Nepal Elephant TB Control and Mgt Action Plan.pdf - Elephant Care ...

Nepal Elephant TuberculosisControl and Management Action Plan(2011-2015)Government of NepalMinistry of Forests and Soil ConservaonDepartment of Naonal Parks and Wildlife ConservaonBabarmahal, Kathmandu, Nepal2011

Nepal Elephant TuberculosisControl and Management Action Plan(2011-2015)Government of NepalMinistry of Forests and Soil ConservationDepartment of National Parks and Wildlife ConservationBabarmahal, Kathmandu, Nepal2011

Copyright © and Published By:Government of NepalMinistry of Forests and Soil ConservaonDepartment of Naonal Parks and Wildlife Conservaon (DNPWC)G.P.O. Box 860, Babarmahal, Kathmandu, NepalTel: +977-1-4220912, 4220850, 4227926Fax: +977-1-4227675E-mail: info@dnpwc.gov.npWebsite: h p://www.dnpwc.gov.npCita!on:DNPWC (2011): Nepal Elephant Tuberculosis Control and Management Acon Plan (2011-2015). Governmentof Nepal, Ministry of Forests and Soil Conservaon, Department of Naonal Parks and WildlifeConservaon, Kathmandu, Nepal.

Table of ContentsSummaryAbbreviaonsDefinionsPart I - TB Management 1-151.1 Introduc!on 11.2 TB Tes!ng Procedures and Management Groups 41.3 Descrip!on of Diagnos!c Methods 101.4 Segrega!on Procedures 101.5 TB Treatment Protocol 111.6 Human Health Considera!ons 15Part II - Acon Plan 17-262.1 Vision 182.2 Goals 182.3 Objec!ves 182.4 Issues, Strategies and Program Ac!vi!es 18Issue I: Risk of tuberculosis to the cap!ve elephants 18Issue II: Risk of transmission of TB from cap!ve to wild popula!onof elephants, rhino and other wildlife species 20Issue III: Risk of transmission of TB from human to elephant andfrom elephant to human 21Issue IV: Possibility of impact of TB on wildlife tourism in Nepal 22Issue V: Insufficient technical and managerial capacity 22Issue VI: Insufficient financial resources 23Issue VII: Inadequate research on elephant and free ranging wildlife species 232.5 Budget 25Bibliography 27-29Appendices 31-40Appendix I: Sample drug dose and cost calcula!ons 32Appendix II: Plan cer!ficates 34Appendix III: Nepal elephant postmortem protocol 35Appendix IV: Contacts 39

SummaryThe Nepal Elephant Tuberculosis (TB) Control and Management Acon Plan (NETCMAP) isbased on a goal of minimizing all factors leading to the risk of TB transmission from capveelephants to the wild. The program will be launched by the Ministry of Forests and Soil Conservaon- Department of Naonal Parks and Wildlife Conservaon (DNPWC) with technicalsupport from naonal and internaonal conservaon partners including the Naonal Trustfor Nature Conservaon (NTNC) Nepal, the Buffer Zone Management Commi ees (BZMCs)Nepal, Elephant Care Internaonal (ECI) USA, World Wildlife Fund (WWF) Nepal, and theHotel Associaon Nepal (HAN). The plan will be implemented under close supervision of aveterinarian with adequate knowledge of animal TB.Issues idenfied in capve elephant TB control and management include: i) Risk of tuberculosisto the capve elephants, ii) Risk of transmission of TB from capve to wild populaonof elephants, rhino and other wildlife species, iii) Risk of transmission of TB from human toelephant and from elephant to human, iv) Possibility of impact of TB on wildlife tourism inNepal, v) Insufficient technical and managerial capacity, vi) Insufficient financial resources,and vii) Inadequate research on elephant and free ranging wildlife species. A range of acvi-es have been proposed to address these issues.All capve elephants in Nepal will be screened for TB. Based on diagnosc test results allelephants will be placed in a management group as described in Secon 1.2 TB TesngProcedures and Management Groups.It is recommended that elephants entering Nepal from India must first be screened for TBusing the Elephant TB Stat-Pak® test. Only elephants that are non-reacve on the screeningtest should ent er Nepal. Elephants that do not have proper documentaon (wri enand photograph) from a licensed veterinarian of pre-entry tesng using the Elephant TBStat-Pak® test will be tested once they are in Nepal. Any elephants that are posive on theElephant TB Stat-Pak® test will be required to leave or undergo treatment as described inthis protocol.Elephants will be treated for TB according to protocols described in Secon 1.5. All elephanthandlers and other staff working in close proximity to elephants will be screened annuallyfor TB using free services available in Nepal. In the case of new elephant handlers, thoroughscreening for TB will be undertaken before they begin work with elephants. Annual cerficateswill be issued to facilies in compliance with this program (Appendix II Plan Cerficates).

All elephants in Nepal will be microchipped. Microchipping will take place at the me of TBtesng for those elephants that are not already microchipped. All privately-owned elephantswill be licensed and registered. Licenses will be issued by DNPWC. License renewalswill be issued in conjuncon with regular TB tesng and documentaon of compliance withthe TB Plan specified in this document. Records of licensure, registraon, and microchipswill be maintained at TB program office at Sauraha. Duplicate records will be maintained atDNPWC headquarters.A full necropsy will be performed on all elephants that die in Nepal. Postmortem examina-on will include a comprehensive gross necropsy, histopathology, and culture for TB. Postmortemexaminaon can provide a definive diagnosis and important informaon for theTB program. Refer to the Nepal Elephant Postmortem Protocol in Appendix III. Completednecropsy reports must be sent to the TB Program Office at NTNC within 30 days.

AbbreviationsAREASBCCBZBZMCCNPCVLDLSODoLSDOTSDNPWCDPPECIHANIAASLAMPMAPIAMOUNATANPNTNCNVSLPAPCRTBUSDAUSFWSWRZSLAsian Rhino and Elephant Acon StrategyBiodiversity Conservaon CentreBuffer ZoneBuffer Zone Management Commi eeChitwan Naonal ParkCentral Veterinary LaboratoryDistrict Livestock Service OfficeDepartment of Livestock ServiceDirectly Observed Therapy Short-CourseDepartment of Naonal Parks and Wildlife ConservaonDual Pathology Pla ormElephant Care InternaonalHotel Associaon NepalInstute of Agriculture and Animal SciencesLoop-mediated Isothermal AmplificaonMul-angen Print ImmunoassayMemorandum of UnderstandingNaonal An-Tuberculosis AssociaonNaonal ParkNaonal Trust for Nature ConservaonNaonal Veterinary Services LaboratoriesProtected AreaPolymerase Chain ReaconTuberculosisUnited States Department of AgricultureUnited States Fish and Wildlife ServiceWildlife ReserveZoological Society of London

DefinitionsAtypical mycobacteria: see non-tuberculous mycobacteriaCulture posi!ve for M. tb complex: Isola!on and iden!fica!on of M. tuberculosis complex organismsfrom any body site using standard mycobacterial methods.Culture posi!ve (M. tb complex) elephant: An elephant from which a M. tuberculosis complex organismhas been isolated. A culture posi!ve elephant is considered posi!ve un!l it has met the treatmentrequirements as outlined in this document.Dual Path Pla"orm (DPP) Vet ® TB test: A field test used to confirm Elephant TB Stat-Pak ® test results.The DPP Vet ® TB test detects an!bodies to specific TB an!gens. Elephants that are reac!ve on the ElephantTB Stat-Pak ® and react on line 2 or lines 1 and 2 of the DPP Vet ® TB test are considered TB-infectedbased on current scien!fic data (Greenwald et al. 2009). Infected elephants may or may not be sheddingTB at the !me of tes!ng. The test was developed by Chembio Diagnos!c Systems Inc (Medford, NY, USA).Elephant TB Stat-Pak ® test: A qualita!ve screening test for TB in elephants which detects an!bodies toM. tuberculosis and M. bovis in elephant sera, plasma or whole blood. During development, the Stat-Pakwas referred to as the “RT” or “Rapid Test.” The Elephant TB Stat-Pak ® test is a USDA-approved product.(See Lyashchenko et al. 2005, Lyashchenko et al. 2006, Greenwald et al. 2009).Exposed: An elephant that 1) has had direct contact (touching or shared air space) with a known TBinfectedelephant or human or 2) that has been kept in a stall adjacent to an infected elephant or which3) has had any other close associa!on with an infected elephant or human.Genotyping assay: A technique for the iden!fica!on and analysis of polymorphism in certain types ofrepeat units in DNA. Restric!on fragment length polymorphism (RFLP) and variable number tandemrepeat (VNTR) are examples of genotyping techniques.Incidence: The rate at which a certain event occurs, for example, the number of new cases of a specificdisease occurring during a certain period.Herd: A group of elephants maintained on common ground. Alterna!vely, two or more groups of animalsunder common ownership or supervision that are geographically separated, but that may have aninterchange or movement of animals or personnel (such as elephants owned by DNPWC).Loop-mediated isothermal amplifica!on (LAMP): A nucleic acid amplifica!on assay that has been usedto detect TB in human pa!ents in Nepal (Pandey et al. 2008).

Mul angen print immunoassay (MAPIA): A lab-based confirmatory test for the Elephant TB Stat-Pak,currently available only in the USA (Lyashchenko et al. 2000, Lyashchenko et al. 2006, Greenwald et al.2009).Mycobacterium: A genus of bacteria in the family Mycobacteriaceae.Mycobacterium avium (M. avium): A non-tuberculous mycobacteria that causes TB in birds and occasionallyhumans and other species. M. avium may be isolated from non-clinically affected elephants andis usually considered as an environmental contaminant.Mycobacterium bovis (M. bovis): The primary causa"ve agent of TB in ca#le, bison, and deer; may alsoinfect other mammals including pigs, humans, primates, elephants, and non-cap"ve ungulates.Mycobacterium tuberculosis (M. tuberculosis): The primary causa"ve agent of tuberculosis in humans;may also affect a variety of animals, including primates, pigs, ca#le, dogs, parrots, elephants, and rhinos.Mycobacterium tuberculosis complex (M. tb complex): A group of mycobacteria which includes M. tuberculosis,M. bovis, M. africanum, M. micro!, M. cane", M. caprae, and M. pinnipedii. A vaccine strainderived from M. bovis (M. bovis BCG) is some"mes included in this group.N-95 mask: A mask that is cer"fied to protect against TB when properly fi#ed.Necropsy: A postmortem (a$er death) examina"on to determine the cause of death.No isolaon: Absence of growth of M. tuberculosis complex organisms from trunk wash, feces, "ssue, orother samples using standard mycobacterial culture methods. Failure to isolate organisms may be due tothe following reasons:i. the animal is not infectedii. the animal may be infected but TB organisms were not present in the sample submi#ed for analysisiii. sampling error (culture overgrowth by contamina"ng organisms, inadequate sample, laboratory error)iv. improperly handled or shipped sampleNon-reacve: A nega"ve finding on a test.Non-tuberculous mycobacteria (NTM): Mycobacteria that do not generally cause granulomas. MostNTM are saprophytes found in soil or water. They are typically non-pathogenic but may occasionallycause disease in humans and animals including elephants. It is also referred to as “atypical” mycobacteriaor “Mycobacteria other Than TB” (MOTT).Nucleic acid amplificaon test: A technique that amplifies en""es such as DNA or RNA.PCR (polymerase-chain reacon): A nucleic acid amplifica"on technique in which specific sequences of

