**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. El ephantTB STAT

- PAK® and MAPIATM Collection Procedure 6. Ancillary

Diagnostic Tests 7. TB Management Groups 8. Principles of Anti

- tuberculosis Therap y 9. Anti

- tuberculosis Drugs 10. Dosages and Routes of Administration 11. Blood Levels 12. Postmortem Examination 13. Employee Safet y 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 I solation 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. M ycobacteria infect a broad range of species including humans, non

human primates, carnivores; marine mammals, psittacine birds, reptiles, fish, artiodactyl ids, pach yderms, 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 b y 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 “m ycobacteriosis” refers to infection with any m yc obacte ria but is generall y used to define disease caused b y non - tuberculous m ycobacteria (NTM). “At ypical mycobacteria” or “mycobacteria other than TB” (MOTT) are other terms used to describe this group. Most NTM are saproph yt es found in soil or water but they may occasionall y cause disease in humans and animals, including elephants. Mycobacterium tuberculosis is the predominant disease

- causing agent in elephants although cases caused b y 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 rapidl y growing m ycobacterium, was isolated from a lung abscess of an elephant that died of chronic respiratory disease (Shojaei 2000). Mycobacteriu m avium is commonly isolated from elephants (Pay eur 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 Guide lines 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 become s 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 b y dissemination of either airborne droplet nuclei or small particles in the respirable size range containi ng 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 b y air currents and may be inhaled b y susceptible individuals who have not had face

to - 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 b y the American Veterinary

Medical Association’s Council on Education Commission for Foreign V eterinary

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, m edicating, 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 scalp el blades, needles, and necrops y knives, may result in exposure to pathogens. (NASPHV 2006) **Culture positive for M.tb**  **complex:** Isolation and identification of M. tuberculosis complex organisms from an y site using standard mycobacterial methods. **Cultur e positive (** 

**M.tb complex) elephant:**  An elephant from which a M. tuberculosis complex organism has been isolated from an y bod y 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:** Enzy me - 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. **Fo mite:**  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 currentl y available for use in elephants). **Genotyping assay:** A technique for the identification and anal ys is of pol ymorphism in certain ty pes of repeat units in DNA. Restriction fra gment length pol ym orphism (RFLP) and variable number tandem repeat (VNTR) are examples of genot yping techniques. **Herd:** A group or groups of elephants, maintained on common ground. Alternativel y, two or more groups of animals under common ownership or su pervision that are geographicall y 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 dis ease 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 MAPIA **TM**
**. Intradermal tuberculin test (skin test):**  The injection of purified protein derivative (PPD) tuberculin into the skin for the purpose of detecting exposure to tuberculosis. In cattle, the test site is either the caudal fold (CFT) or cervical region (e.g. comparative cervical test, CCT) and the t est is read b y 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 200 1, 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 (MAPIA TM** **):** 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): S** ee non - tuberculous mycobacteria. **Mycobacteriosis: A** disease caused by non - tuberculous mycobacteria (NTM).**Mycobacterium :**  A genus in the family M ycobacteriaceae. **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 usuall y considered an environmental contaminant. **Mycobacterium bovis (M. bovis) :**  The primary causative agent of tuberculosis in cattle, bison, and cervids; may

also affect a variet y of mammals includi ng pigs, humans, primates, and non

domestic ungulates. **Mycobacterium tuberculosis (M.tb ):**  The primary causative agent of tuberculosis in humans; may also affect a variet y of animals, including primates, pigs, cattle, dogs, parrots, elephants, and rhinos. **Mycobacterium tuberculosis complex (**  **M.tb complex):**  A group of m ycobacteria 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 l isted 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 is olation:**  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. Improperl y handled or shipped sample **Non -**  **reactive:** Absence of response; in the context o f 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 n ot cause the formation of granulomas. Most NTM are saprophy tes found in soil or water. They are t ypicall y non - pathogenic but may

occasionall y cause disease in humans and animals, including elephants. Also referred to as “at ypi cal” m ycobacteria or “ Mycob acteria 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 b y 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 genot yping assay **Variable number tandem repeat (VNTR):**  A genoty ping 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 whereb y 3 samples are obtained on separate day s. **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 res ults). 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 diseas e 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 with in 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 adequatel y ad dress 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 b y 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 b y 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 shoul**  **d 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 imp ortance. 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 posi tive 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 day s. If possible, collect samples within a seven

- da y 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. L ight 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 sy ringe, instill 60 ml sterile saline into the trunk. Raise the trunk as high as possible to distribute the fluid deeper into the trunk. L owe r 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 a spirate. 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. Fo**  **rcible exhalation**

– Mucous collected without instilling saline into the trunk is acceptable if elephants are trained to forcibl y 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, l eak 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 prefe rred. 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 i n 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, o r 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 Tr ansport 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 Depa rtment of Transportation regulations

