GUIDELINES FOR THE CONTROL OF TUBERCULOSIS IN ELEPHANTS 2010 

UNITED STATES ANIMAL HEALTH ASSOCIATION (USAHA) ELEPHANT 

TUBERCULOSIS SUBCOMMITTEE

8 November 2010 DRAFT REVISION

TABLE OF CONTENTS (topics are bookmarked)

1. Introduction

2. Definitions

3. Annual Testing

4. Culture Collection Procedure

5. ElephantTB STAT-PAK®and MAPIATM Collection Procedure 

6. Ancillary Diagnostic Tests

7. TB Management Groups

8. Principles of Anti-tuberculosis Therapy

9. Anti-tuberculosis Drugs

10. Dosages and Routes of Administration

11. Blood Levels

12. Postmortem Examination

13. Employee Safety and Health

14. Reporting

15. Appendices

Appendix 1. References

Appendix 2. Acknowledgments

Appendix 3. A Trunk Wash Technique for the Diagnosis of TB in Elephants

Appendix 4. Testing Laboratories

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 Form

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)
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, artiodactylids, 

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 

define disease caused by non-tuberculous mycobacteria (NTM). “Atypical mycobacteria” or 

“mycobacteria other than TB” (MOTT) are other terms 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 elephants 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 of TB 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 or 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 animal or person (Siegel 2007).

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 for 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.tb complex: Isolation and identification of M. tuberculosis complex

organisms from any site using standard mycobacterial methods. 

Culture positive (M.tb 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 current 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 MAPIATM (Greenwald et al. 2009). 

ElephantTB STAT-PAK® 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 immunosorbent assay; a test used to detect and measure either antigen 

or antibody.

Exposure: Risk of transfer of an infectious agent from a TB infected elephant(s) or 

contaminated environment through contact (direct, indirect) or airborne modes of transmission. 

Fomite: 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 

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, maintained 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 elephant: an elephant from which Mycobacterium tuberculosis complex has been 

identified through culture, PCR or other molecular techniques or that is reactive on the 

ElephantTB STAT-PAK® Assay and the MAPIATM.

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 72 hours (plus or minus 6 hours) following 

injection. In humans, the test site is the forearm 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.

MultiAntigen Print ImmunoAssay (MAPIATM): A confirmatory test to the ElephantTB 

STAT-PAK® Assay for detection of antibodies to M. tuberculosis and M. bovis in elephant sera 

or plasma (Lyashchenko 2000, 2006, Greenwald 2009). 

Mycobacteria other than 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.tb): The primary causative agent of tuberculosis in humans; 

may also affect a variety of animals, including primates, pigs, cattle, dogs, parrots, elephants, and 

rhinos.

Mycobacterium tuberculosis complex (M.tb complex): A group of mycobacteria which 

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 listed as a 

separate member of this complex.

Mycobacterium Tuberculosis Direct Test (MTD): A nucleic acid amplification test used in the 

diagnosis of TB. The MTD utilizes a technique that replicates RNA from bacteria of the M. 

tuberculosis complex.

No isolation: Absence of growth of M. tb 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 

formation 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 TB” (MOTT).

Nucleic acid amplification test: A technique that amplifies entities such as DNA or RNA.
PCR (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-PAK® 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.

Report date: The date the laboratory reports the results.

Spoligotyping: 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 abnormal 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 elephant: 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-PAK®

Assay performed by a USDA veterinarian trained and certified to perform the test.
3. ANNUAL TESTING 

To adequately address the concerns of TB in the general elephant population, all captive 

elephants must be tested annually by culture and with the ElephantTB STAT-PAK® 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 ElephantTB STAT-PAK® 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-PAK® 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. 

Elephants should be tested within ± 30 days of the established annual test date. Blood for 

ElephantTB STAT-PAK® Assay and culture should be collected within a 2 week period. All 

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 of TB 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. If possible, 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 following methods, the 

trunk wash with bag seems to provide the most effective way to collect samples at this time. 

Samples collected by swab are not 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-filter masks certified by the 

National Institute for Occupational Safety and Health (NIOSH) 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 ml of sterile saline into the trunk

then aspirate. Transfer at least 20 ml 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.

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 -20oC (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 -80oC (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 submission 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 

Aviation Organization Technical Instructions for the Safe Transport of Dangerous Goods by Air 

2009-2010 (http://www.icao.int/icaonet/dcs/9284.html). Also helpful is the 2007 WHO 

document “Guidance on Regulations for the Transport of Infectious Substances 

(http://www.who.int/csr/resources/publications/biosafety/WHO_CDS_EPR_2007_2cc.pdf)

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 Recommendations; Final Rule, 

published June 2, 2006 in the Federal Register.

http://isu1.indstate.edu/terc/infectiousmaterial/pdf/Hazardous%20Materials_Infectious%20substa

nces%20harmonization%20with%20the%20UN%20final%20rule.pdf

http://ecfr.gpoaccess.gov/cgi/t/text/textidx?c=ecfr&rgn=div5&view=text&node=49:2.1.1.3.8&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 Form 10-4, 

Specimen Submission Form. This form is available online in Word or pdf format: 

http://www.aphis.usda.gov/animal_health/lab_info_services/forms_publications.shtml.

Request mycobacterial culture with species differentiation. 

Positive cultures from laboratories that do not have the capability to differentiate M. tuberculosis

complex organisms must be forwarded to NVSL or other qualified laboratories for speciation. 

Culture of mycobacteria requires a minimum of eight weeks. Laboratory reports 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.