nucleic acid (DNA or RNA) are replicated, allowing for detecon of target sequences.Prevalence: The total number of cases of a specific disease in a given populaon at a given me.Quaran ne: Enforced segregaon to prevent the spread of an infecous disease.Reac ve: Presence of response; in the context of serological tesng for TB in elephants, a reacve resultindicates that an angen-anbody reacon has occurred.Segrega on: Separaon from others. In context of this protocol, segregaon at the home faciliesmeans to keep an elephant apart from other elephants by a minimum distance of 15 meters and tomaintain an empty stall on either side of the segregated elephant. Segregaon can also be at a locaonremote from the home facility such as the segregaon facility in Kasara.Sensi vity: A measure of the ability of a test to idenfy infected animal (i.e. the percentage of true posi-ve results). Sensivity is the frequency of a posive or abnormal test result (e.g. a test that is outside ofthe reference interval) when a disease is present (i.e. the percentage of true posive results). Sensivity= [TP ÷ (TP + FN)] X 100 where TP = true posive; FN = false-negave).Shedding: Acve excreon of live TB organisms, usually in respiratory secreons. Indicates that anelephant is infecous to other animals or humans. Shedding is determined by culture, PCR, or othertechnologies that detect TB organisms.Specificity: A measure of the ability of a test to idenfy non-infected animals (i.e. the percentage of truenegavetest results). Specificity is the frequency of a negave or “normal” test result when a disease isabsent (i.e. the percentage of true-negave (TN) test results. Specificity = [TN ÷ (TN + FP)] X 100.Triple sample method: A method of sample collecon whereby 3 samples are obtained on separatedays.Trunk wash and modified trunk wash: Procedures used in elephants to obtain a sputum sample forculture or PCR.TB-infected: An elephant that is reacve on the Elephant TB Stat-Pak ® test and the DPP Vet ® TB test orfrom which TB complex organisms have been idenfied by culture or molecular techniques.TB suspect: An elephant that is reacve on the Elephant TB Stat-Pak ® test.Tuberculin skin test: A screening test for TB used in humans and some animals. The test cannot accuratelydiagnose TB in elephants and it is not recommended.

Part ITB Management

Nepal Elephant Tuberculosis ulosisControl andManagement agemenAction Plan (2011-2015)2015)1.1 IntroduconBackgroundElephants belong to the Family Elephandae inanimal kingdom and are the largest mammaliansamong terrestrial animals. Elephants are believedto have evolved some 50 million years ago. Outof about 350 species under 44 Genera of thisfamily, only two genera (Elephas and Loxodonta)and three species remain today – the Asianelephant (Elephas maximus), the African bushelephant (Loxodonta africana), and the Africanforest elephant (Loxodonta cyclo!s). The Asianelephants are distributed in 13 Asian countries:Nepal, India, Bhutan, Bangladesh, China,Myanmar, Thailand, Indonesia, Laos, Vietnam,Cambodia, Malaysia, and Sri Lanka.Taming of Asian elephant began in the Indusvalley (now in Pakistan) around 4,000 yearsago. Taming of elephants was done for variouspurposes. Elephants were trained and usedin warfare in India, China and Persia over thecenturies. In the past, elephants were extensivelyused across their range in mber industries,parcularly in logging and li!ing large logs, andsafari hunng. Today, elephants in south andsouth-east Asia are used in tourism and protectedarea management. In some places, elephants aresll used in circuses because of their ability tobe trained to perform, and also because of theirimpressive size. Elephants in Hindu communiesare regarded as a symbol of Lord Ganesh.In Nepal, elephants were used for Royal hunngsafaris for Royalty. Elephants were also keptprivately and were used as a symbol of status andmeans of transportaon. A!er the creaon of theProtected Areas, elephants have been extensivelyused in patrolling, surveillance, research andmonitoring, means of transportaon and wildlifetourism. Wild elephants in Nepal are confinedin the protected areas mainly in SuklaphantaWildlife Reserve, Bardia Naonal Park, ChitwanNaonal Park, Parsa Widlife Reserve, and nearbyforest areas. However, elephants from the WestBengal State of India frequently visit the forestareas in Jhapa, Morang, Udayapur and SunsariDistricts in the eastern lowland. A low numberof private elephants are also reported fromKapilbastu, Rautahat and Sunsari Districts.Elephants are prone to various infecous diseasesincluding tuberculosis, anthrax, haemorrhagicsepcaemia, foot-and-mouth disease, rabies,tetanus, encephalomyocardis virus, pox,salmonellosis, elephant endotheliotropic herpesvirus infecon, and others. Among these,tuberculosis (TB) is a chronic, debilitang diseasethat affects capve elephants world-wide. TB is athreat to capve elephants, wild elephants, otherwild and capve animals, and humans.TB was first idenfied in capve elephants inNepal in 2002 (Gairhe 2002). Between 2002 and2009, there were seven deaths in which TB wasdiagnosed among government and NTNC-ownedelephants. Surveillance for TB in elephants in2

Nepal Elephant Tuberculosis Control andManagement Action Plan (2011-2015)Nepal began in 2006. Culture and experimentalserological tests were used to screen elephantsat this me. One of these tests - the ElephantTBStat-Pak® (Chembio Diagnoscs, Inc, Medford,New York, U.S.A. www.chembio.com) wassubsequently licensed by the United StatesDepartment of Agriculture (USDA) in 2007 as ascreening test for TB in elephants.Cultures collected in 2006 and evaluated atthe Naonal Tuberculosis Instute (NTI) inNepal and the Naonal Veterinary ServicesLaboratories (NVSL) in Ames, Iowa, U.S. provednon-diagnosc due to contaminaon, storage,and transport issues. The same problems wereencountered when elephant respiratory sampleswere submi ed to the Nepal An-TuberculosisAssociaon (NATA) in subsequent years. Theseand other issues, such as intermi ent shedding,limit the value of culture as a primary diagnosctechnique. In Sweden, only 7 of 189 respiratorysamples collected from 5 elephants confirmedTB-infected at postmortem were cultureposive(Moller et al. 2005). In Thailand, TB wasisolated from only 2 of 60 respiratory samplescollected from confirmed TB-infected elephants(Angkawanish et al. 2010).Culture as well as molecular diagnosctechniques such as PCR is nonetheless valuableand improving the methods to apply thesetechniques to elephants remains an importantgoal. Collaboraons have been established withthe Nepal An-tuberculosis Associaon (NATA)and the Center for Molecular Diagnoscs –Nepal (CMDN) for this purpose. In 2009-2010,these laboratories confirmed Mycobacteriumtuberculosis, the human strain of TB, as thecausave agent in three elephants in Nepal.TB can be transmi ed from humans to elephantsand from elephants to humans. In 2008, over100 elephant handlers were tested for TB, butno cases of TB were detected. TB is a longtermdisease and previous handlers, no longeremployed may have been the source of infeconfor the three M.tb-infected elephants. Theseelephants may also have acquired TB from otherinfected elephants. It is presumed that elephantsin Nepal may also harbor the bovine strain of TBhowever laboratory capacity to diagnose M. bovisis limited and this has not yet been confirmed.This is an area for further research.Tuberculosis can be caused by a number ofmycobacterial species and many of these bacteriainfect mulple hosts. The recognion of TB inelephants is smulang a shi! in thinking abouthow disease is perceived. Generally zoonoses arethe concern of the public health community butTB is an anthroponosis (an infecous disease inwhich a disease causing agent carried by humansis transferred to other animals) highlighng theneed for involvement of both animal and humanhealth professionals and agencies. A furthercomplicaon in the human community has beena rise in HIV/AIDS; a disease o!en associatedwith tuberculosis which it promotes throughimmune suppression of the human host and bywhich Nepal is increasingly suffering. Therefore,a more holisc view of tuberculosis surveillanceand control is needed. This requires a muchbroader, “One-Health” (Human - Livestock -Wildlife) approach which is gaining momentumin the global health community. This paradigmtakes into account the enre epidemiologyof the disease, examines all the suscepblespecies, carriers and likely drivers of infecon andepidemics. Part of this elephant TB strategy mustbe to integrate with the other health professionsconcerned with livestock and humans, bringingthe perspecve and knowledge gained from theelephant and wildlife experiences. A One HealthNepal iniave is in the conceptual stage and TBis considered a perfect example to provide proofof concept and be er management, diagnosis,control and prevenon of the disease amongstall animal species and most importantly, humanbeings.3

Nepal Elephant Tuberculosis ulosisControl andManagement agemenAction Plan (2011-2015)2015)Treatment for TB amongst elephants began in2008. Exposure to known cases of TB and areacve Elephant TB Stat-Pak ® result were iniallyused as the basis for treatment. The currentdecision tree includes the DPP ® VetTB test (see1.5. TB Treatment Protocol).Roune surveillance of elephants and theirhandlers together with early diagnosis andtreatment are essenal to control TB. It is hopedthat these guidelines will serve as a science-basedmodel for other Asian elephant range countries.The Nepal Elephant Tuberculosis (TB) Controland Management Acon Plan (NETCMAP) isbased on a goal of minimizing the risk of TBtransmission from capve elephants to the wildby managing TB at the capve-wild interface Thisplan outlines methods to diagnose, treat, andmanage TB in elephants in Nepal. The TB controland management program will be launched bythe Ministry of Forests and Soil Conservaon- Department of Naonal Parks and WildlifeConservaon (DNPWC) with technical supportfrom naonal and internaonal conservaonpartners including the Naonal Trust for NatureConservaon (NTNC) Nepal, the Buffer ZoneManagement Commi ees (BZMCs) Nepal,Elephant Care Internaonal (ECI) USA, WorldWildlife Fund (WWF) Nepal, and the HotelAssociaon of Nepal (HAN). The plan will beimplemented under the close supervision of aveterinarian with adequate knowledge on animalTB.1.2 TB Tesng Procedures andManagement GroupsThe TB Plan Veterinary Officer and VeterinaryTechnician will be responsible for tesng allelephants in Nepal.Procedure1. Perform the Elephant TB Stat-Pak ® test2. If Elephant TB Stat-Pak ® test is non-reacvethe elephant is considered TB-free at the meof tesng.3. If Elephant TB Stat-Pak ® test is reacveperform the DPP ® VetTB test.4. If DPP ® VetTB test is non-reacve theelephant is TB-suspect.5. If DPP ® VetTB test is reacve the elephant isconsidered TB-infected.Management groupsGroup 1. TB-free: An elephant that is nonreacveon the Elephant TB Stat-Pak ® test.Connue every other year tesng with theElephant TB Stat-Pak ® . No segregaon or workrestricons.Group 2. TB-suspect. An elephant that is reacveon the Elephant TB Stat-Pak ® test and nonreacveon the DPP ® VetTB test. There are twoopons.Opon 1 (recommended especially for elephantsthat have contact with wild elephants or rhinos):Iniate prophylacc treatment (described insecon 1.5). Elephants in this sub-group shouldbe segregated while they are adjusng totreatment and should not work unl they havecompleted 30 consecuve days of treatment (30full doses). Elephants that have had a knownexposure to a TB infected elephant or human orwhose exposure status is unknown should followOpon 1. Following compleon of treatment,elephants will be designated as Group 2 - Treated.Opon 2: Test with the DPP ® VetTB test at 6months following the first reacve Elephant TBStat-Pak ® result. If the DPP is reacve at this me,the elephant changes to Group 3. If the DPP isnon-reacve at 6 month test again at one year.If the test is non-reacve at one year, monitorannually by the DPP ® VetTB test. Elephants4