– 49 CFR Parts 171, 172, 173 and 175

- Hazardous Materials: In fectious 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/text

idx?c=ecfr&rgn=div5&view=text&node=49:2.1.1.3.8&idno=49 S end samples to NVSLor 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 NVSLor 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 m ycobacteria species suc h 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 m ycobacterium, 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 Roa d 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 hav e 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 ha s not been established (Mikota 2001, L ewerin 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 specificit y for detecting infecte d elephants and monitoring elephants over time. However, ELISA testing is not currentl y 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**  **Mycobacter ium 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 STAT

PAK**®** remains non

- reactive, the elephant continues in Group 1. **GROUP 2: Cul ture 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 ye ar post - exposure, then every 6 months for 2 ye ars, then annuall y thereafter if all cultures remain negative and ElephantTB STAT

- PAK**®** remains non

- reactive.  No travel or public contact until 2 additional non

- reactive ElephantTB STA T
- 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 an y of the f ollow

- 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 MAPIA**TM** / 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 ye ars. Repeat MAPIATM / DPP® every 6 months for the first year if elephantremains 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 an y 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 y ear post exposure, then every 6 months for two y ears 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 peri od, annual testing may resume after 3 years.  No travel or public contact for first ye ar; if results are unchanged at the first y ear, restrictions are removed.  If the culture or MAPIATM/DPP® results change during an y 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 therap y may remain ElephantTB STAT

- PAK® reactive and fall into this group. If appropriate treatment has been documented and approved b y 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 kno**  **wn exposure**  Monitor by

culture (triple sample method) every 3 months for one y ear, 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. I f 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 conversio ns 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 U SDA. 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 an y of the follow

- up testing the individual elep hant will change group. Note: The STAT

- PAK® and MAPIATM /DPP® tests have been shown to be earl y indicators of TB infection. Retrospective studies have shown elephants may be serologicall y reactive months to years in advance of detection b y culture (Greenwa ld 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 y ear post exposure, then every

6 mo nths 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 ye ars, annual serological testing may

resume as described i n 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 m onths of successful documented USDA approved treatment.  If the culture or MAPIATM/DPP® results change during an y of the follow

- up testing the individual elephant will change group.  Culture positive elephants that have completed a course of anti

- tuberculosis therap y may remain ElephantTB STAT

- PAK® reactive and fall into this category . If appropriate treatment has been documented and approved b y USDA, these animals will not have travel/public contact restrictions unless there is a change in their results durin g 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 ST**  **AT -**  **PAK® reactive elephants.** E lephants 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 precisel y known. Numerous variables such as age, genetics, immune status, nutritional condition, other concurrent health problems, and other factors influence the development of di sease 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 under go 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 bas ed 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 analy ses of banked serum samples are strongl y 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 eith er 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 basedon risk. If culture results during an y 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 pr oximity

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

ele ct to administer prophylactic or full treatment after consultation with USDA. Effective proph yl actic therap y 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 adequat e anti

- TB drug levels are achieved in the blood of the elephant under treatment. Acceptable anti

- tuberculosis drugs include isoniazid (INH), rifampin (RIF), p yr azinamide (PZA), ethambutol (ETH), or a fluoroquinolone such as levofloxacin, moxofloxacin, cip rofloxacin, or enrofloxacin. I soniazid 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 t herapy 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 pe riod of 90 day s (i.e. no more than 30 day s 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:**  Admi nister the two anti

- TB drugs dail y for two months (as above, the first 60 doses should be administered within a period of 90 day s). 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 reliabl y. 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 mu st 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 addi tional 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: Py ridoxine 50 mg is administered to human s receiving INH for treatment of active or latent tuberculosis to prevent the development of peripheral neuropath y. Although this side effect has not been reported in elephants, it may be possible. At the discretion of the attendingveterinarian, Vitamin B6 (py ridoxine) can be given prophylacticall y 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, s ide effects, blood levels, and other information. **Monitoring of Prophylactically Treated Elephants** During the 9 months of treatment, elephants should be closel y observed for changes in appetite, behavior, and an y 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, AL T, 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 an y bod y site or sp ecimen. The ElephantTB STAT

- PAK® and MAPIATM/DPP® tests must be performed on b lood 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 fo r 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 safet y should be instituted immediatel y (see Employee Safet y and Health section). Elephants with cultures that yi eld non