Note: Other mycobacteria species such as M. avium, M. kansasii, M. elephantis, and M. 

fortuitum have been isolated from elephants. At this time, there is no substantive evidence that 

these organisms are pathogenic for elephants. However, Mycobacterium szulgai, an unusual 

non-tuberculous mycobacterium, has been associated with pathology in elephants (Lacasse 

2007). 
5. ELEPHANTTB STAT-PAK® ASSAY SAMPLE COLLECTION PROCEDURE

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-PAK® Assay results must be 

submitted for MAPIATM /DPP® testing to: 

Chembio 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 intradermal tuberculin test (skin test) and culture results has not been 

established (Mikota 2001, Lewerin 2005). Therefore, intradermal tuberculin testing cannot be 

deemed reliable for screening or diagnosis and is not recommended.

Enzyme Linked Immunosorbent Assay (ELISA)

A multiple antigen ELISA was developed at the Animal Population Health Institute at Colorado 

State University (Larsen 2000). This test was used for detecting the 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 sole diagnostic test.
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 defined as an elephant from which 

Mycobacterium tuberculosis or Mycobacterium bovis 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 has 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-PAK® non-reactive; no exposure to 

culture positive elephant in past 12 months. 

Monitor annually by culture (triple sample method) and ElephantTB STAT-PAK®(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 STATPAK®

remains non-reactive, the elephant continues in Group 1.

GROUP 2: Culture negative; ElephantTB STAT-PAK®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 STAT-PAK®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 sero-conversion is unknown.

GROUP 3: Culture negative; ElephantTB STAT-PAK®reactive

It is required that blood from elephants with reactive ElephantTB STAT-PAK® results be 

submitted for MAPIATM / DPP® testing (see item 5 above). Based on MAPIATM /DPP® results 

and exposure history, the elephant will fall into one of the following subgroups:

A. Culture negative; STAT-PAK® reactive, MAPIATM/DPP® non-reactive, no 

known exposure

Monitor by culture (triple sample method) every 3 months for the first year after 

becoming ElephantTB STAT-PAK®reactive, then every 6 months for the next 2 

years. Repeat MAPIATM / DPP® every 6 months for the first year if elephant 
remains STAT-PAK® reactive. If all cultures and MAPIATM/DPP® remain 

negative/non-reactive during this period, annual testing may resume.

 No treatment or travel restrictions. 

 If the culture becomes positive or MAPIATM/DPP® becomes reactive during any of 

the follow-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-PAK® reactive, MAPIATM /DPP® non-reactive, known 

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 two years then annually thereafter if all cultures 

remain negative. Repeat MAPIATM/DPP® every 6 months for the first 3 years if 

elephant remains STAT-PAK® reactive. If all cultures and MAPIATM/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 MAPIATM/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-PAK® reactive and fall into this group. If appropriate 

treatment has been documented and approved by USDA, these animals will not have 

travel/public contact restrictions unless there is a change to positive culture and/or 

reactive MAPIATM/DPP® results during follow-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 MAPIATM/DPP® every 3 months for the first year, then 

every 6 months for an additional 2 years if elephant remains STAT-PAK® 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 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 performed, the elephant may be able to travel and have public contact 

after 6 months of successful documented USDA approved treatment.

 If the culture or MAPIATM/DPP® results change during any of the follow-up testing 

the individual elephant will change group.

Note: The STAT-PAK® and MAPIATM /DPP® tests have been shown 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, MAPIATM/ DPP® reactive, known 
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 MAPIATM/DPP® every 3 months for 

the first year, then every 6 months for an additional 2 years if elephant remains 

STAT-PAK® 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 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 performed, the elephant may be able to travel and have public contact 

after 6 months of successful documented USDA approved treatment.

 If the culture or MAPIATM/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 MAPIATM/DPP® will decline 

and may indicate a response to treatment so on-going annual monitoring with 

MAPIATM/DPP® is required for life as changes in MAPIATM 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 

MAPIATM/DPP® testing 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-PAK®reactive, but non-reactive on MAPIATM/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. 

Elephants 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 
on risk. If culture results during any of the follow-up testing become positive, the individual 

elephant will move to Category 4. 

Consideration should be given to minimizing or eliminating contact with the public that would 

result in exposure by contact or aerosol transmission and to providing personal protective 

equipment such as a NIOSH certified N95 respirator /N95 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), rifampin (RIF), 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 first 60 doses should be 

administered within a period of 90 days (i.e. no more than 30 days of “refused medication” 

should occur. It must be documented 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 mg/kg 

daily.

Concomitant use of INH, 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 health 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 INH 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 defined 

as an elephant from which Mycobacterium tuberculosis complex organism has been isolated 

from any body site or specimen. 

The ElephantTB STAT-PAK®and MAPIATM/DPP® tests must be performed on blood from 

culture positive elephants. Serum for MAPIATM/DPP® testing must be submitted regardless of 

ElephantTB STAT-PAK®results.

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 laboratory 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 performed 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 

M. tuberculosis complex organisms is now available at NVSL).

3. Perform ElephantTB STAT-PAK®and MAPIATM 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 MAPIATM 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 treatment has ended to ensure that the animal 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 acknowledged. 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 all 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. 

Treatment is then continued daily for an additional 10 months with 2 drugs to which the isolate is 

susceptible for a total number of doses (with two drugs) of 300. As above, the inclusion of INH 

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 

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 mg/kg daily.