Nepal Elephant Tuberculosis Control andManagement Action Plan (2011-2015)5

Nepal Elephant Tuberculosis ulosisControl andManagement agemenAction Plan (2011-2015)2015)that have had no known exposure to an infectedelephant or human may elect Opon 2. Opon2 may also be suitable for old elephants or asa temporary opon for pregnant elephants.Elephants in this sub-group should not workand must remain segregated from serologicallynon-reacve elephants and from wild elephantsand other wild ungulates unl they have had twonon-reacve DPP tests.Group 2 TB-suspect elephant may or may not beshedding TB organisms. Staff caring for elephantsin this group should consider personal protecon(see secon 1.6).The combinaon of a reacve Elephant TB Stat-Pak ® test and a non-reacve DPP ® VetTB testmay be due to the one of the following:1. Early sero-conversion. The elephant is trulyinfected but seroconversion on the ElephantTB Stat-Pak ® has preceded seroconversion onthe DPP. TB is a chronic disease and the body’sanbody response may vary with the stage ofthe disease which can affect test results. Furtherresearch to be er understand the elephantimmune system is on-going (Landolfi et al. 2009,Landolfi et al. 2010). In almost all cases to dateculture posive (shedding) elephants have beenreacve on both the Elephant TB Stat-Pak ® andthe DPP ® VetTB tests. While elephants thatare Elephant TB Stat-Pak ® reacve and DPP ®VetTB non-reacve are probably less likely tobe shedding they may convert to DPP ® VetTBreacve status and / or become TB cultureposiveand shed TB organisms at any me. Thishas occurred in a several cases including a case inNepal.2. Prior treatment for TB.3. In rare instances, the Elephant TB Stat-Pak ®may produce false posive results. The ElephantTB Stat-Pak ® may cross-react with certain NTMsuch as M. szulgai, which caused the death oftwo African elephants (Lacasse et al. 2007).It has been demonstrated however, that theElephant TB Stat-Pak ® and DPP ® VetTB tests donot react with 11 other NTMs nor with M. avium(Greenwald et al. 2009). In one study, only 4 of147 cases in the U.S. and Europe were consideredto be false-posives (Greenwald et al. 2009).Group 3. TB-infected. An elephant that is reacveon the Elephant TB Stat-Pak ® and the DPP ®VetTB tests and /or from which M. tuberculosiscomplex organisms have been idenfied byculture or molecular techniques.• Segregate according to procedures in Secon1.4.• Collect respiratory samples for culture andmolecular diagnoscs (see procedure below)before iniang treatment.• If culture is posive perform drug sensivitytesng.• Iniate treatment as soon as possible andsuspend work and public contact unl theelephant has completed 90 consecuve daysof treatment (90 full doses). Treat accordingto protocols in secon 1.5• Elephants that have a posive culture areknown to be shedding, are a higher risk totransmit TB and should be segregated for theduraon of treatment.• Instute precauons to protect human health.• Document that treatment is carried out inaccordance with these guidelines. The TBPlan Veterinary Officer will be responsible formonitoring treatment.• Monitor post-treatment with culture andmolecular techniques as the Elephant TB Stat-Pak ® test will remain reacve for some me.6

Nepal Elephant Tuberculosis Control andManagement Action Plan (2011-2015)See secon 1.5 for details.• Elephants that have completed treatment willbe designated as Group 3 - Treated.Note: TB-suspect and TB-infected elephantsshould be treated under most situaons. Thedecision to treat old or pregnant animals shouldbe made on a case by case basis. While veryold elephants with long-standing infecons areunlikely to be cured, treatment may preventshedding and older elephants (>50 years) inNepal have undergone treatment with minimal tono side effects.TB drugs have also been administered to lactangelephants with no side effects to mother or calf.In one case a pregnant elephant under treatmentaborted but it is unknown whether this wasassociated with the TB drugs or other causes.TB drugs are rounely administered to pregnanthumans with no ill effects.If a decision is made to withhold treatment ofa TB-infected elephant, the elephant should bemaintained in permanent segregaon for theremainder of its life and contact with wildlifestrictly prevented. Because of their highly socialnature, visual and auditory proximity to otherelephants is acceptable as long as the distanceprecludes any direct contact with respiratorysecreons, the elephant is cared for by aseparate handler (wearing personal protecon)and separate cleaning utensils are used for theinfected elephant. All untreated TB-infectedelephants are a risk to other elephants, humans,and wildlifeGroup 4. Untested: An elephant that has not yetbeen tested for TB. Untested elephants alreadyin Nepal (such as calves) should be tested assoon as possible. Elephants entering Nepalshould be tested prior to entering Nepal by alicensed veterinarian experienced in performingthe Elephant TB Stat-Pak ® test or by the TB PlanVeterinary Officer.Wri#en documentaon must include theelephant’s name; age; sex; owner’s nameand contact informaon; veterinarian’s nameand contact informaon; and test result. Aphotograph of the elephant and a photographof the test labeled with the elephant’s name anddate of the test must be included. The TB PlanVeterinary Officer has the authority to test newelephants or to re-test elephants to verify testresults upon arrival in Nepal.7

Nepal Elephant Tuberculosis ulosisControl andManagement agemenAction Plan (2011-2015)2015)8

Nepal Elephant Tuberculosis Control andManagement Action Plan (2011-2015)Respiratory Sample Collecon ProceduresThe Nepal Elephant TB Control and ManagementAcon Plan has established collaboraons withthe Nepal An-tuberculosis Associaon, theCenter for Molecular Dynamics-Nepal (CMDN),and the experts in this field to improve methodsto idenfy TB bacterial shedding in elephants.The following procedures may change as newknowledge is gained.Loop-mediated isothermal amplificaon (LAMP)is a molecular technique that has been used todiagnose TB in humans in Nepal (Pandey 2008).It is accurate in humans and may prove to be aneffecve diagnosc tool for elephants but shouldbe performed in conjuncon with culture andPCR. Collect samples in the a ernoon a er theelephant has been acve and moving around.Collecon of trunk wash or liquid nasal dripsample for culture, PCR, and LAMP1. Wear an N95 mask and exam gloves.2. Pre-label a plasc 50 ml screw top centrifugetube with the elephant’s name, project ID#,facility locaon, and date of collecon. Thissample will be for culture.3. Pre-label a 4.5 buffer-filled nunc tube for DNAextracon, PCR, and LAMP.3. Collect standard trunk wash sample or 10ml of a nasal drip sample in a shallow largediameter plasc tub or a large jar.4. Transfer the sample into a plasc 50-mlscrew top centrifuge tube by pouring or use adisposable syringe or pipe!e. If the sample isgrossly contaminated, pour through a gauzesponge.5. Dip a clean sterile swab into the sample andplace the swab in the buffer-filled nunc tube.6. Refrigerate samples unl they can be shipped.7. If plasc pipe!es or syringes have been used,place them in a jar containing bleach. Allowto remain for ~ 10 minutes then dispose intored biohazard container.8. Send samples by courier to CMDN. CMDNwill forward culture samples to NATA andDNA-extracted samples to Dr. Pandey’slab. Samples should reach NATA as soonas possible as prolonged storage me canadversely affect results.Collecon of oropharyngeal swab for culture1. Wear N-95 mask and exam gloves.2. Place a culture!e on the end of the aluminumrod designed for this purpose3. Swab the oro-pharyngeal region.4. Place the swab in the tube and compress theend to release the transport media.5. Label with the elephant’s name, project ID #,collecon date, and facility abbreviaon.6. Refrigerate; ship to CMDN as soon as possible.CMDN will forward to NATA.Collecon of oropharyngeal swab for PCR andLAMP1. Wear N-95 mask and exam gloves.2. Place a sterile co!on swab on the end of aplasc extension rod.3. Swab the oro-pharyngeal region.4. Place the swab in the pre-labeled, buffer-fillednunc tube.5. Label with the elephant’s name, project ID #,facility abbreviaon, and collecon date.6. Refrigerate; ship to CMDN as soon as possible.9

Nepal Elephant Tuberculosis ulosisControl andManagement agemenAction Plan (2011-2015)2015)1.3 Descripon of DiagnoscMethodsa. The Elephant TB Stat-Pak ® test is a bloodanbody test. A reacve test indicatesprobable TB infecon. Confirmaon withother tests is ideal - when possible and asdiscussed above. A non-reacve test indicatesno infecon and a TB-free status at the meof tesng. The Elephant TB Stat-Pak ® test isused as the standard roune TB screeningtest for elephants in Nepal. The test isadministered by the TB Plan veterinarian.b. The DPP VetTB test is a blood anbody testused to confirm the Elephant TB Stat-Pak ® test.The Elephant TB Stat-Pak ® and DPP ® VetTBtests have been shown to be early predictorsof infecon and may be reacve monthsto years in advance of detecon by culture(Greenwald et al. 2009). This provides theopportunity to treat elephants before theyshed and infect other elephants, humans, orwildlife.c. Culture is used to detect elephants that areacvely shedding TB organisms. Respiratorysamples obtained by collecng trunkdischarges are submi!ed to a laboratorythat is qualified to test for mycobacteria(this requires specific procedures). A posiveculture confirms that an elephant is acvelyinfected. Posive isolates should be tested toconfirm if the organisms were M. tuberculosisor M. bovis. Drug sensivity tesng (DST) foran-tuberculosis drugs (isoniazid, rifampin,and ethambutol) should be performed.Note that culture has inherent limitaons.Failure to isolate the organism does notrule out infecon. False-negave resultsmay occur 1) if the elephant is infected butorganisms are not shed on the day of samplecollecon or 2) contaminated trunk washsamples cause other organisms to overgrow.False-negave results allow TB disease toprogress and the infected elephant is a sourceto spread TB to other elephants, humans,or wildlife. Culture is expensive and manysamples may be needed to detect infecon. InSweden, of 189 trunk wash cultures collectedfrom 5 elephants confirmed TB-infected onpostmortem, only 7 samples were posive(Moller et al. 2005).Newer technologies that prove to be morereliable, sensive, or cost effecve fordetecng TB organisms in trunk wash or othersecreons will be incorporated into this planas they become available.d. PCR (polymerase chain reacon) is a type ofnucleic acid amplificaon test (NAAT). NAATsfacilitate the idenficaon of pathogens byamplifying and detecng specific nucleicacid sequences. PCR assays to detect TB inelephants in Nepal are under invesgaon bythe Center for Molecular Dynamics-Nepal (seeKay et al. 2011).e. LAMP (loop-mediated isothermalamplificaon) assay is a type of NAAT thatallows detecon of trace amounts of DNAunder isothermal condions. It has been usedto diagnose TB in humans in Nepal (Pandey etal. 2008) and is under invesgaon for use inelephants.1.4 Segregaon ProceduresSegregaon is an important management tool.Segregaon from wild elephants and otherwildlife (e.g. rhino) is considered a priority toprevent TB transmission to endangered wildlifepopulaons. Segregaon can also minimizetransmission to other capve elephants and tohumans. The goal of segregaon is to preventhealthy animals from coming into contact withrespiratory droplets or other secreons frominfected elephants.10