- tuberculous strains o f m yc obacteria 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 NVSLor other qualified laboratory for m ycobacterial 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, py razinamide, ethambutol,ci profloxacin (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 fo r 2 years then according to the schedule in the group that the elephant falls into post

treatment. 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 b y 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, py razinamide, 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 iso lates are susceptible daily for 2 months. The first 60 doses should be administered within a period of 90 day s (i.e. no more than 30 day s of “refused medication” should occur). Adequate blood levels of all 3 drugs must be demonstrated in two samples coll ected approximately two weeks apart. Serum samples should be collected as soon as the elephant is accepting medication reliabl y. If acceptable levels (see below) are not achieved, the dosage should be adjusted and serum levels tested again (two samples c ollected 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 perio d of 90 day s (i.e. no more than 30 day s 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 reliabl y. 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 dos es 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 andperiods of interruption that may be needed if side effects are noted). Animals that have not completed treatment are considere d 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 (py ridoxine) can be given prophylacticall y 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. L iver tests (AST, AL T, 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 safet y (such a s 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 aero sol 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). **Gro up 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 th e 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 re sistant 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 ty

pically 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 a dditional number of antibiotics results in more rapid clearance of bacteria from the sputum thereb y 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 suscep tible 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 certaint y 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 therap y for humans is currentl y 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 leas t 12 months with 2

- 4 drugs based on the susceptibility pattern (lower numbers of agents are emplo yed 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 currentl y 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, py

razinamide, 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 capreom yci n, ethionamide, cycloserine, and thiacetazone. The fluor oquinolones (FQ; moxifloxacin, ciprofloxacin, levofloxacin, and enrofloxacin) while not considered as 1 st 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 successfull y in salvage regimens. Amikacin, an aminoglycoside (as is streptomycin), is a mainstay in the treatment of non

- tuberculous mycobacterial infection and has been us ed 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 hydrazi de (Isoniazid, INH)**  **Mechanism of action:** INH acts to inhibit cell wall sy nthesis through blockage in the m ycolic acid pathway . The specific target enzy mes are unknown; however, evidence supports a role for the catalase enzy

me, katG , as modifying INH to an active form. Postulated targets of the activated form of INH include ketoacyl sy nthetase and inhA. **Metabolism and excretion:** IN H is acetylated in the liver through the action of N
- acet yl

transferase. The acet yl ated 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 acet yl ation of INH those results in more rapid clearance and lower bioavailability . It is not known whether elephants are pol ym o rphic in this enzy me and differ in the speed of acet yl ation. **Toxicity:** The major adverse effects documented in humans are hepatitis (principally hepatocellular inflammation with a transaminitis) and peripheral neuropathy. Uncommon adverse reactions inclu de headaches, optic neuritis, seizures, psychosis, encephalopathy , twitching, rashes, and gastrointestinal upset. A histamine like reaction can be observed when products with ty

ramine (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 (p yri doxine) is given at a dose of 50 mg daily (~1 mg/kg) to prevent the development of peripheral neuropath y. **To xicity in elephants:**  Observed toxicities of I NH have included inanition, transaminitis, and anemia. Fermented products (mash or other feeds) should likely be avoided to minimize potential histamine reactions. L iver values (SGOT, SGPT, and bilirubin) shou ld be monitored monthly for 2 months and then bimonthly if no liver toxicity

is observed. INH has causedirreversible leukopenia in camels; reversible leukopenia has been observed in one elephant that

was considered as possibly / probabl y related to INH. **Route of administration:** In humans INH is administered orall y. In elephants, I NH is preferentiall y administered as an oral bolus. However, rectal absorption is efficient, yi elding levels similar to oral bolus dosing. In bongo antelope, INH has also be en successfull y administered via intramuscular injection. **Rifampin (RIF) Mechanism of action:**  Rifampin is a semi sy nthetic derivative of rifam ycin, an antibiotic derived from the fungus Streptomyces mediterranei . Rifampin acts to inhibit the DNA

dependent, RNA
- pol ym erase thus blocking formation of messenger RNA (the first step in protein sy nthesis). **Metabolism and excretion:** Rifampin is acetylated in the liver. Both the unaltered and acet yl ated drug is excreted into the bile. Rifampin is then reabsorbe d whereas the acetylated form is not. **Toxicity:** The major toxicity of rifampin is hepatitis. Other side effects include gastrointestinal upset, renal failure, hemoly sis, acute renal failure, and thrombocy topenia. It is avoided in pregnancy

during the first trimester because of possible teratogenicit y . Rifampin is also a strong inducer of the cy tochrome P450 hepatic enzy mes that may