Travel: Elephants in Group 4 should not travel or have public contact (direct or indirect) until 

treatment is completed according to 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 health 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, leukopenia 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 of N-95 masks, gloves, 

etc). Quarantined elephants should be kept out of range from non-infected animals and should 

be monitored for 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, ElephantTB STAT-PAK®/MAPIATM/DPP®), 

ancillary tests and nucleic acid amplification are recommended for data collection.

C. Euthanasia: This 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 11).

Group 5: Untested If an elephant cannot complete procedures as outlined for 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 cannot travel 

nor have public contact until the untested elephant is tested unless approved by USDA.
8. PRINCIPLES OF ANTI-TUBERCULOSIS 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 

prevent the emergence of resistant 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 of TB 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 MAPIATM), 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 susceptibility 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 vast majority of 

cases the index case is not known with certainty and four drugs are given. Moreover, in regions 

or situations when the frequency of resistance exceeds 10%, empiric initial therapy for humans 

consists of five 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 is structured for a 12-month course.
9. ANTI-TUBERCULOSIS DRUGS

Antituberculous agents are divided into first and second line agents. First 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) while not considered as 1st 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 

shown to have significant activity against M. tuberculosis and has been used successfully in 

salvage regimens. Amikacin, an aminoglycoside (as is streptomycin), is a mainstay in the 

treatment of non-tuberculous mycobacterial infection and has been used in salvage regimens 

against MDR-TB. Pharmacokinetic studies of INH, RIF, EMB, and PZA in elephants have been 

published (Maslow et al. 2005a, Maslow et al. 2005 b, Zhu et al. 2005, and Peloquin et al. 

2006).

FIRST LINE AGENTS

Isonicotinic acid 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 INH 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 of INH those results in more rapid clearance and lower bioavailability. It 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 

mg/kg) to prevent the development of peripheral neuropathy.

Toxicity in elephants: Observed toxicities of INH have included inanition, transaminitis, and 

anemia. Fermented products (mash or other feeds) should likely be avoided to minimize 

potential histamine reactions. Liver values (SGOT, SGPT, and bilirubin) should be monitored 

monthly for 2 months and then bimonthly if no 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, INH 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: Rifampin 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 

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 

form 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 bolus although acceptance is low because of the drug’s bitterness. 

Rifampin 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 pyrazinamidase 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 is 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. In 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 formation of arabinogalactose and lipoarabinomannan, which are the dominant lipids 

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, arthralgias, 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 aminoglycoside antibiotic derived from the fungus 

Streptomyces griseus that acts on the 30S ribosome to inhibit protein synthesis.

Toxicity: Similar to other aminoglycosides, streptomycin 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: Streptomycin is administered via intramuscular injection to humans. 

There is no experience in administering streptomycin to elephants.

SECOND LINE AGENTS

Fluoroquinolones: 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 first 
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 al, Moxifloxain-containing regimens of reduced duration produce a stable 

cure in murine tuberculosis, Am J Respir Crit Care Med 2004, 170: 1131-4 ). The antituberculous activity of enrofloxacin, a derivative of ciprofloxacin 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-prolongation. Studies are underway

examining the role of Moxifloxacin in standard treatment and prophylaxis regimens (Burman 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 al. Early bactericidal activity of 

moxifloxacin in treatment of pulmonary tuberculosis: a prospective, randomized study, 

Antimicrob 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). Oral levofloxacin 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 aminoglycoside 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 aminoglycosides 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, L.J., Dubach, J.M. and Phillips, L.G., 1994. 
Pharmacokinetics of amikacin in African elephants. J Zoo Anim. Med 25: 367-375). There is 

no published information regarding amikacin in Asian elephants. Amikacin in one Asian 

elephant given IM 3 times a week at 14 mg/kg yielded good blood levels (acceptable levels in 

elephants unknown) and was eliminated almost 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 elephants. 

Clinicians contemplating the use of agents other than those listed should consult with the USDA 

on an individual basis.

The four first-line drugs used to treat tuberculosis in humans are isoniazid (INH), rifampin (RIF), 

pyrazinamide (PZA) and ethambutol (ETH). 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 / or reduce effects of toxicity. 

Drug Dosage 

(mg/kg)

Route Formulation Target 

conc 

(μg/ml)

Cmax 

(hr)

Isoniazid 5 Oral premixed suspension 3-5 1-2

4 Oral Powder 3-5 0.5-1

4 Rectal premixed suspension 3-5 0.25-0.5

Rifampin 10 Oral only Powder 8-24 2-4

Pyrazinamide 30 Oral or rectal Powder 20-60 1-2

Ethambutol 30 Oral only Powder 2-5 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 

al 2005b, Zhu et al 2005, Peloquin et al 2006). Recent studies have demonstrated that INH 

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 

MAPIATM 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

line agents INH, RIF, PZA, and EMB (Maslow et al 2005a, Maslow et al 2005b, Zhu et al 2005, 

Peloquin et al 2006). 

Blood levels should be determined to measure the maximal concentration of drug (Cmax). While 

INH, PZA, and EMB are rapidly absorbed with a Cmax occurring between 1-2 hrs, drug 

absorption may vary between elephants and may also vary drug to drug. Recent studies have 

demonstrated that INH 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 1hr, 1.5hr, and 2 

hr and for RIF at 2hr, 3hr, and 4hr except if INH is administered rectally and then 15 min and 30 

min blood levels are recommended to accurately measure the Cmax. If the 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 should be assessed. During the initial phase of treatment, time ranges should always 

be assessed to determine 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 anti-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. POSTMORTEM EXAMINATION

It is essential that a post-mortem examination be performed on all 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.org

www.elephantcare.org

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. 