Nepal Elephant Tuberculosis Control andManagement Action Plan (2011-2015)Segregaon should be instuted as soon as testresults indicate. If elephants are to be treated,they should be rested and not used for work ortourism unl they have completed 30 consecuvedays of treatment (Group 2) or 90 consecuvedays of treatment (Group 3). If very old elephantsare to be maintained under permanentquaranne they should never be used for work ortourism.Segregaon should ideally take place in aseparate locaon (such as the Kasara segregaonfacility for government elephants). Privateelephants may be segregated at their homefacilies if the following guidelines are strictlyobserved:a. Segregated elephants should be kept at anappropriate distance (minimum 15 meters)from other animals within the camp andshould not be bred.b. Segregated elephants should be housed suchthat the adjacent stalls on either side areempty. These elephants should be maintainedon a high plane of nutrion. Gram andproduce (carrots, pumpkins, sugar cane etc.)should be added to the diet along with theclose monitoring of their daily raons.c. Separate tools should be used to clean andfeed segregated elephants and they should behandled last.d. Elephant handlers should wear N-95 maskswhen working in close proximity to theelephant and should wash their hands a erhandling, cleaning, or feeding.1.5 TB Treatment ProtocolGeneral ConsideraonsThe government will arrange for treanggovernment-owned elephants. Private ownerswill be responsible for treang privatelyownedelephants. Treatment protocols arebased on established treatment regimenssuccessfully used in the U.S. Treatment plans forindividual elephants will be developed underthe supervision of the TB Plan veterinarian.Treatment protocols will be updated as newinformaon becomes available.The goal is to prescribe TB treatment forelephants in Nepal using the currently availablescienfic informaon with the hope of curingthe disease. The intent is to strive to achieve thecurrently recommended dosages with allowanceto adjust the dosages for each individualelephant as needed to achieve compliance whileminimizing side effects.It is extremely difficult to understand the severityof TB disease that an individual elephant mayhave. It may not be possible to completelycure TB in elephants with advanced disease (>50% of the lung involved). So far, no standardmeasurements have been developed for“complete cure” at this me. See post-treatmentmonitoring guidelines below.Drug doses are best determined by obtaining anaccurate scale weight. A portable weighing scaleis available at the TB Plan office at NTNC. Thescale requires a pla$orm for the elephants tostand on. There are currently pla$orms at NTNC,Kasara, the Chitwan Ha&sar, and Bardia.If scale weighing is not possible, the weight maybe esmated using the chest girth method. Thechest girth should be measured 3 mes andaveraged to ensure accuracy. The chest girth ismeasured immediately behind the front leg. Theweight is calculated from the following formula(Hile et al. 1997):Weight in kg = 18.0 (Heart Girth in cm) – 3336.Group 1 elephants have no work restricons.Group 2 elephants that elect prophylacc11

Nepal Elephant Tuberculosis ulosisControl andManagement agemenAction Plan (2011-2015)2015)treatment should be segregated and restrictedfrom work unl they have completed 30consecuve full doses of an-TB drugs.Group 3 elephants (Elephant TB Stat-Pak ® reacve/ DPP ® VetTB reacve) should be segregatedand restricted from work and public contactunl the elephant has completed 90 consecuvedoses of an-TB drugs. Culture posive elephantspotenally have more advanced disease andshould be segregated and restricted from workfor the duraon of treatment.Majority of the elephants have tendencyof refusing medicaons. The TB drugs arebi er-tasng and many tablets are required.Prophylacc treatment lasts nine months andfull TB treatment lasts one year so it is importantto take me to slowly condion the elephant toaccept a new roune before starng medicaon.Once the elephant accepts the new roune theyare more likely to complete the enre treatmentwithout a problem. Each elephant is different andrequires paence and ingenuity to determine thebest method for each individual elephant. Thefollowing methods have been used successfullyin Nepal. Note that the paral doses suggestedbelow during the condioning period do notcount toward the full dose goal.Method 1. Direct administraon in smallmolasses balls or bananasBegin training elephants to accept small ballsof molasses with no medicaon. These shouldbe small enough to be placed towards the backof the elephant’s tongue so that they swallowthem without chewing. Each elephant shouldreceive about 100-150 small molasses balls eachday in the morning before other food. Once theelephant rounely accepts the un-medicatedmolasses balls, the drugs may be added graduallyas described below. Place only 1-2 pills in eachmolasses ball. Once the medicaons are reliablyaccepted at the full dose it is best to give them allat once in the morning before the elephant eatsa lot of food. If this does not work then the drugscan be divided so that the elephant receives 1/2of the pills in the morning and 1/2 in the evening.Small pieces of banana (with skin), eachcontaining 1-2 pills can be used in place ofmolasses and may work be er for someelephants. Sugar should not be used as itintereferes with the absorpon of INH.Method 2. Direct administraon in large molassesballs (or dana/ kuchi)Some elephants have accepted mulple tabletsin large molasses balls or in their regular dana/kuchi.Method 3. Direct administraon in the mouthA few elephants have been trained to open theirmouths widely and will accept the pills beingtossed directly into their mouths. The pills shouldbe followed by a liter of water to make sure thatthey swallow.Introduce INH-RIF at 80% of the calculated dose.Place 1-2 pills in small molasses balls (or bananapieces). Try to place in the back of the mouth sothat the elephant does not crush and taste thedrugs. Place several pills if using large molassesball or dana/kuchi.Once INH and RIF have been accepted for 3 daysin a row, increase to 90% of the calculated dose.Once INH and RIF at 90% of the calculated doseare accepted for 3 days in a row, increase to 100%of the calculated dose. If there are any problemsnoted at 100% of the dose, back down to 90%.For Group 3 elephants it is important to add EMBas soon as possible a!er the INH and RIF are12

Nepal Elephant Tuberculosis Control andManagement Action Plan (2011-2015)being consumed reliably. Start EMB at 80% of thecalculated dose. If there are no problems aer 3days, increase to 90% of the calculated dose, thento 100% of the calculated dose.Method 4. Rectal administra!on.Rectal administra!on is used only for elephantsthat absolutely cannot be trained to acceptoral medica!ons. This method is more costly.For Group 3 elephants another drug (such asciprofloxacin) must be added or subs!tutedfor RIF as RIF is not absorbed well rectally. Theprocedure is as follows:Protocol for Rectal administra!on of an!-tuberculosis drugs in elephants:a. Place 400 ml of tap water in a jar with a lid.b. Add the calculated number of tablets.c. Shake the bo#le well un!l the drugs arecompletely dissolved. Isoniazid is easilydissolved; ethambutol takes more !me.d. Place a long PD (pregnancy diagnosis) gloveon one hand and a regular exam glove over it.e. Lubricate with soap or other lubricant.f. Aer the elephant lies down in either sternalor sleeping posi!on, remove all the accessiblefecal boluses from the rectum.g. Insert one end of a stomach tube into therectum and slowly pour the drug solu!on intothe funnel at the other end of the tube.h. Drug administra!on is facilitated by squeezingthe pump located in the middle of the tube.i. When drug administra!on is finished clear thetube by flushing it with ~ 100 ml of water.j. Slowly remove the tube and hand.k. Maintain the elephant in the treatmentposi!on for at least 5 minutes to give the drug!me to be absorbed. If the elephant stands uptoo quickly some of the drug may be expelled.l. Aer the elephant stands up, observe to see ifany drug has been expelled.m. Record observa!ons in log book.n. Only fully retained doses count towardsthe required number of doses to completetreatment.Management of side effects: Elephants mayexperience side effects while on TB drugs. Loss ofappe!te is the most common. If loss of appe!teis observed, stop the medica!ons for 1-3 days(or un!l the appe!te improves). When resumingtreatment give 25% of the pills the first day, 50%the next day, 75% the 3 rd day and the full doseon the 4 th day. Be sure and record the change inthe number of pills on the elephant’s treatmentrecord. These par!al doses do not count towardthe full treatment goal.If elephants display side effects that do notresolve by withdrawing medica!ons for a fewdays it is advisable to submit blood for a CBC andserum biochemistry tests. In Chitwan the CancerHospital will receive elephant samples. The TBdrugs may occasionally cause anemia or changesin liver enzymes which can be detected by bloodtests. In elephants that have repeated problemswith loss of appe!te or other side effects, thedrugs can be administered at double the dose butgiven every other day. This is called pulse therapyand has been used successfully in the U.S.Other signs that may occur (less commonly)include: fever, muscle weakness or limb pain,unsteady gait, skin rash, visual or hearingdisturbances. If any of these signs are observed,contact following TB Plan Veterinary Officer.Human health: See Sec!on 1.613

Nepal Elephant Tuberculosis ulosisControl andManagement agemenAction Plan (2011-2015)2015)Record keeping: Keeping accurate records isvery important. Each elephant under treatmentwill have a log book to record how muchmedicaon is consumed and enter commentsabout treatment. The log book will be providedby the TB Plan and the TB Plan Veterinary Officerwill explain how to use the log. Instruct staff tobe honest in reporng whether elephants haveconsumed all or only a poron of the medicaon.This informaon will help the TB Plan VeterinaryOfficer to assess treatment and make anyadjustments if needed. The TB Plan VeterinaryOfficer may review the log book at any me.Upon compleon of treatment the log book willbecome part of the elephant’s permanent recordmaintained in the TB Plan office.Monitoring: The TB Plan Veterinary Officerwill monitor all government elephants undertreatment. Private veterinarians who are treangelephants will report at least monthly to the TBPlan veterinarian who will stay informed of theirtreatment progress, offer advice, and maintaina record on the elephant. All privately ownedelephants that are treated should also have a logbook in which daily notes of the amount of drugoffered and the amount consumed are recorded.At the compleon of treatment a copy of the logbook will become part of the elephant’s record inthe TB Plan office to document compliance withthe program.A. Protocol for Management Group 2: TBsuspectelephants (Prophylac!c treatment)Elephants in Group 2 receive two drugs for 9months.Oral therapy:1. Isoniazid (INH) at a dosage of 5 mg/kg for 270full doses2. Rifampin (RIF) at a dosage of 10 mg/kg for 270full doses Isoniazid and rifampin are availableas a combinaon product. Each tabletcontains 300 mg of INH and 600 mg of RIF.Rectal therapy:1. Isoniazid (INH) at a dosage of 5 mg/kg for 270full doses2. Ethambutol (EMB) at a dosage of 30 mg/kg for270 full dosesPost-treatment monitoringThe Elephant TB Stat-Pak ® test may remainreacve even a!er treatment. Elephants thathave been treated prophylaccally should bemonitored annually using the DPP. If the DPPbecomes reacve the elephant may have been reinfected.An elephant with a reacve DPP shi!s toGroup 3 and should undergo full TB treatment.B. Protocol for Management Group 3: TBinfectedelephantsElephants in Group 3 receive three drugs for twomonths followed by two drugs for another 10months.1. Isoniazid (INH) at a dosage of 5 mg/kg for 360full doses2. Rifampin (RIF) at a dosage of 10 mg/kg for 360full doses3. Ethambutol (EMB) at a dosage of 30 mg/kgfor 60 full doses given the first 2 months oftreatment (60 doses)Elephants in Group 3 must receive 3 drugs fortreatment. Administering fewer drugs has beenshown to result in treatment failure. Other drugsmay be prescribed based on posive cultures anddrug sensivity tesng.The first 60 doses of all three drugs should beadministered within a period of 90 days (i.e.no more than 30 days of refused medicaon14