increase the metabolism of concurrently administered drugs. A prime example is exogenously administered steroids used for in vitro fertilization. For animals being treate d 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 test s, serum creatinine, electrol yt es 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 elephant s rifampin appears to be absorbed well as an oral bolus although acceptance is low because of the drug’s bitterness. Rifampin is not absorbed rectall y; there is no known experience with parenteral administration in elephants or other animals. Urine and fe ces may become orange colored while on this drug. **Pyrazinamide (PZA) Mechanism of action:**  Py razinamide is a s ynthetic antibiotic derived from nicotinic acid. Its mechanism of action is unknown; however the presence of an intact p yr azinamidase is require d. Since Mycobacterium bovis lacks this enzy me, it is resistant to PZA. **Toxicity:** Toxicities observed in humans include arthralgias and arthritis, hy peruricemia, 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, p yrazinamide is administered orall y. In elephants both o ral and rectal dosing have yielded acceptable blood levels. Py razinamide has been successfull y administered to bongo antelope via subcutaneous injection. PZA is should not be given if M bovis infection is suspected since this organism is inherently resist ant to PZA." **Ethambutol (EMB) Mechanism of action:**  Ethambutol is a specific inhibitor of the arabinos yltransferase thereby inhibiting formation of arabinogalactose and lipoarabinomannan, which are the dominant lipids in the M. tuberculosis cell wall. **To xicity:**  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, hy peruricemia , and rarel y anaph yl axis. **Toxicity in elephants:** The toxicity for elephants is currentl y unknown. **Route of administration:** Ethambutol is administered orally to humans and elephants. Rectal administration is irritating and poorl y tolerated resulting in e xpulsion of the drug. Subcutaneous administration has been given successfull y 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 sy nthesis. **Toxicity:** Similar to other aminogl ycosides, streptomycin administration may result in auditory

vestibular and renal toxicity . Specific s ymptoms include ataxia, vertigo, nerve deafness, and rena l failure. Most sy

mptoms are reversible if the drug is discontinued immediatel y after their occurrence. **Toxicity in elephants:** The toxicity for elephants is currentl y 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 streptom ycin to elephants. SECOND LINE AGENTS Fluoroquinolones: Moxifloxacin, Ciprofloxacin, Levofloxacin, Enrofloxacin **Mechanism of action:** Fluoroquinolone antibiotics act to inhibit the topoisomerases DNA gy rase and topoisomerase IV. Both of these enzymes are needed during DNA replication to firstunwind supercoiled DNA and then to again achieve a supercoiled structure of DNA. Of the

commerc iall y 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 E L et al, Moxifloxain

- containing regimens of reduced duration produ ce a stable cure in murine tuberculosis, Am J Respir Crit Care Med 2004, 170: 1131

- 4 ). The anti

tuberculous activit y of enrofloxacin, a derivative of ciprofloxacin is unknown. Gatifloxacin also has excellent in vitro activity against strains of TB, althou gh the drug was recentl y withdrawn due to reports of antibiotic associated diarrhea and QT

- prolongation. Studies are underway examining the role of Moxifloxacin in standard treatment and proph yl axis 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 elep hants:**  The toxicity for elephants is unknown. **Route of administration:** These agents are administered either orall y or intravenousl y (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. Amik acin **Mechanism of action:** Amikacin is an aminogl yc oside antibiotic that acts on the 30S ribosome to inhibit protein sy

nthesis. I solates that are resistant to streptomycin may be susceptible to amikacin. **Toxicity:** Similar to other aminogl ycosides amikacin administration may result in auditory

vestibular and renal toxicity . Specific s ymptoms include ataxia, vertigo, nerve deafness, and renal failure. Most sy

mptoms are reversible if the drug is discontinued immediatel y after their occurrence. **Toxicity in elephants:**  The toxicity for elephants is currentl y 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 c onducted (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 give n 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 (person al 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), py razinamide (PZA) and ethambutol (ETH). Second

- line drugs used in cases of drug intolerance or multi

- drug resistant organisms include amikacin and a fluoroquinolo ne. 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 directl y 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 som e experimentation may be required to determine a workable regimen for each individual elephant. Isoniazid and PZA can be given either orall y or rectally. Rifampin and ethambutol should only be administered orally

(effective blood levels of rifampin canno t be achieved with rectal administration and ethambutol is quickly expelled when given rectall y). 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 Py razinamide 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 demons trated that INH achieves Cmax much more quickl y than previously thought when administered rectall y Dosages are considered as estimates with the goal of achieving target serum concentrations as listed in #10 below without causing significant side effects th at 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 cons idered 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 1 st line agents INH, RIF, PZA, and EMB (Maslow et al 2005a, Maslow et al 2005b, Zhu et al 2005, Peloquin et al 2006). Bloo d levels should be determined to measure the maximal concentration of drug (Cmax). While IN H, 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 previousl y 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 det ermined 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 accuratel y measure the Cmax. If the first measured time point represents the greatest level for an y drug, then Tmax may have already

passed and earlier time points should be assessed. Conversel y, 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 point s should be assessed. During the initial phase of treatment, time ranges should alway s be assessed to determine the true Tmax. NOTE: Target blood levels for anti

