Journal of Zoo and Wildlife Medicine 32(1): 1?16, 2001 Copyright 2001 by American Association of Zoo Veterinarians EPIDEMIOLOGY AND DIAGNOSIS OF MYCOBACTERIUM TUBERCULOSIS IN CAPTIVE ASIAN ELEPHANTS {ELEPHAS MAXIMUS) Susan K. Mikota, D.V.M., Linda Peddie, D.V.M., James Peddie, D.V.M., Ramiro Isaza, D.V.M., M.S., Freeland Dunker, D.V.M., Gary West, D.V.M., William Lindsay, D.V.M., R. Scott Larsen, D.V.M., M.S., M.D. Salman, B.V.M.S., Ph.D., Delphi Chatterjee, Ph.D., Janet Payeur, D.V.M., Ph.D., Diana Whipple, M.S., Charles Thoen, D.V.M., Ph.D., Donald S. Davis, Ph.D., Charles Sedgwick, D.V.M., Richard J. MontaU, D.V.M., Michael Ziccardi, D.V.M., M.P.V.M., and Joel Maslow, M.D., Ph.D. Abstract: The deaths of two Asian elephants (Elephus maximus) in August 1996 led the United States Department of Agriculture to require the testing and treatment of elephants for tuberculosis. From August 1996 to September 1999, Mycobacterium tuberculosis infection was confirmed by culture in 12 of 118 elephants in six herds. Eight diagnoses were made antemortem on the basis of isolation of M. tuberculosis by culture of trunk wash samples; the remainder (including the initial two) were diagnosed postmortem. We present the case histories, epidemiologic characteristics, diagnostic test results, and therapeutic plans from these six herds. The intradermal tuberculin test, enzyme-linked immunosorbent assay serology, the blood tuberculosis test, and nucleic acid amplification and culture are compared as methods to diagnose M. tuberculosis infection in elephants. Key words: Elephant, Elephus maximus, Loxodonta africana, tuberculosis, Mycobacterium tuberculosis, tuberculin test. INTRODUCTION Tuberculosis (TB) has been recognized for over 2,000 yr as a disease of elephants.''-^ It has been most frequently reported in Asian elephants {Ele- phas maximus)}'^'^^-"-^^-^'^-^^'^^ Only one case in Af- rican elephants (Loxodanta africana) has been con- firmed by culture,'^?^'?''^ and all cultures in both spe- cies have involved Mycobacterium tuberculosis. A retrospective North American zoo study noted eight deaths from TB among 379 elephants. Four of these From the Audubon Center for Research of Endangered Species, 14001 River Road, New Orleans, Louisiana 70131, USA (Mikota); 15333 Rains Court, Moorpark, California 93021-3211, USA (Peddie); the Department of Clinical Sciences, College of Veterinary Medicine, Kansas State University, 1800 Denison Avenue, Manhattan, Kan- sas 66506, USA (Isaza); the San Francisco Zoological Gardens, One Zoo Road, San Francisco, California 94132- 1098, USA (Dunker); Feld Entertainment, 8607 West- wood Center Drive, Vienna, Virginia 22182, USA (West, Lindsay); the Center of Veterinary Epidemiology and An- imal Disease Surveillance Systems, College of Veterinary Medicine and Biom?dical Science (Larsen, Salman), and Department of Microbiology (Chatterjee), Colorado State University, Fort Collins, Colorado 80523, USA; the Unit- ed States Department of Agriculture, Animal and Plant Health Inspection Service, National Veterinary Services Laboratories, 1800 Dayton Road, Ames, Iowa 50010, USA (Payeur); the Zoonotic Diseases Research Unit, United States Department of Agriculture, Agricultural Re- search Service, National Animal Disease Center, 2300 Dayton Road, Ames, Iowa 50010, USA (Whipple); the cases occurred prior to 1941, and three were con- firmed by culture.-^ Clinical signs of TB in elephants are comparable to those observed in humans and may include weight loss, anorexia, weakness, dyspnea, and coughing."23.29.3i.32 Definitive antemortem diagnostic techniques for TB in elephants have limitations. The intradermal (i.d.) tuberculin test, currently the accepted standard Department of Microbiology, Immunology, and Preven- tive Medicine, College of Veterinary Medicine, Iowa State University, 16th Street S.W., Ames, Iowa 50011, USA (Thoen); the Department of Veterinary Pathobiology, Tex- as A&M University, Highway 60 West, College Station, Texas 77843-4467, USA (Davis); the Los Angeles Zoo, 5333 Zoo Drive, Los Angeles, California 90027, USA (Sedgwick); the Smithsonian National Zoological Park, 3001 Connecticut Avenue, N.W., Washington, D.C. 20008-2598, USA (Montali); the Lincoln Park Zoo, PC. Box 14903, Chicago, Illinois 60614, USA (Ziccardi); and the Infectious Diseases Section, Veterans Affairs Medical Center, and University of Pennsylvania, University and Woodland Avenues, Philadelphia, Pennsylvania 19104, USA (Maslow). Present addresses: (West): San Antonio Zoological Gardens and Aquarium, 3903 North Saint Mary's Street, San Antonio, Texas 78212-3199, USA; (Larsen): Environmental Medicine Consortium, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, North Carolina 27606, USA; (Ziccardi): Wildlife Health Center, University of California, Davis, California 95616, USA. Correspon- dence should be directed to Dr. Peddie. 