Nepal Elephant Tuberculosis Control andManagement Action Plan (2011-2015)should occur). If more than 30 days of interruptedtreatment occur, treatment must start over. Onlyfull doses count toward the total of 60 doses.Treatment is connued for an addional 10months with INH-RIF. The total number of dosesfor the enre treatment is 360 full doses. Theenre treatment should be completed within 15months. This allows for refused medicaon daysand periods of interrupon that may be needed ifside effects occur.Compleon of Treatment: Treatment is completewhen an elephant has consumed 60 full dosesof EMB and 360 full doses of INH-RIF. There iscurrently no single test that confirms cure. Weightgain, improved exercise tolerance, improvedbreathing, and other signs may be used toevaluate treatment response. Other parametersthat signify a response to treatment are underinvesgaon.Post Treatment Policy: A"er successfulcompleon of the prescribed treatment (nogreater than 15 months) an elephant will bereleased back into its home facility and/orresume normal acvity. For the first two yearspost-treatment, respiratory samples for culture,PCR, and LAMP should be submi#ed every 6months (triple sample method). A"er two years,respiratory samples for culture, PCR, and LAMPshould be submi#ed annually (triple samplemethod).Secon 1.6 Human HealthConsideraonsTB can be transmi#ed between elephants andhumans (Michalak et al. 1998). It is recommendedthat elephant handlers and other exposed staffwear N-95 masks when working in close contactwith TB posive elephants. N-95 masks are specialmasks that are cerfied to be protecve against TB.The mask must be secured ghtly around the faceto be effecve. Wearing masks for the duraonof treatment is ideal; at minimum masks shouldbe worn for the first 3 months. Handlers shouldbe instructed to wash their hands a"er workingdirectly with suspect or infected elephants. All newelephant handlers or other staff working in closecontact with elephants should be screened for TBbefore starng employment. An annual humanTB screening program for all staff is essenalas exposure from outside sources may occur atany me. Human TB tesng services are widelyavailable in Nepal. Documentaon of staff TBtesng will be required for elephant owners orfacilies to receive Cerficates of Compliance.Note that the Elephant TB Stat-Pak ® may remainposive even a"er treatment, so cannot be usedto determine re-infecon or treatment failuresin elephants that have been treated. The MAPIAdeclines with treatment (Lyashchenko et al.2006) and it is expected that the intensity of thereacon on the DPP ® VetTB test may also changepost-treatment, however further research inthis area is needed. Post-treatment monitoringguidelines may change as new informaon ortests become available.15

Nepal Elephant Tuberculosis ulosisControl andManagement agemenAction Plan (2011-2015)2015)16

Part IIAction Plan

Nepal Elephant Tuberculosis ulosisControl andManagement agemenAction Plan (2011-2015)2015)2.1 VisionThe vision of the Acon Plan is to ensuretuberculosis free capve elephants andcontribute in conserving biodiversity at all levelsin lowland landscape of Nepal.2.2 GoalsThe goals of this plan are to: i) eliminate TB incapve elephants as well as among the elephanthandlers and other staff working in closeproximity to elephants, ii) prevent transmissionof TB to wildlife, and iii) safeguard tourism frompotenal risk of elephant TB.2.3 Objec!vesThe objecves of this plan are to:i) instute a sustainable program to diagnoseand treat TB among capve elephants andtheir handlers in Nepal,ii)establish an effecve elephant healthmonitoring system,iii) establish a TB educaon program forelephant staff,iv) educate tourists, hotel owners, localcommunies and media,v) integrate the plan into livestock and humanTB surveillance and control iniaves andensure regular reporng to and from thesesectors, with collaborave One Healthacvies supported.2.4 Issues, Strategies and ProgramAc!vi!esIssue I: Risk of tuberculosis to thecapve elephantsStrategy: Instute and enforce a sustainabletesng, segregaon, and treatment programfor all capve elephants and their handlers, andprevent TB-suspect or infected capve elephantsfrom entering Nepal .Program Ac!vi!es1. Program managementThe Plan will be managed by DNPWC incollaboraon with NTNC, WWF Nepal,BZMC and HAN, Chitwan. DNPWC willseek connued technical support fromElephant Care Internaonal (ECI). TB PlanOffice will be placed at NTNC’s BiodiversityConservaon Center (BCC) at Sauraha,Chitwan.2. TesngAll capve elephants in Nepal will be TBtested every one to two years in accordancewith the procedures in Secon 1.2.18

Nepal Elephant Tuberculosis Control andManagement Action Plan (2011-2015)3. Prevenng untested elephants fromentering NepalPreven!ng infected elephants from enteringNepal is one of the most effec!ve methodsto control TB. The prac!ce of obtainingelephants from outside Nepal is stronglydiscouraged. All elephants that do enterNepal must have cer!ficate of a nonreac!veElephantTB Stat-Pak ® test performedwithin the previous 30 days by a licensedveterinarian. Wri$en documenta!on mustinclude the elephant’s name; age; sex;owner’s name and contact informa!on;veterinarian’s name and contact informa!on;and test results. A photograph of the testlabeled with the elephant’s name and dateof the test must be included. The TB PlanVeterinarian will have the authority to retest(if tested previously) elephants to verifytest results upon arrival in Nepal.TB Plan Veterinary Officer will be availableto travel with private owners planning toprocure new elephants. The private ownerwill bear the cost of tes!ng and travelexpenses for TB Plan Veterinarian. TB PlanVeterinarian will have the authority totest elephants that enter Nepal untestedimmediately and independently. ElephantTB Stat-Pak ® reac!ve elephants must leaveNepal. All new elephants must be microchippedfor iden!fica!on.4. Elephant eventsAll elephants par!cipa!ng in events such aselephant races or polo must have cer!ficateof a current non-reac!ve Elephant TB Stat-Pak® test or comple!on of prophylac!ctreatment (including post-treatment tes!ngas described under Post-Treatment Policy insec!on 1.5).5. SegregaonAll elephants that are reac!ve on theElephant TB Stat-Pak® and DPP ® VetTBtests will be segregated to prevent poten!altransmission of TB to other elephants,humans, or wildlife according to segrega!onguidelines given in segrega!on procedures.6. TreatmentElephants will be treated for TB as prescribedabove in Sec!on 1.5 using the same drugsshown to be effec!ve for humans. Elephantsare treated for a longer period of !me as ameasure of safety as there is s!ll much thatis unknown about TB in this species.7. Grazing praccesEfforts will be made to prevent elephantsfrom grazing in close proximity to cap!veca$le or buffalo to minimize the risk of TBtransmission.8. Dung disposalThe current prac!ce of burning elephantdung in close proximity to elephantstables will be discon!nued, as smoke is arespiratory irritant and adversely affects thefunc!on of the lungs of both the elephantsand their handlers. A prac!cal mechanismwill be developed and implemented for dungburning at a sufficient distance to preventsmoke from reaching the elephants and theircaretakers or alterna!ve methods of dungdisposal will be ins!tuted.9. One Health working groupEstablish a One Health Nepal working groupwith local wildlife, medical and veterinary19

Nepal Elephant Tuberculosis ulosisControl andManagement agemenAction Plan (2011-2015)2015)authories and clinicians to devise acomprehensive plan to idenfy pockets ofinfecon and control or eradicate TB fromthe local environment.10. Human TB screeningHumans are a potenal source of TBfor elephants. Handlers and other staffmembers working in close proximity toelephants will be TB tested annually. Newemployees will be tested before beginningwork. The TB posive staff members willnot be allowed to handle or care for theelephants unl they have completed fourweeks of DOTS treatment. Medicaon will bestarted as soon as the person is diagnosedTB posive.11. Cap!ve elephant postmortem examina!onAll capve elephants that die will receive acomprehensive postmortem examinaonconducted by the TB Plan Veterinarian.Nepal elephant postmortem protocolworksheet is given in Appendix III.12. MonitoringThe feeding, working, and grazing of TBposive elephants will be closely monitoredby the owner and the TB Plan veterinaryofficer.13. Adop!on of animal welfare codeThe five freedom codes of animal welfarewill be always taken into consideraon. Thefive freedoms are:i. Freedom from hunger and thirstii.iii.Freedom from thermal and physicaldiscomfortFreedom from injury and diseaseiv.Freedom to express most normalpa$erns of behaviourv. Freedom from fear and distressIssue II: Risk of transmission of TB fromcap!ve to wild popula!on of elephants,rhino and other wildlife speciesStrategy: Control TB among capve elephantsand instute pracces to prevent contactbetween capve TB-suspect or infected elephantsand wildlife species.Program Ac!vi!es1. Segregate and treat infected elephantsAll infected elephants will be segregated andtreated as per guidelines described underIssue I.2. Prevent contactAll possible measures will be applied toprevent the contact between capve TBsuspector infected elephants and freeranging wildlife species. Use electric fencesor other praccal methods to preventcontact between capve TB-suspect orinfected elephants and free ranging wildelephants, rhinos and other large as well asmedium sized ungulates. Capve TB-suspector infected elephants will be restricted tospecified grazing areas. The segregaonplan should be approved by the TB Planveterinary officer.3. Educate handlersA regular TB control and managementeducaon program will be launched amongelephant handlers, caretakers and theirfamily members. For this, a praccal guidehighlighng the causes of TB, symptoms,20

Nepal Elephant Tuberculosis Control andManagement Action Plan (2011-2015)impact of TB on human as well as animalhealth, importance of prevenng TBtransmission to the wild and effecvemeasures to manage TB will be developed,presented orally, and printed in the locallanguage to educate persons directly orindirectly associated with the capveelephants.4. Postmortem wildlife surveillanceThorough postmortem examinaons of allungulates that are found dead in the park,buffer zone community forests and nearbyareas will be conducted by experiencedveterinarians. For this, ssues will becollected and deposited in 10% bufferedformalin even if carcasses are decomposedand will be submi!ed for histopathology orssue bank. Any lesions suspicious for TBshould be placed in a secure container (noformalin), and submi!ed fresh (preferable)or frozen for TB culture. Efforts, whereverpossible, will be made to collect postmortemblood, separate and bank serum in the TBPlan lab freezer.Full postmortem reports (or copies) will befiled in the TB Plan Office, respecve Parkheadquarters and DNPWC. A MOU will besigned among DNPWC, NTNC, IAAS, CVL,ECI, WWF, and Tu$s to effecvely facilitateimplementaon of a comprehensive TBmanagement program and minimize the riskof transmission from capve elephants tofree ranging elephants and other ungulates.5. Opportunis!c TB tes!ng in wild elephantsBlood samples from any elephant that isimmobilized will be collected and ElephantTB Stat-Pak® test (and DPP if indicated) willbe performed. Serum will also be banked forfuture use.6. Opportunis!c TB tes!ng in other speciesOpportunisc TB test will also be performedamong other wild ungulates or carnivoresthat are captured and trapped for otherpurposes. While the Elephant TB Stat-Pak®test is licensed for elephants, it has beenused successfully in a number of otherspecies, including rhinos and wild felidsIssue III: Risk of transmission of TB fromhuman to elephant and from elephantto humanStrategy: Minimize the risk of transmission fromelephant to human and human to elephantthrough an integrated TB management program.Program Ac!vi!es1. Develop an ac!on plan from the One Healthworking group and implement, to dealwith the root cause of TB infec!on in theenvironment, animals and people.Research will be necessary and surveillanceacross the environment to establish themain causes and drivers of TB and theseneeds to be followed up by a controland management plan involving all keyauthories in public and animal health.2. Enforcing elephant tes!ng, segrega!on andtreatment planAs described in Program Acvies underIssue I and Issue II.3. Human TB screeningAll elephant handlers (Phanits, Pachhuwasand Mahouts), and other staff workingin close proximity to elephants will be TBtested prior to employment and retested21