It is recommended that a trained veterinary pathologist direct the necropsy if possible. In 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 information is excerpted from the SSP Elephant Necropsy Protocol: 

Protective equipment for tuberculosis cases - Mandatory

Respiratory protective equipment should be available during any elephant necropsy procedure 

regardless of the historical TB testing status of the animal. In animals with an unknown, suspect, 

or positive TB test history, respiratory protection should be considered mandatory. OSHA 

standards (29CFR1910.134) 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 positive air pressure respirators (PAPRs) 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 half-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. PAPRs 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.gov/SLTC/tuberculosis/standards.html 

2. Guidelines for Preventing the Transmission of Mycobacterium tuberculosis in

Health-Care Settings, 2005: 

http://www.cdc.gov/nchstp/tb/Federal_Register/New_Guidelines/TBICGuidelines.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 

fixation (in buffered 10% formalin). In addition, search thoracic organs carefully for early stages 

of TB 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 elephants 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 intradermal 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, BCG 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 

BCG, but should instead be considered as positive for exposure to TB.

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. tuberculosis complex 

(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 transfer 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 TB), disinfection procedures, and the use of separate implements for infected 

animals. The local public health department should be contacted for further guidelines.

Measures to protect staff from infected animals should include the use of respiratory (N95) 

HEPA 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/ohs/biosfty/bmbl5/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 

pharmacokinetics 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., Lyashchenko, K. Comparison of trunk 

wash results matched to multiantigen print immunoassay (MAPIATM) 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, 5th ed. U.S. Government Printing Office, 

Washington, D.C. 143-147. http://www.cdc.gov/od/ohs/biosfty/bmbl5/BMBL_5th_Edition.pdf

Davis, M. 2001. Mycobacterium tuberculosis risk for elephant handlers and veterinarians. Appl 

Occup Environ Hyg. 16: 350-353.

Greenwald, R., Lyashchenko, O., Esfandiari, J., Miller, M., Mikota, S., Olsen, J.H., Ball, 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 16(5): 605-612. 

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 of the 39th international symposium of the European zoo and wildlife medicine. 

Vienna, Austria. May 12-16, 1999. 121-124.

Lacasse, C., Terio, K., Kinsel, M.J., 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 

africana). J. Zoo Wildl. Med. 38 (1): 101-107.

Landolfi, J.A., Mikota, S.K., Chosy, J., Lyashchenko, K.P., Giri, K., Gairhe, K., Terio, 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, J.A., Schultz, S.A., Mikota, S.K., Terio, 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, R.J. and Triantis, J. 2000. 

Evaluation of a multiple-antigen enzyme-linked immunosorbent assay (ELISA) for detection of 

Mycobacterium tuberculosis 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 

immunoassay for the development of serological diagnosis of infectious disease. Journal of 

Immunological Methods 242: 91-100.

Lyashchenko, K., Miller, M., and Waters, W.R. 2005. Application of MAPIATM (Multiple 

Antigen Print Immunoassay) and rapid lateral flow technology for tuberculosis testing of 

elephants. Proc American Association of Zoo Veterinarians, 64-65.

Lyashchenko, K.P., 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, J., Riddle, H., 

and Peloquin, C.A. 2005. Population pharmacokinetics of isoniazid in the treatment of 

Mycobacterium tuberculosis among Asian and African elephants (Elephas maximus and 

Loxodonta africana). J Vet Pharmacol Ther. 28(1): 21-27.

Maslow, J.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, J. Tuberculosis and other mycobacteria as zoonoses. 1997. Proceedings American 

Association of Zoo Veterinarians. 110-115.

Michalak, K., Austin, C., Diesel, S., Bacon, J. M., Zimmerman, P., and Maslow, J. N. 1998.

Mycobacterium 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,J., 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., Miller, 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, R.S., 

Salman, M.D., Chatterjee, D., Payeur, J., Whipple, D., Thoen, C., Davis, S., Sedgwick, C., 

Montali, R.J., Ziccardi, M., and Maslow, J. 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.J. 2000. Tuberculosis in elephants in North 

American. Zoo Biology 19: 393-403.

Moller, T., 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 Symposium119-124.

Moller,T., Roken,B., Petersson,L., Vitaud,C., Lyashchenko,K.. 2005. Preliminary results of a 

new serological test for detection of TB-infection (Mycobacterium tuberculosis) in elephants 

(Elephas maximus and Loxodonta africanum) - Swedish Case studies. Verh.ber.Erkrg.Zootiere. 

42, 173-181. 

Montali, R.J., Mikota, S.K., and Cheng, L.I. 2001. Mycobacterium tuberculosis in zoo and 

wildlife species. Revue Scientifique et Technique Office International des Epizooties 20(1): 

291-303.

Montali, R.J., Spelman, L.H., Cambre, R.C., Chatterjee, D. and Mikota, S.K. Factors influencing 

interpretation of indirect testing methods for tuberculosis in elephants. 1999. Proc. Amer. 

Assoc. Zoo Vet. 109-112.

Murphree, R., Dunn, J.R., Warkentin, J.V., Schaffner, W., and Jones, T.F. 2010. Mycobacterium 

tuberculosis infection among employees of an elephant refuge. 2010 National Tuberculosis 

Conference, 06/22–6/24, 2010, Atlanta, GA. 

