WHO monographs on selected medicinal plants (detail) VOLUME 1, World Health Organization Geneva 1999 http://whqlibdoc.who.int/publications/1999/9241545178.pdf WHO monographs on selected medicinal plants Definition Herba Echinaceae Purpureae consists of the fresh or dried aerial parts of Echinacea purpurea (L.) Moench harvested in full bloom (Asteraceae) ( 1 ). Synonyms Brau
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99090eb.pm6Zoo Biology 19:393–403 (2000)
Tuberculosis in Elephants in North
Susan K. Mikota,1 R. Scott Larsen,2 and Richard J. Montali3*
1Audubon Center for Research of Endangered Species, New Orleans, Louisiana2Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado 3Department of Pathology, Smithsonian National Zoological Park, Washington, DC Within the past 4 years, tuberculosis (TB) has emerged as a disease of concernin elephants. The population of elephants in North America is declining [Wiese,1997], and transmissible diseases such as TB may exacerbate this trend. Guide-lines for the Control of Tuberculosis in Elephants, which require the screeningof all elephants for TB, were instituted in 1997 [USDA, 1997; 2000]. BetweenAugust 1996 and May 2000, Mycobacterium tuberculosis was isolated from 18of 539 elephants in North America, indicating an estimated prevalence of 3.3%.
Isolation of the TB orga nism by culture is the currently recommended test toestablish a diagnosis of TB; however, culture requires 8 weeks. Further researchis essential to validate other diagnostic tests and treatment protocols. Zoo Biol19:393–403, 2000.
Key words: Loxodonta africana; Elephas maximus; tuberculosis; bacterial disease
Tuberculosis (TB) was first described in elephants more than 2,000 years ago by ancient Ayurvedic physicians in Ceylon [Iyer, 1937; McGaughey, 1961]. In re-cent times, a case in London [Garrod, 1875] was followed by sporadic reports through-out the twentieth century [Narayanan, 1925; Baldrey, 1930; Gutter, 1981; Saunders,1983; Chandrasekharan, et al., 1995]. Although it is thought that elephants are sus-ceptible to Mycobacterium bovis [Dannenberg, 1978; Schmidt, 1986], Mycobacte-rium tuberculosis (M. tuberculosis) has been identified as the causative agent in allcases in which bacteria have been isolated. To date, most reported cases of TB haveoccurred in captive Asian elephants (Elephas maximus). Two suspected cases in Af- R.S. Larsen is currently at the Environmental Medicine Consortium, Department of Clinical Sciences,College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina.
*Correspondence to: Richard J. Montali, Department of Pathology, Smithsonian National ZoologicalPark, Washington, DC 20008-2598. E-mail: [email protected] Received for publication December 1, 1999; Accepted June 20, 2000.
2000 Wiley-Liss, Inc.
Mikota et al.
rican elephants (Loxodonta africana) in Uganda and Israel were not confirmed byculture [Woodford, 1982; Gorovitz, 1962]. M. tuberculosis was isolated from an Af-rican elephant in France [Urbain, 1938]. In a retrospective medical study of 379elephants in North American zoos, eight elephants died of TB between 1908 and1994 [Mikota et al., 1994]. It is likely that this figure would have been higher hadprivately owned elephants been included in the survey.
RECENT CASES (1996–2000)
Between August 1996 and June 2000, samples from 539 elephants were sub- mitted to the National Veterinary Services Laboratory (NVSL, Ames, IA) for myco-bacterial culture [J. Payeur, personal communication]. Seventeen elephants from eightherds in Illinois, California, Arkansas, Missouri, and Florida were diagnosed withTB. At the time of diagnosis, three elephants resided in American Zoo and AquariumAssociation accredited zoos and 14 in private facilities. There had been known pre-vious contact between elephants in five of the herds. Three elephants demonstratedclinical signs that could be caused by TB. M. tuberculosis was isolated from 12elephants pre-mortem and five elephants post-mortem. Restriction fragment lengthpolymorphism was performed on M. tuberculosis isolates from 11 elephants. Fivedistinct M. tuberculosis strains were identified by this analysis [D.L. Whipple, 1997;personal communication].
Based on the 539 elephants in the NVSL database and the 532 elephants iden- tified by the North American Regional Studbook keepers [M. Keele and D. Olson,personal communication], the prevalence of TB in elephants in North America isestimated to be 3.3%.