Nepal Elephant Tuberculosis ulosisControl andManagement agemenAction Plan (2011-2015)2015)on an annual basis. TB tesng by sputumis available free of charge at numeroushealth clinics in Nepal. Posive sputumtests will be followed by other diagnosctests and/or DOTS treatment as determinedby human health care professionals at thetesng facility. If infected individuals areidenfied, their families will also be tested.Further invesgaon will be managed byappropriate public health agencies. Elephanthandlers undergoing DOTS treatment willnot have contact with elephants unl theyhave completed four weeks of treatment.Proof of current elephant handler tesngmust be presented for a facility to receive aCerficate of Parcipaon (Appendix V).4. Educaon programDevelop a TB educaon program. Apreventave health program will minimizeTB risk for elephant handlers, theirfamilies, and tourists. This program willinclude appropriate bio-safety praccesand informaon about TB. Handlers ofsegregated and/or infected elephants willbe given addional training. If deemednecessary, protecve measures (such asspecial clothing and masks) may be providedby the TB Plan Office.Issue IV: Possibility of impact of TB onwildlife tourism in NepalStrategy: Instute and enforce the NepalElephant Tuberculosis Control and ManagementAcon Plan; educate tourists, tour agencies, andthe media.Program Ac!vi!es1. Cerficaon programIn conjuncon with bi-annual elephant TBtesng, DNPWC will issue a cerficate of TBPlan compliance that parcipang faciliescan display to tourists. Addional cerficateswill be issued for individual elephants thathave completed treatment. Templates ofplan cerficate are given in Appendix II.2. Educate tourists and tour agenciesNTNC TB Program Office staff and DNPWCwill collaborate to create and distributea brochure for tourists and tour agenciesexplaining how the Nepal ElephantTuberculosis Control and ManagementAcon Plan will safeguard the health ofelephants and tourists.3. Educate the mediaNTNC TB Plan Office staff and DNPWC willcollaborate with local media (both audioand visual) to highlight the ways in whichthe Nepal Elephant Tuberculosis Controland Management Acon Plan safeguardselephant and tourist health. A shortdocumentary may also be prepared toillustrate the Plan.Issue V: Insufficient technical andmanagerial capacityStrategy: Develop partnerships to strengthentechnical and managerial capacies.Program Ac!vi!es1. Strengthen capacityVeterinary facilies parcularly targeted toTB control will be developed and capacityof park, zoo, Buffer Zone veterinary clinicveterinarians will be enhanced throughhands-on-training, workshop and higherdegree study programs. Partnership withnaonal and internaonal experts andorganizaons will also be expanded to22

Nepal Elephant Tuberculosis Control andManagement Action Plan (2011-2015)hold training workshops to encourageveterinary diagnosc and clinical skills forthe surveillance of TB and other diseases.2. Naonal level collaboraonLong term collaboraon will be establishedwith NATA to improve respiratory samplecollecon and culture techniques, andwith IAAS and CVL to improve pathologydiagnosis. Partnerships will also be builtwith other organizaons in Nepal to developmolecular screening techniques for capveelephants and other free ranging wildlife TB.3. InternshipsOne or two graduates from IAAS may beinvited annually for a short internshipperiod (3-6 months) under the NETCMAP todevelop a cadre of young veterinarians inwildlife medicine.Issue VI: Insufficient financial resourcesStrategy: Create a sustainable fundingmechanism to support the Nepal TB Control andManagement Acon Plan.Program Ac!vi!es1. Annual budgetAnnual recurring budget for the NepalElephant Tuberculosis Control andManagement Acon Plan will be prepared.2. PartnershipLong term financial commitment will besought from naonal and internaonalconservaon partners including NTNC,WWF Nepal, ECI, ZSL, USFWS etc. to ensurefinancial sustainability of the NETCMAP.3. Basket fundA basket fund to ensure a regular supplyof financial resources will be created atthe field level. Potenal sources for thebasket fund would be concessionaire feefrom the lodges inside Chitwan and BardiaNPs, and revenue diverted back to thecapve elephant bearing Protected Areas(Koshitappu, Parsa and Suklaphanta WRs;Chitwan, Banke and Bardia NPs).4. Fund raising capacity buildingFund-raising capacity of DNPWC and partnerorganizaons will be developed to sustainlong term financial health through a series ofgrant-wring training workshops.Issue VII: Inadequate research onelephant and free ranging wildlifespeciesStrategy: Develop partnerships with naonaland internaonal researchers and researchorganizaons to promote elephant and freeranging wildlife TB research.Program Ac!vi!es1. Research collaboraonEstablish collaboraons under the OneHealth Nepal iniave to bring the varioushealth sectors together and establish a proofof concept study into the TB ecology in theenvironment of the Terai Naonal Parks andbuffer zone human and animal communies.Collaboraons will be established to developtests that can determine the stage ofdisease; quickly and economically detectshedding; measure blood levels of TB drugsin elephants; confirm cure; and determine23

Nepal Elephant Tuberculosis ulosisControl andManagement agemenAction Plan (2011-2015)2015)if elephants have genec suscepbility orresistance to TB. Addional studies will focuson epidemiology, pathology, and improvingtreatment methods.A serum bank has been established at NTNC- BCC at Sauraha that is a resource for newstudies. Collecon of blood and urine will beconnued by the TB Plan Veterinary Officerat the me of regular TB tesng so that thisresource will connue to grow for furtherresearch. Elephant Care Internaonal willconnue to coordinate this effort. A researchreview commi ee will be established toreview and approve proposals.2. Affilia onsAffiliaons with researchers establishedby ECI will be connued. Collaboraon willalso be established with researchers inother countries by sharing informaon anddata base generated from the ongoing TBprogram. ECI will help to coordinate thiseffort.3. Molecular screeningPartnerships will be established withnaonal and regional instuons to developmolecular screening techniques for elephantand other free ranging wildlife TB.24

Nepal Elephant Tuberculosis Control andManagement Action Plan (2011-2015)2.5 BudgetIssues/Ac!vi!es Year 1 Year 2 Year 3 Year 4 Year 5 Total (NRs)Issue I: Risk of tuberculosis to the capve elephantsi. Program veterinarians salaryand allowanceii. Salary for veterinary technicianiii. Tesng of capve elephantsusing ElephantTB Stat-Pak®iv. Tesng of capve elephantsusing DPP VetTB every twoyears for EelephantTB Stat-Pak®reacve elephantsv. Microchipping of new elephantsvi. Segregaon site establishmentand managementvii. Treatment of reacve elephants(for about 8 elephantsevery year)viii. Supplements for the elephantsix. Human TB tesng for handlersof TB infected elephantsx. Purchase of post mortemexaminaon suppliesXi. Regulaon on free movementof elephant (transborderand in country)865,000 950,000 990,000 1,050,000 1,125,000 4,980,000300,000 350,000 380,000 400,000 450,000 1,880,000450,000 500,000 550,000 1,500,000150,000 180,000 250,000 580,00050,000 50,000 50,000 150,0001,200,000 50,000 50,000 1,300,0001,800,000 1,900,000 1,950,000 2,050,000 2,100,000 9,800,00040,000 50,000 60,000 70,000 80,000 300,00060,000 60,000 60,000 60,000 60,000 300,00070,000 30,000 30,000 30,000 30,000 190,000300,000 500,000 600,000 300,000 300,000 2,000,000Issue II: Risk of transmission of TB from capve to wild populaon of elephants, rhinos and other wildlifespeciesi. Educaon to handlers onTB transmission from capveelephants to other wild animalsii. Opportunisc TB tesng inwild elephants (Purchase ofElephantTB-Stat Pak® and DP-PVetTB) and other animals250,000 300,000 150,000 150,000 150,000 1,000,00015,000 30,000 50,000 50,000 50,000 195,000Issue III: Risk of transmission of TB from human to elephant and from elephant to humani. Educaon program handlers'families and local stakeholderson transmission of TB fromhuman to elephant and fromelephant to humanAcon plan for one healthprogram150,000 170,000 180,000 150,000 190,000 840,000500,000 300,000 300,000 300,000 300,000 1,700,00025

Nepal Elephant Tuberculosis ulosisControl andManagement agemenAction Plan (2011-2015)2015)Issues/Acvies Year 1 Year 2 Year 3 Year 4 Year 5 Total (NRs)Issue IV: Possibility of impact of TB on wildlfie tourism in Nepali. Cerficate program 150,000 25,000 25,000 25,000 25,000 250,000ii. Educaon to tourist and touragencies150,000 250,000 200,000 150,000 150,000 900,000iii. Educaon to the media 75,000 70,000 50,000 50,000 50,000 295,000Issue V: Insufficient technical and managerial capacityi. Capacity building program forVeterinarian and Managersii. Laboratory expenses at na-onal level laboratoriesIssue VI: Insufficient financial resourcesi. Daily office managementcostsii. Computer, camera, printer,staonary, fax etciii. Vehicle purchase and management1,500,000 1,200,000 700,000 300,000 300,000 4,000,0001,500,000 750,000 750,000 750,000 750,000 4,500,0001,200,000 1,400,000 1,600,000 1,800,000 2,000,000 8,000,000350,000 150,000 150,000 150,000 150,000 950,0004,500,000 450,000 450,000 450,000 450,000 6,300,000Issue VII: Inadequate research on elephant and free ranging wildlife speciesi. Expenditure on the collabora-ve research1,000,000 1,000,000 1,000,000 1,000,000 1,000,000 5,000,000TOTAL 16,625,000 9,935,000 10,455,000 9,285,000 10,610,000 56,910,00026