National Association of State Public Health Veterinarians (NASPHV), Veterinary Infection 

Control Committee. 2006. Compendium of veterinary standard precautions: Zoonotic disease 

prevention in veterinary personnel. 

http://www.nasphv.org/Documents/VeterinaryPrecautions.pdf

Oh, P., Granich, R., Scott, J., Sun, B., Joseph, 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, J.G., and Whipple, D.L. 2002. Mycobacterial isolations 

in captive elephants in the United States. Ann N Y Acad Sci 969: 256-258.
Peloquin, C.A., 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 of rifampin in elephants for 

the treatment of tuberculosis. J Vet Pharmacol Ther. 29: 581-586.

Peloquin, C.A. 2003. Clinical pharmacology of the anti-tuberculosis drugs. In 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 antimycobacterial drugs. 

Clinics in Chest Medicine 18: 79-97.

Ryan, C.P. 1997. Tuberculosis in circus elephants. Pulse Southern California Veterinary Medical 

Assoc. 8.

Siegel JD, Rhinehart E, Jackson M, Chiarello L, and the Healthcare Infection Control Practices 

Advisory Committee, 2007 Guideline for Isolation Precautions: Preventing Transmission of 

Infectious Agents in Healthcare Settings, June 2007. 

http://www.cdc.gov/ncidod/dhqp/pdf/isolation2007.pdf

Zhu, M., Maslow, J.N., Mikota, S.K., Isaza, R., Dunker, F., and Peloquin, C.A. 2005. Population 

pharmacokinetics of pyrazinamide in elephants. Journal of Veterinary Pharmacology and 

Therapeutics 28: 403-409.
APPENDIX 2. ACKNOWLEDGMENTS

The following individuals have contributed to the historical development of these 

Guidelines:

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 memorium)

Dr. Ramiro Isaza, University of Florida – Gainesville 

Dr. Barbara Kohn, 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 

Subcommittee:

Dr. Chuck Massengill, Missouri Department of Agriculture

Dr. Susan K. Mikota, Elephant Care International

Dr. Michele Miller, Palm Beach Zoo

Dr. Kathy Orloski, USDA, APHIS, 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 2010 Guidelines:

Dr. Joel Maslow, University of Pennsylvania

Dr. Denise Sofranko, USDA (regulatory advisor only) 
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.gov/ac/ElephTBGuidelines2000.html). Compliance with this 

policy 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, allow 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, 

1 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 within 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. 

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 1 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 allowed 

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 ml). The 

sample should contain visible mucus from the inside of the trunk and often contains dirt and food 

particles that are normally 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 oC) 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 decisions 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 animal’s trunk can be handled 

though a set of bars. This method still requires that the animal is fully cooperative and, therefore, 

usually 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 

first 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 confirm 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 not routinely overgrow or mask the growth of pathogenic 

mycobacteria, although no 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., Chatterjee, D., Payeur, J., Whipple, D., Thoen, C., Davis, D.S., Sedgwick, 

C., Montali, R.J., Ziccardi, M., and Maslow, J. Epidemiology and diagnosis of Mycobacterium 

tuberculosis in captive Asian elephants (Elephas maximus). J Zoo Wildl Med 2001 Mar. 32 (1): 

1-16. 
3. Michalak, K., Austin, C., Diesel, S., Bacon, J.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.
APPENDIX 4. TESTING LABORATORIES

CULTURES, ANTIMICROBIAL SENSITIVITY, GENOTYPING

USDA APHIS VS

National Veterinary Services Laboratories (NVSL) 

1920 Dayton Avenue 

Ames, IA 50010

Lab web site: http://www.aphis.usda.gov/animal_health/lab_info_services/diagnos_tests.shtml

Dr. Janet Payeur 

Scientific Outreach Coordinator 

(515) 337-7003 Fax: (515) 337-7397

Email: Janet.B.Payeur@aphis.usda.gov

Dr. Beth Harris

Head, Mycobacteria and Brucella Section

(515) 337-7362 Fax: (515) 337-7315

Email: Beth.N. Harris@aphis.usda.gov

Dr. Suelee Robbe-Austerman

Veterinarian, Mycobacteria and Brucella Section

(515) 337-7837 Fax: (515) 337-7315

Email: Suelee.Robbe-Austerman@aphis.usda.gov

Send trunk washes to NVSL either frozen or on icepacks by overnight express (Federal 

Express 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 submitted for culture, tissues should be frozen and sent on ice packs 

overnight. Lesioned tissues should be split and 1⁄2 should be sent to the histopathology lab so 

PCR can be run to see if the tissue is compatible for tuberculosis. There is no charge for 

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 

formalized 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 Gen 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, 16s 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 
complex organisms for $112.00 per isolate. Please contact NVSL at (515) 337-7388 for test 

schedule. 

To establish an account at NVSL for billing, contact Connie Osmundson (515) 337-7571 or 

Email: Connie.J.Osmundson@aphis.usda.gov .

(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.aphis.usda.gov/animal_health/lab_info_services/diagnos_tests.shtml

Mycobacteriology Laboratory at National Jewish Medical and Research Center

National Jewish Medical and Research Center

Director: Leonid Heifets, M.D.

1400 Jackson St.

Denver, CO 80206

(303) 398-1384

E-mail: heifetsl@njc.org

For price list, shipping instructions, and requisition form: 

http://www.nationaljewish.org/research/clinical-labs/about/learn/mycobac/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.