Confirmed cases of TB in elephants have typically been identified on post- mortem examination as ante-mortem signs are frequently absent. Chronic weightloss, anorexia. and weakness may occur [McGaughey, 1961; Gutter, 1981; Saunders,1983], and dyspnea and coughing are sometimes observed [Seneviratna et al., 1966;Pinto et al., 1973]. Exercise intolerance is most likely to be observed in workinganimals [K.U. Mar, personal communication]. In Ceylon, ancient Ayurvedic elephantphysicians regarded ventral edema as a sign of incurable lung disease [Pinto et al.,1973]. More recently, ventral edema has been observed in some TB-infected elephants;however, it may have been caused by concurrent congestive heart failure, anemia, orother medical conditions [Seneviratna et al., 1966; Pinto et al., 1973].
Several techniques have been used to diagnose mammalian TB. Methods such as culture, acid fast smears, fluorescent smears, and nucleic acid amplification techniquesdirectly detect the bacterial organism. Indirect methods such as serological assays, thegamma-interferon test (GIT), and the intra-dermal tuberculin test detect antigen-anti-body or cellular reactivity to mycobacterial antigen [Mikota and Maslow, 1997].
In humans and domestic animals, the intra-dermal tuberculin skin test is the primary screening method to detect infection with M. tuberculosis or M. bovis. The Tuberculosis in Elephants
intra-dermal tuberculin test is the official ante-mortem test in cattle, bison, anddeer and is incorporated into the U.S. Department of Agriculture (USDA) eradica-tion guidelines [Essey and Davis, 1997; USDA, 1994; 1999]. The blood TB test(BTB), enzyme-linked immunosorbent assay (ELISA), and GIT have been used todiagnose TB infection in a limited number of species [Griffin and Cross, 1989;Rothel et al., 1992; Gaborick et al., 1996]. These indirect methods have not beenvalidated in most non-domestic species nor have they been uniformly administeredor consistently interpreted [Montali and Hirschel, 1990]. The limitations of apply-ing such diagnostic tests to non-domestic species have been discussed [Hietala andGardner, 1999].
Intra-dermal tuberculin testing is used in conjunction with slaughter surveil- lance to control TB in domestic cattle [Thompson et al., 1998]. Obviously, this methodshould not be applied to species such as the elephant. The current gold standard forthe ante-mortem diagnosis of M. tuberculosis infection in elephants is mycobacterialculture of respiratory secretions obtained by trunk “wash” [USDA, 1997; 2000]. El-ephants must be conditioned to permit the trunk wash procedure, which consists ofthe instillation of 60 mL of sterile saline into one or both nostrils, application of a 1-gal plastic bag over the end of the trunk, elevation of the trunk to distribute thesaline, and collection of a forcibly exhaled sample into the bag [Isaza and Ketz,1999]. Because the TB organism can be shed intermittently, three samples are col-lected on separate days.
To date, intra-dermal and serologic tests have frequently shown poor correla- tion with mycobacterial culture results in elephants [Montali et al., 1998]. One el-ephant that died from disseminated pulmonary M. tuberculosis infection had negativeresults when tested with human and bovine purified protein derivative (BPPD) tu-berculins [Saunders, 1983]. Furthermore, four of five M. tuberculosis culture-posi-tive elephants diagnosed since 1996 have demonstrated negative intra-dermaltuberculin responses to BPPD or balanced tuberculins. Eight of 30 M. tuberculosisculture-negative elephants had suspect tuberculin responses [Mikota, 1999].
Nucleic acid amplification techniques (NAAT) such as PCR detect mycobacte- ria by amplification of DNA or RNA [de Wit et al., 1990; Clarridge et al., 1993;Liébana et al., 1995; Roberts et al., 1996]. Advantages of NAAT include rapid turn-around time (hours) and the capability of detecting low numbers of organisms. Thesetechniques have been reported to have high specificity for M. tuberculosis complexorganisms (M. tuberculosis, M. bovis, M. africanum, M. microti), but mycobacterialspecies cannot be differentiated. Since both live and dead organisms are detected,NAAT is of limited value in monitoring response to therapy.