- TB drugs in elephants have not been rigorousl y established. Until further studies can be c onducted, 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 tha t 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 cur

rent 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 necrops y if possible. Inthe event of an elephant necropsy (electiv e 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 L ake, FL 3 2830, 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 Necrops y Protocol: **Protective equipment for tub erculosis cases**  **- Mandatory**  Respiratory

protective equipment should be available during any elephant necrops y 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 necrops y. According to the draft CDC guidelines for the prevention of transmission of tuberculosis in health care settings, respiratory protective devices used for prote ction 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 usuall y be met by suppl yi ng 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/ TBI CGuidelines.pdf **Necropsy procedures** All elephants undergoing necropsies should have a careful examination of the tonsillar regions and submandibular l ymph nodes for tuberculous appearing lesions. These ly mph nodes may

be more easil y visualized followi ng removal of the tongue and lary ngeal structures during the dissection. All ly

mph nodes should be carefull y evaluated for lesions since other sites may also be infected (ex. reproductive or gastrointestinal tract). Collect any nodes that appear caseous or granulomatous for m yco bacterial and standard bacterial culture (freeze or ultrafreeze), and fixation (in buffered 10% formalin). In addition, search thoracic organs carefully for earl y stages of TB as follows: after removal of the lungs and trachea, lo cate 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 l ymph node and submit for TB culture to NVSL or a laboratory experienced in mycobacterial culture and ident ification ( **even if no lesions are evident** ). Submit sections in formalin for histopathology . Carefull y 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 mo**  **re)** se ctions of an y suspicious lesions. Open the trachea and look for nodules or plaques and process as above. Regional thoracic and tracheal ly mph nodes should also be examined and processed accordingly . Split the trunk from the tip to its insertion and take samples of an y plaques, nodules or suspicious areas for TB diagnosis as above. Look for and collect possible extra - thoracic TB lesions, particularl y if there is evidence of advanced pulmonary TB.**13. EMPLOYEE HEALTH AND SAFETY**

**All employees that are i n 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. Conversel y, those declining treatment are followed yearl y with a chest radiograph and clinical evaluation to determine whether they

have developed active disease. A pos itive 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 Societ y has published guidelines for the interpretation of intradermal testing. If inoculation with BCG occurred more than 10 ye ars 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 fr om 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 st ate 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 ani mals 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 (N 95 HEPA filtered masks, certified by the National Institute for Occupational Safet y 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 facilit y 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 5 th Edition of Biosafet y 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 READ ING**  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): 19 3
- 203. Ball, R.L ., Dumonceaux, G., Olsen, J.H., Burton, M.S., Ly ashchenko, K. Comparison of trunk wash results matched to multiantigen print immunoassay (MAPIATM) in a group of captive Asian elephants (

Elephas maximus ). 2006. Proceedings American Associati on of Zoo Veterinarians. 303

- 304. Centers for Disease Control and Prevention and National Institutes of Health. 2007. Biosafety in Microbiological and Biomedical Laboratories, 5 th ed. U.S. 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., Pay eur, J.B., Harris, B., Sofranko, D., and Waters, W.R., Lyashchenko, K. 2009. Highl y accurate antibody assays for earl y and rapid detection of tuberculosis in African and Asian elephants. Clinical and Vaccine Immunology 16(5): 605

- 612. Is aac, R. The elephant trunk wash

– An update. Elephant Managers Association Annual Conference. Orlando, Florida. November 9

- 11, 2001. Is aza, R, and Ketz, C. A trunk wash technique for the diagnosis of tuberculosis in elephants. Proceedings of the 39th international s ymposium 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 aty pical m ycobacteriosis caused b y Mycobacterium szulgai associated with mortality in captive African elephant s ( 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 sy stemic cy tokine 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

cy tokine quantitative, real

- time RT

- PCR assay s for characterization of Asian elephant immune respo nses. Vet Immunol Immunopathol. Sep 15; 131(1

- 2): 73

- 78. Larsen, R.S., Salman, M.D., Mikota, S.K., I saza, R., Montali, R.J. and Triantis, J. 2000. Evaluation of a multiple

- antigen enzy me - 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 elep hants 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 24 2: 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., Pay eur, 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 earl y diagnosis, and monitoring of treatment. Clin Vaccine Immunol 13(7): 722

- 732. Maslow, J.N., Mikota, S.K., Zhu, M., Is aza, 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 m ycobacteria as zoonoses. 1997. Proceedings American Association o f 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: 2 83
- 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 ran ge countries. Proceedings International Elephant Conservation and Research Sy mposium,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., I saza, R., Dunker, F., West, G., Lindsay , W., Larsen, R.S., Salman, M.D., Chatterjee, D., Pay eur, 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., L arsen, 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., Ly ashchenko, K., 2006. The elephant Rapid Test (RT) the future diagnostic test for TB ( M. tuberculosis ) in elephants? Ca ll for a validation study in Europe. Proceedings International Elephant Conservation and Research S ymposium119

- 124. Moller,T., Roken,B., Petersson,L., Vitaud,C., Lyashchenko,K.. 2005. Preliminary results of a new serological test for detection of TB

- infec tion ( 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 emplo ye es 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/Veterinary Precautions.pdf Oh, P., Granich, R., Scott, J., Sun, B., Joseph, M., Stringfield, C., Thisdell, S., Staley , J., Workman