HISTOPATHOLOGY

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

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 APHIS NVSL Pathobiology Laboratory

1920 Dayton Avenue

Ames, IA 50010

(515) 337-7521

Fax (515) 337-7527

Lab web site: http://www.aphis.usda.gov/animal_health/lab_info_services/diagnos_tests.shtml
Dr. Art Davis 

Director of Pathobiology Laboratory

(515) 337-7526

Email: Arthur.J.Davis@aphis.usda.gov

Dr. Mark Hall

Head Pathological Investigations

(515) 337-7927

Email: Mark.Hall@aphis.usda.gov

Send formalin 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 the testing can be coordinated and results 

combined on one form.

ANTI TB DRUG LEVELS

Infectious Diseases Pharmacokinetics Laboratory (IDPL) 

National Jewish Medical and Research Center

1400 Jackson St.

Denver, CO 80206 

Web site: http://www.nationaljewish.org/research/clinical-labs/about/learn/infectious/idpl.aspx

Refer to the above website for specimen handling instructions and to download

Requisition forms.

Infectious Disease Pharmacokinetics Lab, College of Pharmacy, and Emerging Pathogens 

Institute

Charles Peloquin, Pharm.D.

Professor and Director

University of Florida

1600 SW Archer Rd., Rm P4-33

PO Box 100486

Gainesville, FL 32610-0486

Tel: 352-273-6266

Fax: 352-273-6804

peloquin@cop.ufl.edu

Call or email for information on sample submission.

The National Veterinary Services Laboratories

USDA APHIS NVSL

1920 Dayton Avenue

Ames, IA 50010

Web site: http://www.aphis.usda.gov/animal_health/lab_info_services/diagnos_tests.shtml
Dr. David Kinker

Head, Serology Section

515-337-7950

Email: David.R.Kinker@aphis.usda.gov

Call before shipping samples for current prices.

Chembio Diagnostic Systems, Inc.

3661 Horseblock Road

Medford, NY 11763

Tel: 631-924-1135 

Fax: 631-924-6033

Email: customerservice@chembio.com

Call Chembio before shipping samples for current prices on veterinary products such as 

ElephantTB STAT-PAK®, MAPIATM or DPP®.
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 Mycobacteria

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 (MB) section.

Warning: Mycobacterium bovis, M. tuberculosis and M. avium are pathogenic to humans 

and they are a Class III pathogen. All procedures must be performed in a Class II or Class 

III biological safety cabinet.

2. Materials

2.1 50 ml sterile conical centrifuge tubes

2.2 15 ml sterile conical centrifuge tubes

2.3 Sorvall RC 3BP refrigerated centrifuge 

2.4 N-Acetyl-L cysteine (NALC); Sigma catalog number A-7250

2.5 NaOH-Sodium citrate solution; (NVSL #10687)

2.5.1.1 Dissolve 29 gm of sodium citrate dehydrate in 1000 ml of Super Q 

H2O. Dissolve 40 gm of sodium hydroxide pellets in 1000 ml of 

Super Q H2O. Combine the 2 solutions and dispense as requested. 

Autoclave for 15 minutes at 121 °C.

2.6 Sterile distilled water

2.7 Johne’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 BHI w/out dextrose (BBL # 250220) 

with 1000 ml Super Q H2O. Bring to a rolling boil. Dispense as 

requested. Autoclave 20 min. at 121°C for flasks, 15 min. for

tubes.

2.7.2 Super QH2O 1000 ml

2.7.3 Nalidixic Acid (NVSL #40153) 10 ml

2.7.3.1 Mix 10 g of nalidixic acid with 500 ml of distilled H2O. Add 10N 

NaOH drop by drop until solution clears and QS to 1000 ml. Filter 
sterilize solution thru a 0.22μm filter into sterile jug with bell end. 

Dispense (wearing gloves), 20 ml into a 50 ml sterile conical tube. 

Caution – chemical is carcinogenic. 

2.7.4 Vancomycin (NVSL #40151) 10 ml

2.7.4.1 Combine 9.346 gm with 1000 ml of distilled Super Q H2O and mix 

well. Filter sterilize. Dispense into 50 ml sterile tubes in 20.5 ml 

amounts.

2.7.5 Amphotericin B (NVSL #40154) 5 ml

2.7.5.1 Add 10 ml warm sterile distilled H2O to a 100 mg Amphotericin B 

(Fungizone). Shake gently until dissolved and dispense as 

requested. 

2.7.6 Combine BHI broth and Super QH2O. Autoclave for 20 min at 121°C. 

Cool to 50°C. Remove 25 ml of cooled broth and discard. Add Nalidixic 

Acid, Vancomycin, and Amphotericin B. Mix well. Dispense in tubes 

and cover with foil because the Amphotericin B is light sensitive. Store in 

-20°C freezer. Media is good for 3 months.

2.8 37oC CO2 incubator preferred

2.9 Media set-up (one tube of each per sample):

2.9.1 Middlebrook 7H10 w/glycerol; NVSL #10941 or BBL Middlebrook and 

Cohn 7H10 Agar tubes (BBL #220959).

2.9.2 Middlebrook 7H11 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 12B medium vial with Panta and Erythromycin (32 μg/ml)

2.10 1 ml tuberculin syringes

2.11 5 ml syringes

2.12 Slant trays, media tube baskets

2.13 Vortex

2.14 Sterile swabs

3. 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 ml of sterile distilled water into a 50 ml 

conical centrifuge tube; this sample will 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 ml conical centrifuge tube for storage. 

3.2.1 These 15 ml centrifuge tubes are stored at -20 ± 2o 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 

-20o + 2 ° C freezer for at least 6 months. 
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 of MBSOP0008.

3.2.3 Samples from cases in which mycobacteria have been isolated will be 

retained for one year and stored in a -20o C freezer.