There are at present two amplification assays that are approved by the U.S. Food and Drug Administration for commercial use in human clinical and public health labo-ratories: the Amplicor MTB PCR (Roche Molecular Systems, Branchburg, NJ) and theAmplified M. tuberculosis Direct Test (MTD ) (Gen-Probe, San Diego, CA). Theseassays have demonstrated specificities of ≥95%, but they have been less sensitive thanculture for diagnosis of M. tuberculosis infection in humans [Dalovisio et al., 1996].
Validation has not yet been reported in elephants. Between August 1996 and August1999, the NVSL performed the MTD on 612 elephant samples. The MTD correctlydiagnosed six of nine culture-positive samples and 559 of 581 culture-negative samples.
Results of 23 samples were inconclusive [J. Payeur, personal communication]. Usingculture as the gold standard, the MTD demonstrated a sensitivity of 66.6% and a speci- Mikota et al.
ficity of 96.2%. Validation of this test is still needed, and, at this time, the MTD andother NAATs are considered ancillary tests for detecting TB in elephants.
Serologic testing for TB has been performed using ELISA techniques with a non-species–specific protein A that is labeled with horseradish peroxidase. Early in-vestigation of this technique in a herd of five elephants, which included one culture-positive animal, was described by Thoen and colleagues . The antigens usedwere heat-killed cells of M. bovis, BPPD, and purified protein derivative of M. avium(APPD). Two tuberculin-test positive elephants showed substantial seroreactivity toheat-killed M. bovis and BPPD, whereas two of three tuberculin-test negative ani-mals showed seroreactivity only to heat-killed M. bovis. Between 1997 and 1998, during the evaluation of a herd of four culture-nega- tive elephants, it was proposed that tuberculin exposure may influence serologic andBTB analysis [Montali et al., 1998]. The antigens used in this serologic investigationwere BPPD, M. tuberculosis culture filtrate protein, M. avium culture filtrate protein,APPD, and a lipoarabinomannan antigen (LAM) derived from M. tuberculosis. El-ephants showed minimal to zero reactivity to these antigens before intra-dermal tu-berculin injection, but dramatic increases in seroreactivity after injection. Elephantsare constantly exposed to saprophytic Mycobacterium species and can be colonizedby non-tuberculous mycobacteria (as well as M. avium) due to their behavior ofbathing and dusting using their trunks. These organisms do not appear to cause clini-cal disease, but it has been hypothesized that such exposure accounts for these seem-ingly non-specific reactions to mycobacterial antigens [Montali et al., 1998].
Recently, a multiple-antigen ELISA was evaluated for its ability to detect M . tuberculosis infection in captive elephants [Larsen et al., 2000]. Serum samples werecollected from 32 Asian and 15 African elephants, and a panel of six antigens wasused to determine seroreactivity. The antigens included M. bovis culture filtrate (CF),PPD, M. bovis modified protein 70, two LAM antigens from strains of M. tuberculo-sis, and APPD. Discriminant analysis was used to determine the linear combinationof antigens that accurately predicted the true infection status of the most animals,and the resulting classification functions were used to calculate the percentage ofanimals that were correctly classified. Of 47 elephants, seven of the Asians wereinfected (culture-positive); 25 Asians and 15 Africans were considered non-infected(culture-negative). Criteria for elephants designated as non-infected were: 1) trunkwashes within 4 months of serum sampling that were negative for mycobacterialculture of M. tuberculosis or M. bovis; 2) no contact with elephants or other animalsthat had been diagnosed with M. tuberculosis of M. bovis within the past 5 years; 3)no intra-dermal tuberculin testing within the 6 months before sampling; and 4) notravel outside the institution in the previous 5 years. The specificity and sensitivityof the multiple-antigen ELISA, with 95% confidence intervals, were 100% (91.9–100%) and 100% (54.4–00%), respectively. The limitations inherent to this studysuggest that much additional research is needed regarding the use of this ELISA;however, the results also indicate that this multiple-antigen ELISA may be a goodscreening test for elephants [Larsen et al., 2000].