- Malcolm, D., Borenstein, L., L ehnkering, 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. M ycobacterial 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 sele ction 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. L ondon, 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 Gu ideline for Isolation Precautions: Preventing Transmission of In fectious Agents in Healthcare Settings, June 2007 **.** http://www **.** cdc.gov/ncidod/dhqp/pdf/isolation2007.pdf Zhu, M., Maslow, J.N., Mikota, S.K., I saza, R., Dunker, F., and Peloquin, C.A. 2005. Population pharmacokinetics of p yrazinamide in elephants. Journal of Veterinary Pharmacology and Therapeutics 28: 403

- 409.**APPENDIX 2. ACKNOWLEDGMENTS**

**The following individuals have con tributed 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, APHI S, Animal Care Dr. Scott Larsen, University of California, Davis Dr. William A. L indsey , Feld Inc. Dr. Konstantin Lyashchenko, Chembio Diagnostic Sy stems, Inc. Dr. Joel Maslow, University of Pennsylvania Dr. Bob Mey er, USDA, APHI S, Veterinary Services Dr. Susan K. Mikota, Elephant Care International Dr. Richard Montali, Dr . C. Douglas Page, Jacksonville Zoo Dr. L inda 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 .A nimal 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. Kath y Orloski, USDA, APHI S, Veterinary Services Dr. Janet B. Payeur, USDA, APHI S, 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 I saza, 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 d angerous 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 shed ding tuberculosis organisms whereas a negative result is non

- diagnostic. **Introduction** Tuberculosis in Asian elephants ( Elephas maximus ) has been sporadically r eported in the literature for man y ye ars (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/ElephTBGuidelines200 0.html). Compliance with this policy r equires 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 tr unk wash technique as the authors are currentl y 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 man y 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. Ino ur 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 sy ringe, 1 gallon plastic zip lock ty pe bags (heav y duty), and sterile, 50 ml, screw top, plastic jar or centrifuge tube. As long as attention is given t o 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 si mply

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 tr unk 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 anattempt to obtain a representative sample of the na sal 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 r estrained by

the handlers so that the tip is held up. The 60 ml sy ringe 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 f ound inside the trunk. The collection of moderate amounts of foreign material does not i nvalidate 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 carefull y transferred into a labeled container. Id eall y, the sample is refrigerated and sent directly t o a laboratory

f or processing and mycobacterial culture. If the sample cannot be sent directly f or culturing, it may

b e frozen in a regular freezer (

- 20 to
- 10 o C) until it can be sent to the laboratory . Often the recommended three d aily c ultures samples are collected and frozen until all samples are collected and the batch of samples can be sent to the laboratory together. **Discussion** Id entification of a M. tuberculosis infected animal has significant management implications to both the animal and the collection. Management of the infected animal may r equire 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 e xpensive. Inthe 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 on ly d iagnostic 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 b e very d angerous to the handlers. This is particularly true when attempted on an uncooperative elephant, because an y attempts to manually

r estrain 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 safet y of the procedure. In some cases, with potentially d angerous or unpredictable animals, an increased level of handler safet y can be obtained by h aving the animal lie in sternal or lateral recumbency prior to sample collection. This technique does not guarantee safet y or successful sample collection, as it still requires cooperation of the animal and does not replace adequate training. Inthe c ase 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 full y cooperative and, therefore, usually requires extensive training prior to the collection. A seco nd safet y 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 muc us 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 m ethod of detecting infected animals has several limitations that are best illustrated by e xamination of the underlying biological as sumptions. The first assumption is that most infected elephants have respiratory i nfections. Although the literature suggests that most infected elephants have respiratory i nfection, there have been no comprehensive necrops y studies to confirm these observ ations. 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. Iti s unknown what proportion of elephants can carry l atent 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 t esting period. Currently i t is unknown if s hedding animals pass organisms periodicall y or continuously
. Finall y, 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 o vergrow or ma sk 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 infec ted; 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 onl y detect animals shedding tuberculosis through the trunk, and not detect all ani mals that are infected. However, with time and repeated cultures of all animals in the population, it may b e possible to detect and treat most of the elephants shedding infectious organisms. If these animals are then treated properl y and shedding of organi sms 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., I saza, R., Dunker, F., West, G., L indsay , W., Larsen, R.S., Salman, M.D., Chatterjee, D., Pay eur, J., Whipple, D., Thoen, C., Davis, D.S., Sedgwick, C., Montali, R.J., Ziccardi, M., and Maslow, J. Epidemiology and d iagnosis 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 Day ton 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.Pay eur@aphis.usda.gov **Dr. Beth Harris** Head, M ycobacteria and Brucella Section (515) 337