3.3 Allow the 10 - 12 ml of the trunk wash in the 50 ml conical tube to stand 

undisturbed for 15 – 20 minutes to allow sediment to settle to the bottom. Or 

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, 10oC, using the Sorvall RC 3BP centrifuge.

3.4 Slowly pour the supernate, trying not to disturb the sediment, into a sterile 50 ml 

conical centrifuge tube.

3.5 Prepare the N-Acetyl-L-cysteine (NALC)/NaOH- sodium citrate solution 

according to the following proportions:

Volume (ml) NaOH- sodium citrate a(ml) NALC (g)

 50 50 0.25

 100 100 0.50

 150 150 0.75

 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

3.6 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. 

3.7 Vortex or vigorously hand shake for 20 + 5 seconds

3.8 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.

3.8.2 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. 

3.9 Add enough sterile distilled water to the NALC/wash solution to fill the 

centrifuge tube

3.10 Centrifuge the water/NALC/wash solution for 20 minutes at 6000g and 10oC.

3.11 Carefully pour off and discard the supernatant. 

3.12 Re-suspend the sediment with 2 mls of sterile distilled water and 1 ml of the 

Johne’s antibiotic mixture.
3.12.1 Vortex the re-suspended mixture for 20 + 5 seconds.

3.13 Incubate overnight at 37 ± 2oC.

3.14 Vortex or vigorously hand shake the incubated sample for 20 + 5 seconds.

3.15 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 tubes are Middlebrooks 7H10 w/glycerol, 

Middlebrooks 7H11 w/ glycerol, Mycobactosel 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.

3.16 Prepare the BACTEC 12B bottles.

3.16.1 A 5 ml syringe is used to add 2 ml of Erythromycin to the reconstituting 

fluid supplied for the PANTA solution for a final concentration of 

32 μg/ml.

3.16.2 Using a new 5 ml syringe, add 5 ml of the reconstituting fluid to the vial 

of PANTA. 

3.16.3 Using a 1 ml tuberculin syringe, each vial of the BACTEC media is 

inoculated with 0.2ml of the reconstituted supplement.

3.17 Inoculate the BACTEC media with your sample.

3.17.1 Using a 1 ml tuberculin syringe, inoculate the BACTEC media with 

≤ 0.5 ml of the sample

3.18 Place the inoculated solid media on a slant rack and incubate overnight at 

37 ± 2oC in 10% CO2, if available.

3.18.1 Incubation in an atmosphere of CO2 will encourage earlier growth.

3.18.2 After being incubated overnight on a slant rack, the media tubes can be 

stored in the 37oC incubator in an upright position.

3.19 Place the inoculated BACTEC bottles in a locked 37oC incubator.

3.20 Read the media tubes as outlined below and record the results on the appropriate 

worksheets:

3.20.1 Solid Media

3.20.1.1 Read tubes weekly for weeks 1-7 after inoculation.

3.20.1.2 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.

3.20.1.3 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.
3.20.2.3 BACTEC bottles that have a growth indicator value of 300 

or higher are examined by acid fast staining. See the 

current version of MBSOP2210 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 

of MBSOP2007 and MBSOP2008 for these procedures.

4. References

4.1 Della-Latta, P. and I. Weitzman. 1998. Mycobacteriology. p. 169-204 In H.D. 

Isenberg (ed.), Essential Procedures for Clinical Microbiology. American 

Society for Microbiology, Washington, D.C.

4.2 Zimbro, M.J. and D.A. Power. 2003. Difco & BBL Manual – Manual of 

Microbiological Culture Media. Becton, Dickinson and Company, Sparks, MD.
APPENDIX 6. CONTACTS FOR QUESTIONS

DIAGNOSIS 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 Dumonceaux

The Florida Aquarium

701 Channelside Drive

Tampa, Florida 33602

Cell: 813-465-9234 

Work: 813-367-4055

Email: gdumonceauz@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: smikota@elephantcare.org

Website: www.elephantcare.org

ELEPHANT TB STAT–PAK® ASSAY AND MAPIATM

Konstantin Lyashchenko, Ph.D.

Research Director, Mycobacterial Immunology

Chembio Diagnostic Systems, Inc.

3661 Horseblock Road

Medford, NY 11763

Tel: 631-924-1135, ext.111

Fax: 631-924-6033

Email: klyashchenko@chembio.com
REGULATORY

Dr. Denise Sofranko

USDA-APHIS-Animal Care 

Field Specialist for Elephants

Voice Mail: 240-461-9142

Email: Denise.M.Sofranko@aphis.usda.gov

2150 Centre Avenue 

BLDG B Mail Stop #3-W11 

Ft. Collins, CO 80562-8117 

HUMAN HEALTH, 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.maslow@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: 321-229-9363

Email: Scott.P.Terrell@disney.com

INTERNET

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)
APPENDIX 7. SOURCES FOR ANTI-TUBERCULOSIS DRUGS 

There are various veterinary compounding pharmacies that have experience with formulations 

for elephants. Please contact one of the consultants in Appendix 6 for information. Select 

veterinary compounding pharmacies are also listed on www.elephantcare.org.
APPENDIX 8. Elephant Serum Bank Submission Form

Institution/owner: _______________________________________________________________