The major pathology in elephants infected with M. tuberculosis infections oc- curs primarily in the lungs and thoracic lymph nodes with lesser involvement of Tuberculosis in Elephants
extra-thoracic sites [Seneviratna et al., 1966; Pinto et al., 1973, Gutter 1981; Saunders1983; Michalak et al., 1998; R.J. Montali, unpublished observations, 1999]. As inother animals, tubercular lesions in elephants appear to vary with the staging of thedisease. In the less extensive cases, firm granulomatous nodules, sometimes withcaseous foci, are noted in the bronchial lymph nodes and pulmonary tissue. Elephantswith extensive involvement of both lungs (≥66%) usually die with severe caseo-calcareous and cavitating lesions. These often result in large pulmonary abscessesfrom which M. tuberculosis and opportunistic bacteria such as Pseudomonasaeruginosa have been isolated. Tenacious, mucopurulent, bronchial plugs are alsocommon in advanced TB; bronchial and other thoracic lymph nodes are markedlyenlarged and usually show a proliferative response with less caseation than the pul-monary lesions.
The pathologic descriptions “caseous” and “mucopurulent” are based on post- mortem observations of the lungs and upper respiratory tract. At post-mortem, theseverity of the disease is determined and a time frame ascribed. Active, early lesions,which conceivably can occur within weeks to months, are usually localized and lim-ited in size and scope. Advanced lesions may involve major portions of one or bothlung lobes and become mineralized over a period of months to years. Both, however,may have areas that are caseous or mucopurulent.
Characteristic histologic findings include epithelioid granulomas with some gi- ant cell formation in the earlier lymph node and pulmonary lesions and extensivecaseous and pyogranulomatous pneumonia in the advanced forms. Though sparse,acid-fast bacilli are more easily found in central areas of caseation in the lungs butare typically rare in the lymph nodes.
Bronchial and tracheal tuberculous plaques and caseous and mucopurulent exu- date in the nasal passages have been noted in both the early and late stages of TB,suggesting that the shedding of mycobacteria may occur at any stage of the disease.
Less extensive tuberculous lesions were observed in the mesenteric lymph nodes,liver, kidneys, adrenals, and spleen in some of the more advanced cases. These le-sions suggest that in disseminated cases shedding may occur by routes other than therespiratory system.
There is little information in the literature regarding treatment of elephants for TB. In one report, an Asian elephant was treated prophylactically with isoniazid (INH)after a suspect intradermal tuberculin test [Devine et al., 1983]. Another suspectedcase of TB was treated with streptomycin administered intramuscularly on alternatedays for 4 weeks [Chandrasekharan et al., 1995]. Current treatment protocols havebeen extrapolated from human treatment regimens [American Thoracic Society, 1994]and are still under investigation for efficacy in elephants.
Anti-TB drugs recently used in elephants includes INH, pyrazinamide (PZA), rifampin (RIF), and ethambutol (ETH). These drugs have been administered to elephantsin food, by direct oral administration, and rectally. Oral delivery has been challenging,as many elephants refuse oral medications. Direct oral administration can be achievedin some elephants by conditioning the animals to accept a bite block and oral syringe[L. Peddie and J. Peddie, personal communication]. For other elephants, rectal drugadministration techniques (including suppositories) have been developed.
Mikota et al.
Blood levels of INH, consistent with human therapeutic values, can be achieved in elephants by direct oral or rectal administration. Blood levels of RIF can be achievedorally, but not rectally [Dunker and Rudovsky, 1998]. PZA appears to be absorbedby either route [S.K. Mikota, unpublished data]. When anti-TB drugs are adminis-tered in food, blood levels are variable and this route of administration is not recom-mended. Anti-TB drug doses for individual elephants should be determined bymeasuring blood-level response. Elephants should be weighed before and through-out treatment.
The current recommended treatment for known infected elephants consists of INH and RIF daily for 2 months, then every other day for 10 months. A thirddrug, such as PZA, is given daily for the first 2 months of treatment. As a start-ing dose, INH can be given orally or rectally at a dose of 2.5–5.0 mg/kg. Al-though humans typically achieve a blood level of 3–5 µg/mL of INH at 2 hours[C.A. Peloquin, personal communication], some elephants became ill when theirblood levels were in this range. An INH blood level of 1–2 µg/mL is recom-mended for elephants.
RIF can be initiated orally at a dose of 7.5–10.0 mg/kg orally. Human 2-hour blood levels for this drug are 8–24 µg/mL. PZA can be initiated at a dose of 25–35mg/kg orally or rectally. Human 2-hour levels for PZA are 20–60 µg/mL. Supple-mentation with vitamin B6 (pyridoxine) at a daily dose of 1 mg/kg is recommendedto prevent possible peripheral neuropathy, a condition that has been associated withINH therapy in humans [Goldman and Braman, 1972].