- 7362 Fax: (515) 337

- 7315 Email: Beth.N. Harris@aphis.usda.gov **Dr. Suelee Robbe -**  **Austerman** Veterinarian, M ycobacteria 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.** Itis also helpful to call or email NVSLcontacts when sending TB suspects to schedule testing and relay any relevant history

of the case. NVSLTrunk wash cost: $98 per sample for processing which includes a Gen Probe® DNA probe on an y 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 anal ys is, spolioty

ping and VNTR genoty

ping. DNA fingerprinting of M. tuberculosis or M. bovis isolates is also available. Antimicrobial susceptibility testing is available for M. tuberculosiscomplex organisms for $112.00 per isolate. Please c ontact NVSLat (515) 337

- 7388 for test

schedule. To establish an account at NVSLfor 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/an imal_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 in structions, and requisition form: http://www.nationaljewish.org/research/clinical

- labs/about/learn/mycobac/index.aspx Serum sample submission: it is import ant 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. **HISTOPA THOLOGY**  **Scott P. Terrell, DVM, Diplomate ACVP** SSP Pathology

Advisor Disney ’s Animal Kingdom 1200 N Savannah Circle Ba y 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 an y gross lesion and complete set of tissues including lung, liver, spleen, mesenteric ly

mph nodes, bronchial l ymph nodes and other major organs. Use leak proof container. **USDA APHIS NVSL Pathobiology Laboratory**  1920 Day ton 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 L aboratory (515) 337

- 7526 Email: Arthur.J.Davis@aphis.usda.gov **Dr. Mark Hall** Hea d Pathological Investigations (515) 337

- 7927 Email: Mark.Hall@aphis.usda.gov Send formalin sections of an y gross lesion and target tissues (lung, liver, mesenteric and bronchial ly

mph 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 Medi**  **cal 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 Veteri nary Services Laboratories**  USDA APHI S NVSL 1920 Day ton 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 b y 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: My**  **cobacterium 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 cy steine (NALC); Sigma catalog number A

- 7250 2.5 NaOH
- Sodium citrate solution; (NVSL#10687)

2.5.1.1 Dissolve 29 gm of sodium citrate deh ydrate in 1000 ml of Super Q H 2 O. Dissolve 40 gm of sodium hydroxide pellets in 1000 ml of Super Q H 2 O. 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 vancom ycin, amphotercin B, and nalid ixic 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 H 2 O. Bring to a rolling boil. Dispense as requested. Autoclave 20 min. at 121°C for flasks, 15 min. for tubes. 2.7.2 Super QH 2 O 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 H 2 O. Add 10N NaOH drop b y drop until solution clears and QS to 1000 ml. Filtersterilize solution thru a 0.22μm filter into sterile j ug with bell end. Dispense (wearing gloves), 20 ml into a 50 ml sterile conical tube. Caution

– chemical is carcinogenic. 2.7.4 Vancom ycin (NVSL#40151) 10 ml 2.7.4.1 Combine 9.346 gm with 1000 ml of distilled Super Q H 2 O 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 H 2 O to a 100 mg Amphotericin B (Fungizone). Shake gently until dissolved and dispense as requested. 2.7.6 Combine BHI broth and Super QH 2 O. Aut oclave for 20 min at 121°C. Cool to 50°C. Remove 25 ml of cooled broth and discard. Add Nalidixic Acid, Vancom ycin, and Amphotericin B. Mix well. Dispense in tubes and cover with foil because the Amphotericin B is light sensitive. Store in

- 20°C freez er. Media is good for 3 months. 2.8 37oC CO 2 incubator preferred 2.9 Media set

- up (one tube of each per sample): 2.9.1 Middlebrook 7H10 w/gl ycerol; NVSL #10941 or BBL Middlebrook and Cohn 7H10 Agar tubes (BBL #220959). 2.9.2 Middlebrook 7H11 w/gl ycerol; NVSL #10942 or BBL S even 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 Ery thromycin (32 μg/ml) 2.10 1 ml tuberculin sy ringes 2.11 5 ml sy

ringes 2.12 Slant tray s, media tube baskets 2.13 Vortex 2.14 Sterile swabs **3. Procedures**  3.1 Carefull y 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 b y 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 Alternatel y: 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 Sorval l 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

- Acet yl

- L
- cy steine (NALC)/NaOH

- sodium citrate solution according to the following proportions: Volume (ml) Na OH
- sodium citrate a (ml) NAL C (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% sodi um citrate b. Allow NAL C 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 NAL C solution. 3.7 Vortex or vigorousl y 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 NAL C solution may

need to remain in contact with the sample for up to 20 + 5 minutes. 3.8.2 NAL C is a mucoly tic agent and this procedure reduces or eliminates contaminating bacteria while releasing M ycobacteria 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 m inutes at 6000g and 10oC. 3.11 Carefull y 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 In cubate overnight at 37 ± 2oC. 3.14 Vortex or vigorousl y 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/gl yc erol, Middlebrooks 7H11 w/ glycerol, M ycobactosel 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 sy

ringe is used to add 2 ml of Ery thromycin 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 sy ringe, add 5 ml of the reconstituting fluid to the vial of PANTA. 3.16.3 Using a 1 ml tuberculin sy ringe, 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 Plac e the inoculated solid media on a slant rack and incubate overnight at 37 ± 2oC in 10% CO 2 , if available. 3.18.1 In cubation in an atmosphere of CO 2 will encourage earlier growth. 3.18.2 After being incubated overnight on a slant rack, the media tubes can be st ored 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 M yc obacteria 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 b y 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. BAC TEC 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. Is enberg (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 L imo View Lane Hohenwald, TN 38462 Tel: 931