Nepal Elephant Tuberculosis Control andManagement Action Plan (2011-2015)BibliographyIn addion to the literatures cited, this list also contains selected TB references. For complete TBbibliography, please visit: www.elephantcare.orgAnonymous 2008. Guidelines for the Control of Tuberculosis in Elephants (hp://www.elephantcare.org/protodoc_files/2008/TBGuidelines.pdf).Abraham, D. and J. Davis. 2008. Revised trunk wash collec on procedure for cap ve elephants in a rangecountry se ng, Gajah, Newsleer of the Asian Elephant Specialist Group of the IUCN, 28:53-54. hp://www.asesg.org/PDFfiles/Gajah%2028%20Aug%2008.pdfAlexander, K.A., E.Pleydell, M.C.Williams, E.P.Lane, J.F.C.Nyange and A.L.Michel. 2002. Mycobacteriumtuberculosis: An Emerging Disease of Free-Ranging Wildlife. Emerg. Infect. Dis. 8: 598-601.Angkawanish, T., W. Wajjwalku, A. Sirimalaisuwan, S. Mahasawangkul, T. Kaewsakhorn, K. Boonsri,V.P.M.G. Ruen. 2010. Mycobacterium tuberculosis infec on of domes cated Asian elephants, Thailand.Emerg. Infect. Dis. 16, 1949-1951.Davis, M. 2001. Mycobacterium tuberculosis risk for elephant handlers and veterinarians. Appl. Occup.Environ. Hyg. 16: 350-353.Dumonceaux, G. and S. Mikota. 2006. Tuberculosis treatment protocols and complica ons for elephants.Proceedings Interna onal Elephant Conserva on and Research Symposium 84-85.Gairhe, K. 2002. A case of Tuberculosis in cap ve elephants in Nepal. A report submied to Departmentof Na onal Parks and Wildlife Conserva on, Kathmandu (unpubl.)Greenwald,R., O. Lyashchenko, J. Esfandiari, M. Miller, S. Mikota, J. H. Olsen, R. Ball, G. Dumonceaux, D.Schmi, T. Moller, J. B. Payeur, B. Harris, D. Sofranko, W. R. Waters and K. P. Lyashchenko. 2009. Highlyaccurate an body assays for early and rapid detec on of tuberculosis in African and Asian elephants.Clin. Vaccine Immunol. 16: 605-612.Hile, E.M., H. F. Hintz and N. Hollis. 1997. Predic ng body weight from body measurements in Asianelephants (Elephas maximus). J. Zoo Wildl. Med. 28: 424-427.Kay, M., M. Linkie, J. Trian s, M.D. Salman and R.S. Larsen 2011. Evalua on of DNA extrac on techniquesfor detec ng Mycobacterium tuberculosis-complex organisms in Asian elephant trunk washes. J. Clin.Microbiol. 49(2): 618-623.27

Nepal Elephant Tuberculosis ulosisControl andManagement agemenAction Plan (2011-2015)2015)Lacasse, C., K. Terio, M. J. Kinsel, L. L. Farina, D. A. Travis, R. Greenwald, K. P. Lyashchenko, M. Miller andK. C. Gamble. 2007. Two cases of atypical mycobacteriosis caused by Mycobacterium szulgai associatedwith mortality in cap!ve African elephants (Loxodonta africana). J. Zoo. Wildl. Med. 38: 101-107.Landolfi, J.A., S. A. Schultz, S. K. Mikota and K. A. Terio. 2009. Development and valida!on of cytokinequan!ta!ve, real !me RT-PCR assays for characteriza!on of Asian elephant immune responses. Vet.Immunol. Immunopathol. 131: 73-78.Landolfi, J.A., S.K. Mikota, J. Chosy, K.P. Lyaschenko, K. Giri, K. Gairhe, and K.A. Terio. 2010. Comparisonof systemic cytokine levels in Mycobacterium spp seroposi!ve and seronega!ve Asian elephants (Elephasmaximus). J. Zoo. Wildl. Med. 41: 445-455.Lewerin, S. S., S. L. Olsson, K. Eld, B. Roken, S. Ghebremichael, T. Koivula, G. Kallenius and G. Bolske.2005. Outbreak of Mycobacterium tuberculosis infec!on among cap!ve Asian elephants in a Swedishzoo. Vet. Rec. 156: 171-175.Lyashchenko, K., M. Singh, R. Colangeli and M. L. Gennaro. 2000. A mul!-an!gen print immunoassay forthe development of serological diagnosis of infec!ous disease. Journal of Immunological Methods 242:91-100.Lyashchenko, K., M. Miller, and W.R. Waters. 2005. Applica!on of mul!ple an!gen print immunoassayand rapid lateral flow technology for tuberculosis tes!ng of elephants. Proceedings of AmericanAssocia!on of Zoo Veterinarians Annual Mee!ng. 64-65.Lyashchenko, K. P., R. Greenwald, J. Esfandiari, J. H. Olsen, R. Ball, G. Dumonceaux, F. Dunker, C. Buckley,M. Richard, S. Murray, J. B. Payeur, P. Andersen, J. M. Pollock, S. Mikota, M. Miller, D. Sofranko and W.R. Waters. 2006. Tuberculosis in elephants: an!body responses to defined an!gens of Mycobacteriumtuberculosis, poten!al for early diagnosis, and monitoring of treatment. Clin. Vaccine Immunol. 13: 722-732.Maslow, J.N., S. K. Mikota, M. Zhu, H. Riddle and C. A. Peloquin. 2005. Pharmacokine!cs of ethambutol(EMB) in elephants. J. Vet. Pharmacol. Ther. 28: 321-323.Maslow, J.N., S. K. Mikota, M. Zhu, R. Isaza, L. R. Peddie, F. Dunker, J. Peddie, H. Riddle and C. A. Peloquin.2005. Popula!on pharmacokine!cs of isoniazid in the treatment of Mycobacterium tuberculosisamong Asian and African elephants (Elephas maximus and Loxodonta africana). Journal of VeterinaryPharmacology and Therapeu!s 28(1): 21-27.Michalak, K., C. Aus!n, S. Diesel, M. J. Bacon, P. Zimmerman and J. N. Maslow. 1998. Mycobacteriumtuberculosis infec!on as a zoono!c disease: transmission between humans and elephants. Emerg. Infect.Dis. 4: 283-287.28

Nepal Elephant Tuberculosis Control andManagement Action Plan (2011-2015)Michel, A.L., R. G. Bengis, D. F. Keet, M. Hofmeyr, L. M. de Klerk, P. C. Cross, A. E. Jolles, D. Cooper,I. J. Whyte, P. Buss and J. Godfroid. 2006. Wildlife tuberculosis in South African conservaon areas:Implicaons and challenges. Veterinary Microbiology 112: 91-100.Mikota. S.K. and J. N. Maslow. 2011. Tuberculosis at the human-animal interface: An emerging diseasefor elephants. Tuberculosis 91: 208-211.Mikota, S. K., M. Miller, G. Dumonceaux, K. Giri, K. Gairhe, K. Hamilton, S. Paudel and B. Vincent. 2006.Elephant tuberculosis diagnosis: implicaons for elephant management in Asian range countries.Proceedings of American Associaon of Zoo Veterinarians. 142-143.Mikota, S.K., L. Peddie, J. Peddie, R. Isaza, F. Dunker, G. West, W. Lindsay, R. S. Larsen, M. D. Salman,D. Cha!erjee, J. Payeur, D. Whipple, C. Thoen, D. S. Davis, C. Sedgwick, R. Montali, M. Ziccardi and J.Maslow. 2001. Epidemiology and diagnosis of Mycobacterium tuberculosis in capve Asian elephants(Elephas maximus). J. Zoo Wildl. Med. 32: 1-16.Mikota, S. K. 2008. Tuberculosis in elephants, In: Fowler, M. E. and R. E.Miller (eds.). Zoo and Wild AnimalMedicine, Current Therapy 6th edion. Saunders/Elsevier, St. Louis. 355-364.Mikota, S.K., G. Dumonceaux, M. Miller, K. Gairhe, K. Giri, J. V. Cheeran, D. Abraham, K. Lyashchenko, S.Larsen, J. Payeur, R. Waters, and G. Kaufman. 2006. Tuberculosis in elephants: An update on diagnosisand treatment; implicaons for control in range countries. Proceedings of Internaonal ElephantConservaon and Research Symposium. 110-118.Moller, T., B. Roken, L. Petersson, C. Vitaud and K. Lyashchenko. 2005. Preliminary results of a newserological test for detecon of TB-infecon (Mycobacterium tuberculosis) in elephants (Elephasmaximus and Loxodonta africanum) - Swedish Case studies. Verh. ber. Erkrg. Zoo!ere. 42: 173-181.Pandey, B.D., A. Poudel, Y. Tomoko, A. Tamaru, N. Oda, Y. Fukushima, B. Lekhak, B. Risal, B. Acharya,B. Sapkota, C. Nakajima, T. Taniguchi, B. Phetsuksiri and Y. Suzuki. 2008. Development of an in-houseloop-mediated isothermal amplificaon (LAMP) assay for detecon of Mycobacterium tuberculosis andevaluaon in sputum samples of Nepalese paents. Journal of Medical Microbiology 57: 439–443.Peloquin, C.A., J. N. Maslow, S. K. Mikota, A. Forrest, F. Dunker, R. Isaza, L. R. Peddie, J. Peddie andM. Zhu. 2006. Dose selecon and pharmacokinecs of rifampin in elephants for the treatment oftuberculosis. J. Vet. Pharmacol. Ther. 29: 581-585.Zhu, M., J.N. Maslow, S.K. Mikota, R. Isaza, F. Dunker, H. Riddle and C.A.Peloquin. 2005. Populaonpharmacokinecs of pyrazinamide in elephants 564. J. Vet. Pharmacol. Ther. 28: 403-409.29

Nepal Elephant Tuberculosis ulosisControl andManagement agemenAction Plan (2011-2015)2015)30

Appendices

Nepal Elephant Tuberculosis ulosisControl andManagement agemenAction Plan (2011-2015)2015)Appendix ISample Drug Dose and Cost Calculaons (Based on May 2010 Prices)TB drug cost / tablet:Isoniazid (INH); 300 mg = Rs 2Isoniazid-Rifampin combinaon (INH-RIF); 300 mg INH + 600 mg RIF = Rs 8.6Ethambutol (EMB); 800 mg = Rs 6.1The following sample calculaons are for a 3000 kg cow:A. Prophylacc Treatment for Group 2 Elephants: two drugs for nine months1. Oral protocolINH-RIF:Dose /day: 5mg/kg/day x 3000 kg = 15,000 mgTablets/day: 15,000 mg / 300 mg / tablet = 50 tabletsTotal # of tablets: 50 tablets / day X 270 days = 13,500 tabletsTotal cost: 13,500 tablets @ Rs 8.6 /tablet = Rs 116,100 (plus replacements for wasted drugs).2. Rectal protocol (more expensive; use only if oral therapy cannot be managed)INH:Dose /day: 5mg/kg/day x 3000 kg = 15,000 mgTablets/day: 15,000 mg / 300 mg / tablet = 50 tabletsTotal # of tablets: 50 tablets / day X 270 days = 13,500 tabletsTotal cost: 13,500 tablets @ Rs 2 /tablet = Rs 27,000 (plus replacements for wasted drugs).EMB:Dose /day: 30mg/kg/day x 3000 kg = 90,000 mgTablets/day: 90,000 mg / 800 mg / tablet = 112 tabletsTotal # of tablets: 112 tablets / day X 270 days = 30,240 tabletsTotal cost: 30,240 tablets @ Rs 6.1 /tablet = Rs 184,464 (plus replacements for wasted drugs).Total cost: Rs 211,464 (plus replacements for wasted drugs).32