Side effects of treatment may include anorexia, lethargy, and colitis. Leukope- nia was observed in one elephant receiving INH and RIF; the condition resolvedafter treatment was temporarily stopped and dosages adjusted [L. Peddie and J. Peddie,personal communication]. Elevations of liver enzymes have been observed in asso-ciation with INH toxicity in humans and elephants.
Of 11 living M. tuberculosis culture-positive elephants, six are currently re- ceiving anti-TB drugs including one elephant that is undergoing a second course oftreatment. Five elephants that completed treatment in December 1997, June 1998,December 1998, October 1999, and April 2000, are presently culture negative. Allculture-positive elephants ceased shedding organisms shortly after treatment was ini-tiated and remained culture-negative during the treatment period.
GUIDELINES FOR THE CONTROL OF TB IN ELEPHANTS
In 1996, in response to the TB-related deaths of two privately owned el- ephants, an Elephant Tuberculosis Advisory Panel was formed. This panel, whichwas composed of USDA and zoo veterinarians, cooperated with the National Tu-berculosis Working Group for Zoo and Wildlife Species to develop Guidelines forthe Control of Tuberculosis in Elephants [USDA, 1997; 2000; www.aphis.usda.gov/ac/acindex.html]. These guidelines specify criteria for the testing, surveillance, andtreatment of elephants for TB. In January 1998 and January 2000, they weredistributed by the Animal Care Division of the USDA Animal Plant Health In-spection Service (USDA-APHIS) to all licensed elephant exhibitors regulated bythe Animal Welfare Act. The guidelines require annual testing (three trunk cul-tures) of all elephants and strongly recommend submission of samples for ancil-lary diagnostic tests.
Tuberculosis in Elephants
According to the guidelines, elephants are placed in one of four groups based on culture results and exposure history. Group A elephants have negative cultureresults and no known exposure to a culture-positive animal in the previous 5 years.
These animals are cultured annually and have no travel restrictions while they re-main culture negative for M. tuberculosis. Culture-negative elephants exposed to aculture-positive animal 1–5 years previously (group B) are cultured quarterly andhave no travel restrictions. Culture-negative elephants exposed to a culture-posi-tive animal within the previous 12 months (group C) may be monitored by culture(three-sample method), every other month for 1 year, with no travel permitted or,alternatively, may be treated, with travel permitted after 2 months if cultures re-main negative. Culture-positive elephants (group D) are not permitted to traveluntil at least 6 months of treatment have been completed and two negative cultureshave been demonstrated. Figure 1 illustrates the sequence of events for each of thefour groups.
A thorough post-mortem examination should be performed on all elephants that die or are euthanized. Lungs and lymph should be closely examined for evidence ofTB. Cultures for TB should be submitted on all elephants even if gross lesions areabsent. A necropsy protocol for elephants may be accessed at the above website.
IMPLICATIONS FOR HUMAN HEALTH
After the diagnosis of TB in the Illinois herd, all personnel were tuberculin tested by the Illinois Department of Health. Of 22 handlers, 11 were tuberculin testpositive. Eight of the 11 were positive on the initial test, indicating the possibility ofprior exposure; three individuals converted during the investigation. One handlerhad culture-positive TB. The isolate from this individual matched that of the Illinoiselephants [Michalak et al., 1998]. The original source of infection (for both elephantsand humans) is unknown.
The apparent low incidence of TB in African elephants and the absence of reports of TB in free-ranging elephants suggest that this is primarily a disease ofhumans and that elephants are accidental hosts. Nonetheless, M. tuberculosis can betransmitted between elephants and humans and must be considered zoonotic [Maslow,1997; Michalak et al., 1998]. Humans are most likely to contract TB when they haveprolonged contact with infected individuals. It is likely that this is also the case withelephants, suggesting that handlers with close, daily contact with infected animalsare at greatest risk. Elephant handlers and other personnel in contact with elephantsshould be tested for TB annually following established human testing protocols. Allnew employees should be tested before contact with elephants and anyone with ac-tive TB should not have contact with elephants. Some zoos have developed elephant/people interaction protocols to limit direct visitor contact with elephants, a practicethat should be encouraged [Montali, 1999].
The ante-mortem diagnosis of TB in elephants continues to be problematic.