1 JOURNAL OF ZOO AND WILDLIFE MEDICINE domestic animal screening test, has been validated only in domestic cattle, bison, and Cervidae and has been poorly correlated with culture results in several species."?^''''' Other indirect methodologies, such as s?rologie assay and cellular responsiveness to mycobacterial antigens, also lack validation in nondomestic species. Radiographie thoracic evalu- ation is feasible only in young elephants. Bison and Cervidae are the only nondomestic ungulates officially regulated under the National Cooperative State?Federal Bovine Tuberculosis Eradication Program, which is based on slaughter- surveillance to control Mycobacterium bovis.^^"^^ With elephants and other endangered species, treat- ment with intent toward cure is preferable to test and slaughter. Treatment is expensive, however, and may be associated with fatal, idiosyncratic effects in some species,* so accurate diagnosis of both ac- tive and latent disease is of paramount importance. Subsequent to the diagnoses of M. tuberculosis in two Asian elephants in 1996, an Elephant Tu- berculosis Advisory Panel was formed. Through the efforts of this panel, the United States Depart- ment of Agriculture (USDA), Animal and Plant Health Inspection Service (APHIS), Veterinary Ser- vices, and the National Tuberculosis Working Group for Zoo and Wildlife Species (formed in February 1997 and comprised of veterinarians from the American Association of Zoo Veterinarians and USDA), an elephant TB screening protocol was es- tablished.^^ This protocol, requiring the testing of elephants for TB, was sent to all licensed exhibitors in the United States in January 1998. We review the presentation and clinical course of M. tuberculosis infection in 12 elephants from six affected herds. Culture results are compared with indirect methods of TB diagnosis including i.d. tuberculin testing, s?rologie responsiveness de- termined by enzyme-linked immunosorbent assay (ELISA) and lymphocyte stimulation, as well as di- rect testing by amplification to detect mycobacterial nucleic acids. We briefly describe treatment regi- mens and efficacy, although these findings will be reported and discussed elsewhere. HISTORY OF AFFECTED HERDS Herd A On 3 August 1996, a 47-yr-old female Asian el- ephant (Al) died in California after anesthesia for a suspected dental problem. Weight loss had been noted since October 1995. At necropsy, dissemi- nated, caseous, granulomatous lesions were ob- served in the cervical, tracheobronchial, and me- diastinal lymph nodes. The serosal surfaces of the liver, stomach, and peritoneum were covered with white, miliary, firm nodules. Numerous firm, white, nodular lesions were identified in the lungs, with less than 10% functional parenchymal mass re- maining. Acid-fast bacteria were observed in 9 of 26 tissue smears. Culture samples from multiple tis- sues yielded M. tuberculosis. Four elephants traveling with elephant Al were en route to the home facility when a 26-yr-old fe- male Asian elephant (A2) died on 6 August 1996. This animal was asymptomatic and in fair body condition. Postmortem examination revealed se- vere, multifocal, granulomatous pneumonia with numerous abscesses, caseation, and granulomatous lymphadenitis. Culture of lung tissue from this el- ephant also yielded M. tuberculosis. In September 1996, a 37-yr-old male Asian ele- phant (A4) died suddenly, with a history of foot problems, weight loss, and inappetance preceding death. An enlarged heart and pericardial effusion were the most significant postmortem findings. His- tologie examination showed no evidence of tuber- culosis, and mycobacteria were not isolated. Eigh- teen elephants (A3, A5?A21) remained in the