Nepal Elephant Tuberculosis Control andManagement Action Plan (2011-2015)B. Full Treatment for Group 3 Elephants: three drugs for two months followed by two drugs for 10months1. Oral protocolINH-RIF:Dose /day: 5mg/kg/day x 3000 kg = 15,000 mgTablets/day: 15,000 mg / 300 mg / tablet = 50 tabletsTotal # of tablets: 50 tablets / day X 360 days = 18,000 tabletsTotal cost: 18,000 tablets @ Rs 8.6 /tablet = Rs 154,800 (plus replacements for wasted drugs).EMB:Dose /day: 30mg/kg/day x 3000 kg = 90,000 mgTablets/day: 90,000 mg / 800 mg / tablet = 112 tabletsTotal # of tablets: 112 tablets / day X 60 days = 6720 tabletsTotal cost: 6720 tablets @ Rs 6.1 /tablet = Rs 40,992 (plus replacements for wasted drugs).Total cost: Rs 195,7922. Rectal protocol (more expensive; use only if oral therapy cannot be managed)INH:Dose /day: 5mg/kg/day x 3000 kg = 15,000 mgTablets/day: 15,000 mg / 300 mg / tablet = 50 tabletsTotal # of tablets: 50 tablets / day X 360 days = 18,000 tabletsTotal cost: 18,000 tablets @ Rs 2 /tablet = Rs 36,000 (plus replacements for wasted drugs).EMB:Dose /day: 30mg/kg/day x 3000 kg = 90,000 mgTablets/day: 90,000 mg / 800 mg / tablet = 112 tabletsTotal # of tablets: 112 tablets / day X 360 days 40,320 tabletsTotal cost: 40,320 tablets @ Rs 6.1 /tablet = Rs 245,952 (plus replacements for wasted drugs).Ciprofloxacin: 5-10mg/kg (empirical dose) or Amikacin (injectable): to be discussed with TB PlanVeterinary Officer on a case-by-case basis.33

Nepal Elephant Tuberculosis ulosisControl andManagement agemenAction Plan (2011-2015)2015)Appendix IIPlan CerficatesFor compliance with a regular tes!ng program:Cerficate of ParcipaonThis is to cerfy that Name of Hotel or Owner has tested the elephants andmahouts for tuberculosis in compliance with the guidelines of the NepalElephant Tuberculosis Management Program administered by Department ofNaonal Parks and Wildlife Conservaon.Date: …/…./…..........................SignatureFor elephants that have completed treatment:Cerficate of Compleon of Elephant TB TreatmentThis is to cerfy that Name of Elephant (program ID….) owned byName of Hotel or Owner has completed treatment for tuberculosisin compliance with the guidelines of the Nepal Elephant TuberculosisManagement Program administered byDepartment of Naonal Parks and Wildlife Conservaon.Date: …/…./…..........................Signature34

Nepal Elephant Tuberculosis Control andManagement Action Plan (2011-2015)Appendix IIINepal Elephant Postmortem ProtocolSpecial considera!ons for TB-infected elephants or TB suspectsA thorough search for lesions of tuberculosis (TB) is encouraged in all elephant necropsies especiallyelephants that are known to be reacve on the Elephant TB Stat-Pak ® and DPP Vet ® TB tests. ElephantTB is likely to be caused by M. tuberculosis or M. bovis which are contagious to humans. Wear properprotecve apparel, and contain any suspicious organs or lesions as soon as possible. All involved personnelshould wear N-95 masks.Approach: During examinaon of an elephant with unknown, suspicious, or posive TB test history, disseconof the thoracic cavity should always be performed last, and should be done by two people withN-95 face masks and other protecve clothing. All other personnel should be dismissed from the areabefore the thoracic cavity is entered. A!er the abdominal viscera have been removed, the diaphragmcan be cut from its costo-sternal a"achments and the lungs palpated from a caudal approach for tuberculosisnodules, as the lobes are being separated from the closely adhered visceral and parietal pleura.The heart, lungs, and associated structures may then be removed “en bloc”.Necropsy procedures: Carefully examine the tonsillar regions and sub-mandibular lymph nodes for TBlesions. These lymph nodes may be more easily visualized following removal of the tongue and laryngealstructures during the dissecon. All lymph nodes should be carefully evaluated for lesions sinceother sites may also be infected (ex. reproducve or gastrointesnal tract). Take any nodes that appearcaseous or granulomatous for culture (freeze or place in sodium borate) and histopathology (in buffered10% formalin).Search thoracic organs carefully for early stages of TB as follows: a!er removal of the lungs and trachea,locate the bronchial nodes at the juncon of the bronchi from the trachea. Secon the nodes and collectin formalin even if no lesions are present. If lesions are present, place half of the lymph node in a 50 mlscrew-top tube and submit for culture.Carefully, palpate the lobes of both lungs from the apices to the caudal borders to detect any firm B-Bshot to nodular size lesions. Take numerous (5 or more) secons of any suspicious lesions. Open the tracheaand look for nodules or plaques and process as above. Regional thoracic and tracheal lymph nodesshould also be examined and processed accordingly. Split the trunk from the p to its inseron and takesamples of any plaques, nodules or suspicious areas for TB diagnosis as above. Look for and collect possibleextra-thoracic TB lesions, parcularly if there is evidence of advanced pulmonary TB.35

Nepal Elephant Tuberculosis ulosisControl andManagement agemenAction Plan (2011-2015)2015)Nepal Elephant Postmortem WorksheetElephant name: _________________________________TB Program ID # __________Owner / locaon: ________________________________Age:__________Sex:__________Esmated or actual weight (Kg): _____Country of origin: ___________Phanit: ______________________Capve Born ___Wild Caught ___Unknown ___Pachhuwa: _______________ Mahout: _____________Date /me / locaon of death: ______________________________________________________Date /me/ locaon of postmortem:__________________________________________________Post mortem interval /condion:_____________________________________________________History:Gross examinaon (Describe abnormal; indicate N= normal or NE = not examined):1. External (physical and nutrional condion, skin, body orifices, temporal gland)2. Musculoskeletal (muscles, bones, joints)3. Body cavies (fat stores, pleura, fluid, thymus, lymph nodes)4. Spleen and Liver5. Respiratory system (trunk passages, pharynx, larynx, trachea, bronchi, lungs, lymph nodes;(Examine lungs, trachea, tonsil area, and bronchial lymph nodes for evidence of TB; submit any lung lesionsfor TB culture; culture bronchial lymph nodes for TB even if normal in appearance)6. Cardiovascular (heart, pericardial sac, vessels, valves, chambers)7. Digesve (mouth, teeth, tongue, esophagus, stomach, small intesne, cecum, large intesne, rectum,pancreas, mesenteric lymph nodes)8. Urinary (kidneys, ureters, bladder, urethra)9. Reproducve (testes/ovaries, uterus, cervix, penis/vagina, urogenital canal, prostate, seminal vesicles,bulbo-urethral gland, mammary gland, placenta).10. Endocrine (thyroids, parathyroids, adrenals, pituitary)11. Central nervous system (brain, meninges, spinal cord)12. Sensory organs (eyes, ears)36

Nepal Elephant Tuberculosis Control andManagement Action Plan (2011-2015)Check list for fixed ssues: Preserve all ssues in group I and as many ssues in groups II and II as possible.Use 10% buffered formalin at a ra o of ~ 1 part ssue to 10 parts solu on. Cut ssues no thickerthan 0.5 cm. Take 2-3 sets. Submit one set for histopathology and bank 2 sets. Freeze 3-5 cm blocks ofssue from lesions and major organs (lung, liver, kidney, spleen, heart) in small plas c bags or n foil.Freeze post mortem serum (from heart); urine; and any abnormal fluid accumula ons.Group I__ heart (sec ons from auricles, ventricles and valves)__ lung (specimens from several lobes)__ trachea (may be site for TB)__ liver (3 representa ve specimens and bile duct)__ kidney (cortex and medulla of each kidney)__ adrenal gland__ spleen (representa ve cross sec on with capsule)__ lymph nodes (submandibular, bronchial, mesenteric, and tonsillar ssue)__ pancreas__ stomach (several specimens from all areas)__ intes nes (3cm representa ve specimen from each region)__ thyroid gland (and parathyroid if possible)Group II__ urinary ssue (ureters, urethra, bladder cross sec on including mucosa)__ uterus and ovaries (transverse sec ons of cervix, uterine horn, and ovaries)__ testes and epididymus__ prostate, seminal vesicles, bulbo-urethral gland37

Nepal Elephant Tuberculosis ulosisControl andManagement agemenAction Plan (2011-2015)2015)Group III__ esophagus__ tongue__ salivary gland__ thymus__ temporal gland__ sciac nerve secon__ muscle__ bone marrow__ skin (secons of abdomen, lip and ear pinna)__ eye (whole eye, incise sclera to allow entry of fixave)__ mammary gland__ brain, pituitary gland,__ spinal cord (secons from cervical, thoracic and lumbar)__ neonates (umbilical stump and surroundingFluids collected: __ serum __ urine __ peritoneal fluid__ other _____________Cultures collected: ________________________________________Tissues sent for histopathology to (Pathologist name, address, email): _________________________________________________________________________________________________Veterinarian and Assistants: ______________________________________________________Preliminary Diagnoses: _____________________________________________________38

Nepal Elephant Tuberculosis Control andManagement Action Plan (2011-2015)Appendix IVContactsCentral Commiee1. Naonal Trust for Nature Conservaon (NTNC)Dr. Shant Raj Jnawali (srjnawali@ntnc.org.np)2. World Wildlife Fund - NepalDr. Christy Williams (christy.williams@wwfnepal.org)Dr. Ghana Gurung (Ghana.gurung@wwfnepal.org)3. Department of Naonal Parks and Wildlife Conservaon (DNPWC)Dr. Maheswar Dhkal (maheshwar.dhakal@gmail.com)Dr. Kamal Gairhe (kamalgairhe@hotmail.com)Field Commiee1. Buffer Zone Management Commi"ee (BZMC)2. Hotel Associaon Nepal (HAN)3. Naonal Trust for Nature Conservaon (NTNC)Naresh Subedi (nareshsubedi@gmail.com)Dr. Jeewan Thapa (lifethapa@hotmail.com)Research Commiee1. Elephant Care InternaonalDr Susan Mikota (smikota@elephantcare.org)2. Instute for Agriculture and Animal ScienceDr. I.P. Dhakal (ipdhakal@yahoo.com)3. Tu#s Center for Conservaon Medicine, Tu#s UniversityDr. Gretchen Kaufman (Gretchen.kaufman@tu!s.edu)4. WWF - Nepal (AREAS)Dr. Christy Williams (christy.williams@wwfnepal.org)5. The Royal Veterinary CollegeDr. Richard Kock (rkock@rvc.ac.uk)39

Nepal Elephant Tuberculosis ulosisControl andManagement agemenAction Plan (2011-2015)2015)Laboratories1. Nepal An-Tuberculosis Associaon; Genetup (TB cultures)Dr. Bhawana, Chief Medical Officer; Phone: 4270483; Email: nata@genetup.wlink.com.npBhagwan Das Maharjan, Lab in Charge; Phone: 270483; Email: nata@genetup.wlink.com.np2. B.P. Koirala Memorial Cancer Hospital (biochemistries and histopathology)Dr. Chin Bahadur PunHead and Consultant Pathology DepartmentBharatpur, ChitwanPhone: 977-56-524501 Ext 2107; Fax: 977-56-523747; Email: cbpun99@hotmail.com3. Instute of Agriculture and Animal Science (Histopathology)Dr. D.K. Singh; Phone: 9845023132; Email: dksnl@yahoo.com4. Chembio Diagnosc Systems, Inc. Medford, NY, USAElephant TB Stat-Pak® and DPP Vet ® TB test testsOrdering informa!on: Bobbie CocoPhone: 1-631-924-1135 x100; Fax: 1-631-924-6033Email: BCoco@chembio.com5. Center for Molecular Dynamics - NepalDibesh Karmacharya, Interna!onal Director; Email: dibesh@cmdn.orgSamer Dixit, PhD - Country Director Phone: 977-9802030789Email: s.dixit@cmdn.org.npwww.cmdn.org.np40