Although identification of M. tuberculosis definitively establishes the presence ofinfection, failure to isolate the organism does not rule out infection. Mycobacteriaare slow-growing organisms and culture typically requires 8 weeks. Clearly, better Mikota et al.
Elephant management groups for TB surveillance [from USDA, 2000]. (For protocol, see Guidelines for the Control of Tuberculosis in El- ephants [USDA, 2000] www.aphis.usda.gov/ac/acindex.html).
Tuberculosis in Elephants
diagnostic tests are needed. Preliminary ELISA results are promising, but many moresamples (both culture positive and negative) must be analyzed before this test (orany other serologic test) can be validated. Institutions holding elephants are stronglyencouraged to submit samples for ancillary diagnostic tests so that valuable researchdata may be collected.
Although there is optimism that infected elephants in the North American popu- lation have been successfully treated, only long-term monitoring will confirm this.
Pharmacokinetic studies are needed to further evaluate anti-TB drugs and to validatetherapeutic protocols. It is essential that elephants that undergo treatment be observedfor possible side effects. Blood levels of anti-TB drugs must be documented andcorrelated with treatment outcome.
Information on the pathophysiology and staging of TB in elephants is needed.
A necropsy should be performed on all elephants that die and a thorough search forTB lesions should be conducted, even if the disease is not suspected. The elephantnecropsy protocol (available on the USDA website) outlines appropriate samples tosubmit for laboratory evaluation. In cases of euthanasia, a diagnostic workup for TB(including ancillary tests as outlined in the guidelines) should be performed pre-mortem so that results can be correlated with postmortem findings.
A reporting mechanism has been established whereby annual culture and ancil- lary test results are submitted to the American Association of Zoo Veterinarians fortabulation. Compliance has been poor for this critically needed information. We en-courage the cooperation of the zoo community to comply with this reporting mecha-nism so that we may further our understanding of TB in elephants.
1. Reported cases of TB in elephants have been caused by M. tuberculosis (the agent of human TB). Although TB has been reported more frequently in Asianelephants, it is unknown whether there is a true species predilection.
2. Isolation of M. avium and non-tuberculous mycobacteria from elephant trunk wash samples is common, but these organisms have not been associated with clinicaldisease.
3. Isolation of M. tuberculosis is currently the only definitive test to diagnose TB in elephants, although ancillary tests such as NAAT and ELISA may be useful.
4. The intra-dermal tuberculin test is unreliable for diagnosing TB in elephants.
5. It is possible to deliver dosages of anti-TB drugs that achieve blood levels consistent with therapeutic levels in humans; however, the long-term efficacy of cur-rent treatment protocols remains to be documented.
6. Shedding of TB organisms generally ceases when elephants receive ad- 7. Transmission of TB between an elephant and a human has been reported.
Handlers in close daily contact with infected elephants are at greatest risk.
8. Elephants may be at risk of contracting TB from infected humans. Han- dlers should undergo periodic TB screening to minimize risks to elephant health.
9. Complete post-mortem examination should be performed on all elephants that die. A thorough search for TB lesions should be conducted even if TB is notsuspected.
10. Zoos are encouraged to establish protocols for elephant-visitor interactions.
Mikota et al.
Many individuals have been involved in addressing the complex issues associ- ated with TB in elephants. We acknowledge the following individuals who have con-tributed to our knowledge of the epidemiology, diagnosis, and treatment of this diseasein elephants: Joel Maslow, M.D. Ph.D., Freeland Dunker, D.V.M., Gary West, D.V.M.,Ramiro Isaza, D.V.M., William Lindsay, D.V.M., Jim Peddie, D.V.M., Linda Peddie,D.V.M., Wilbur Amand, V.M.D., Mitch Essey, D.V.M., Werner Heuschele, D.V.M.,Janet Payeur, D.V.M., Diana Whipple, Miava Binkley, D.V.M., Delphi Chatterjee,Ph.D., Mo Salman, B.V.M.S., Ph.D., Scott and Heidi Riddle, Gary and Kari Johnson,John Cuneo, and the dedicated elephant handlers who have been involved with thetreatment of infected elephants.
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Formatting Your Paper for Evolutionary Computation B. A. Author Department of Science, My University, MyTown, Zip, Country D. C. Author2 Department of Science, My University, MyTown, Zip, Country Abstract The abstract goes here. It should be about 200 words and give the reader a summaryof the main contributions of the paper. Remember that readers may decide to read ornot to re