Submitter: _____________________________________________________________________

Address: _____________________________________________________________________ 

______________________________________________________________________________

Tel: __________________ Fax: _________________ Email: _______________________

Animal Information

Asian [ ] African [ ] ISIS# ______ Studbook # ____________

Name ____________ Age: _________ [ ] actual [ ] estimate 

Sex: [ ] male [ ] female

SAMPLE COLLECTION INFORMATION

Date of sample collection: ___________ Time of collection: __________

Site of sample collection: [ ] ear vein [ ] leg vein [ ] other: ___________

Health status of animal: [ ] normal [ ] abnormal

Fasted: [ ] no [ ] yes – how long ______________

Weight ________________ [ ] actual [ ] estimated

Type of restraint: [ ] manual [ ] anesthetized/sedated [ ] behavioral control

Temperament of animal: [ ] calm [ ] active [ ] excited 

Type of blood collection tube: 

[ ] no anticoagulant (red-top) 

[ ] EDTA (purple)

[ ] heparin (green) 

[ ] other: ___________________

Sample handling: [ ] separation of plasma/serum by centrifugation 

(Check all that apply) [ ] stored as whole blood 

[ ] frozen plasma

[ ] other – describe _______________________

TB EXPOSURE STATUS

[ ] Known infected animal

[ ] Known exposure to culture positive source within the past 12 months

[ ] Known exposure to a culture positive source within the past 1-5 years

[ ] No know exposure to a culture positive source in the last 5 years 

TREATMENT INFORMATION

Is elephant currently receiving any medication or under treatment? [ ] yes [ ] no

If yes, please list drugs and doses: _________________________________________________

_____________________________________________________________________________

_____________________________________________________________________________

_____________________________________________________________________________

Time between blood collection and last treatment: _________________________
53

Ship samples overnight frozen with shipping box marked “PLACE IN 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 Summit Blvd., West Palm Beach, FL 33405

Phone: 561-833-7130 ext 224; Cell: 561-727-9630; 

Email: mmiller@palmbeachzoo.org
54

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.

I understand that all results and recommendations regarding the individual elephant will be kept 

confidential.

_____ Yes, I agree to allow the SSP to use our sample for designated research and testing 

results.

_____ No, I do not consent to the use of our sample and test results unless specified.

____________________________________ _______________________

Signature, title Date

____________________________________ _______________________

Printed name Phone number

__________________________________________ ________________________

Institution Email address

______________________________________________________________________________

Address

______________________________________________________________________________

______________________________________________________________________________

Comments: ___________________________________________________________________
55

Appendix 9. TB Management Groups

TB Management Group 1

TB Management Group 2

Group 1: Culture 

negative; STAT-PAK®

non-reactive; no exposure 

past 12 months

No treatment or travel 

restrictions

If exposed to untested 

elephant in previous 3 

months repeat STAT-PAK®

in 3 months

Group 2: Culture 

negative; STAT-PAK®

non-reactive; exposed to 

culture positive animal in 

past 12 months

Culture q 3 months for 1 

year post-exposure; then q 6 

months for 2 years then 

annually if cultures negative 

and STAT-PAK® nonreactive

No travel or public contact 

until 2 non-reactive STATPAK® tests at 3 and 6 

months post-exposure
56

TB Management Group 3A

TB Management Group 3B

Group 3 A: Culture negative; 

STAT-PAK® reactive; 

MAPIATM/DPP® nonreactive; no known exposure

Culture q 3 months 1st year; q 6 

months next 2 years then 

annually if cultures negative and 

MAPIA TM/DPP® remains nonreactive; repeat 

MAPIATM/DPP® q 6 months for 

1st year

No treatment or travel 

restrictions

Group 3 B: Culture negative; 

STAT-PAK® reactive; 

MAPIATM/DPP® nonreactive; known exposure to 

culture positive elephant 

(no time limit)

Culture q 3 months 1st year; q 6 

months next 2. Repeat 

MAPIATM/DPP® q 6 months 

1st 3 years. Resume annual 

testing if cultures negative and 

MAPIA®/DPP® non-reactive 

after 3 years

No travel or public contact 1st 

year; restrictions removed if 

results unchanged. If results 

change, elephant will change 

group.
57

TB Management Group 3C

Group 3C: Culture negative; STATPAK® reactive; MAPIATM/DPP®

reactive; no known exposure

Culture q 3 months 1st year; q 6 months 

for life. Repeat MAPIATM/DPP® q 3 

months 1st year; q 6 months next 2 years. 

If cultures negative after 3 yrs, resume annual 

serological testing according to these 

Guidelines 

No travel or public contact until the 1st 

year of testing has been completed. Consider treatment - see text
58

Management Group 3D

Group 3D: Culture negative; 

STAT-PAK® reactive; 

MAPIATM/DPP® reactive; known 

exposure to culture positive 

elephant (no time limit)

Culture q 3 months 1st year; 

q 6 months for life. Repeat 

MAPIATM/DPP® q 3 months 1st year; 

q 6 months next 2 years. If cultures 

negative after 3 yrs, resume annual 

serological testing according to these 

Guidelines.

No travel or public contact until 

1st year of testing has been 

completed.

Consider treatment - see text
59

Management Group 4

Group 4: Culture 

positive for

M. tuberculosis

complex

Perform STAT-PAK® and 

MAPIATM/DPP®; submit positive 

cultures to NVSL for genotyping; 

request antibiotic sensitivity testing

Initiate treatment -

refer to text

Culture q 2 months for 

1st 6 months then q 6 

months for life

No travel or public 

contact until 

treatment 

completed 

according to these 

Guidelines

Maintain in 

permanent quarantine 

- refer to text

No travel or public 

contact until 

treatment is 

completed 

according to these 

Guidelines.

Euthanasia - refer to 

text