeptive; STAT-PAK
DOD-reactive; exposed t~
cliiture positive an Imal in
palt 12 mOD hs

Culture q 3 months for I

year post-exposure; then q 6
months for 2 years then
annually if cultures negative

and STAT-PAK@nonreactive

No travel or public con'tact

until 2 non-reactive 51 T
PAK tests at 3 and 6
months post-exposure

Elephant TB Guidelines 48

TB Management Group 3A

Group 3 A: Cullure Deprive;

STAT-PAKe reactive;
MAPIAT!I'IJ)PN BODreactive; DO kaowli exposare

Culture q 3 months 1st year; q 6

months next 2 vears then
annually if cultures negative and
MAPIA TMIDPP remains nonreactive: repeat
MAPIA ThlfDPP<il q 6 months for
1st year

No treatment or tray I
restrictions

TB Management Group 3B

Group 1 B: Culture Dqative;

STATPA K II> readive;
MAPIATM/DPP DO&reactive; knowD exposure to
cultun! po..<ritive eletthant
(no time limit)
I

I
Cultur q 3 months 1st y~ar: q 6
months ne. t 2. Repeat
MAPIAl'MIDPP q 6 months
Ist 3 years. Resume annual
testing if cultures negative and
MAPIAIDPP non-reactive

after 3 years

1
No travel or public contact 1st
year; restrictions removed if
results unchanged. If results
change, elephant will change
group.

Elephant TB Guidelines 49

TB Management Group 3C

Group 3C: Culture nept va; STATPAK react ive; MAPIATMID.PP

reactive; no known exposare

Culture q 3 months 1st year; q 6 months

for life. Repeat MAPlA TMIDP~ q 3
months I st year; q 6 months next 2 years.
If cultures negati ve after 3 yrs. resume annual
sera-logical testing according to these
Guidelines

No lravel or public contact until the 1st

year of testing has been completed.

Consider treatment - see text

Elephant TB Guidelines 50

Management Group 3D

Grottp 3D: Cui .u.re negative;

STAT-PAK reactive;
MAPlATMIDPP reactive; kllown

exposure to C11lture positive

eleplwlt (no time limit)

Culture q 3 months 1sf year;

q 6 months for life. Repeat
MAPIA TM/DPP q 3 months 1st year;
q 6 months next 2 years. If cultures
negative after 3 yrs. resume annual
serological testing according to hese
Guidelines.

No travel or public contact until

1st year of testing has been
completed.

Consider treatment - see text

Elephant TB Guidelines 51

Management Group 4
Group 4: Culture
positive f()r
M. tuberculnm
CoalpleX

I
Perfonn STAT-PAK and

MAPIA TMIDf"P: submit positive

cultures to NVSL for genotyp-ing;
request antibioti1:: sensitivity testing

Initiate tre&tmet1[
efer to text

Maintain in
pemlanent qUM8Jttine
- refer to text

Culture q 2 m{)nths for

1st 6 months then q 6
mont.hs for life

\
No travel or public
contact until

No travel or pub-lie
contact until

treatment

treatment is
completed
according to these

completed
according to these
Guidelines

Guidelines.

Euthanasia - refer to
text