- 796
- 7102 Cell: 931

- 628
- 5962 Email: smikota@elephantcare.org Website: www.elephantcare.org **ELE PHANT TB STAT** 

**– PAK** **® ASSAY AND MAPIA**  **TM Konstantin Lyashchenko, Ph.D.**  Research Director, M yco bacterial Immunology Chembio Diagnostic S ys tems, 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, D**  **VM, Diplomate ACVP** SSP Pathology

Advisor Disney ’s Animal Kingdom 1200 N Savannah Circle Ba y 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 A sian [ ] African [ ] ISIS# ______ Studbook # _________

___ Name ____________ Age: _________ [ ] actual [ ] estimate Sex: [ ] male [ ] female SAMPLE COLLECTION IN FORMATION **Date of sample collection: ___________ Time of collection: __________**  **Site of sample collection:** [ ] ear vein [ ] leg vei n [ ] 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 appl y) [ ] 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 currentl y receiving an y 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.org54 **Consent Form for Use of Serum by Elephant SSP** I give consent for the serum submitted to the Elephant Species Survival Plan (SS P) 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 b y the SSP veterinary advisor in providing health

- related recommendat ions 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 c onsent 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: Cultur**  **e negative; ST** 

**AT -**  **PA K** **® non**  **- reactive;**  **no exposur e**  **past 12 months**

No treatment or travel restrictions If exposed to untested elephant in previous 3 months repeat ST

AT
- PA K ® in 3 months

**Group 2: Cultur e**  **negative; ST**

**AT -**  **PA K** **® non**  **- reactive;**  **exposed to cultur**  **e positive animal in past 12 months** 

Culture q 3 months for 1 ye ar post - exposure; then q 6 months for 2 years then annually i f cultures negative and ST AT
- PA K ® non reactive No travel or public contact until 2 non

- reactive ST

AT
PA K ® tests at 3 and 6 months post

- exposure56



### TB Management Group 3A

**TB Management Group 3B Group 3**  **A: Cultur e negative;**  **ST AT**  **- PA**  **K ®**  **reactive; MAPIA** **TM**

**/DPP ®**  **non**   ** reactive;** **no known exposur**  **e**

Culture q 3 months 1st year; q 6 months next 2 years then annually i f cultures negative and MAPIA TM/DPP ® remains non

reactive; repeat MAPIA TM/DPP® q 6 months for

1st year No treatment or travel restrictions

**Group 3 B: Cultur e negative;**  **ST AT**  **- PA**  **K ®**  **reactive; MAPIA** **TM**

**/DPP ®**  **non**   ** reactive;** **known exposur**  **e to cultur**  **e positive elephant (no time**  **limit)**

Culture q 3 months 1st year; q 6 months next 2. Repeat MAPIA TM/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 ye ar; restrictions removed if results unchanged. If results change, elephant will change

group.57



### TB Management Group 3C

**Group 3C: Cultur e negative; ST**  **AT****** **PA K** **® reactive;**  **MAPIA TM**  **/DPP ®**  **reactive; no known**  **exposur e** 

Culture q 3 months 1st year; q 6 months for life. Repeat MAPIA TM/DPP ® q 3 months 1st year; q 6 months next 2 years . If cultures negative after 3 yrs, resume annual serological testing according to these

Guidelines

No travel or public contact until the 1st year of testing has been completed. Consider treatment

- see text58



### Management Group 3D

**Group 3D: Cultur e negative;**  **ST AT**  **- PA**  **K ®**  **reactive; MAPIA** **TM**

**/DPP ®**  **reactive; known**  **exposur e to cultur**  **e positive elephant (no time limit)** 

Culture q 3 months 1st year; q 6 months for life. Repeat MAPIA TM/DPP ® q 3 months 1st year; q 6 months next 2 years. If cultures negative after 3 y

rs, resume annual serological testing according to these

Guidelines.

No travel or public contact until 1st year of testing has been completed. Consider treatment

- see text59



### Management Group 4 **Group 4: Cultur e**  **positive for M**  **. tuberculosis**  **complex**

Perform ST

AT
- PA K ® and MAPIA TM/DPP ® ; submit positive cultures to NVSL

f or genoty ping; request antibiotic sensitivity t esting

In itiate 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