herd: one male Asian elephant (age 3 yr), 15 female Asian elephants (18?55 yr), and two female African elephants (16 and 18 yr). Elephant facility A consisted of a large barn sub- divided into small, medium, and large elephant holding areas. A small (3 m X 9 m) entry area was situated between the medium and large barns, and an enclosed exercise area was situated between the small and large barns. Ventilation ducts located in the ceiling were continuous between barns. Be- cause of the northern location of the facility, the elephants were housed indoors for 3-4 mo during the winter Approximately 14 animals were kept in a line in the larger of the barns. Two to five animals were housed in the medium barn. The elephants were exercised together in a large outdoor paddock. The young Asian elephant male was housed in the small barn. Twelve elephants from herd A, including el- ephants Al and A2 (which typically traveled to- gether) and A3 (which traveled in a separate group), were leased to various circuses. Six animals did not travel. Two cases of M. tuberculosis infection had been documented in herd A prior to this study period. The first case occurred in an 11-yr-old female Asian elephant that died in California in 1983.^' The sec- ond case was a 46-yr-old female Asian elephant that died in 1994. After the diagnosis of TB in el- ephants Al and A2, the remaining 18 elephants were evaluated in October 1996 and December MIKOTA ET AL.?ELEPHANT TUBERCULOSIS 1996. Mycobacterium tuberculosis was isolated from a trunk wash culture and a pharyngeal swab culture taken from a 46-yr-old female Asian ele- phant (A3). Treatment of the herd was initiated in December 1996; trunk cultures were collected quar- terly during this period. Subsequent diagnostic evaluations were conducted in August 1997, Janu- ary 1998, and December 1998. HerdB In March 1997, a 30-yr-old female Asian ele- phant (Bl) died of severe fibrinonecrotic entero- colitis. Fecal cultures yielded Salmonella typhimu- rium, which was considered to represent the etio- logic agent. Prior to death, the only significant clin- ical problem noted for this animal was chronic arthritis of the left hip. At necropsy, several small focal granulomas were found in the thoracic and abdominal cavities. Histology showed necrotizing, granulomatous lymphadenitis and multinucleated giant cells. Culture of these tissues yielded M. tu- berculosis. The remainder of the herd consisted of a 39-yr- old female Asian elephant (B2), a 12-yr-old male Asian elephant (B3), and two female African ele- phants, ages 32 and 37 yr (B4 and B5, respective- ly). Housing at facility B consisted of a large open- sided indoor barn area and a large dirt ?oor outdoor area. Within herd B, the four females were housed together and had extensive contact. Contact with the remaining male Asian was limited to interaction across a fence. After the diagnosis of M. tuberculosis in elephant Bl, trunk wash samples for culture were obtained from the remaining herd members in July 1997. Follow-up cultures were obtained every 1?2 mo for the next 6 mo and then quarterly. Prophylactic treatment of the herd was initiated. No other M. tuberculosis-infected animal was detected. Herd C Herd C consisted of a 29-yr-old female Asian elephant (Cl), a 32-yr-old female Asian elephant (C2), and two female African elephants, ages 30 and 37 yr (C3 and C4, respectively). Elephant Cl joined herd C in March 1997; prior to that time it was in herd B. The two Asian elephants (Cl and C2) shared a barn and exercise area. The two Af- rican elephants (C3 and C4) were housed together in a separate area of the zoo, and there was no contact between the two groups. Trunk wash samples for culture were obtained from elephants Cl and C2 in March 1997. Myco- bacterium tuberculosis was isolated from elephant C1. Therapeutic treatment of elephant C1 and pro- phylactic treatment of elephant C2 were initiated in May 1997. Follow-up cultures of elephants Cl and C2 were obtained on average every 4?6 wk through August 1999 and continue on a monthly schedule. The African elephants (C3 and C4) were initially cultured in January 1998. Mycobacterium. tuberculosis was not isolated from elephant C2, C3, or C4. HerdD Trunk wash samples for culture were taken from this group of elephants in May 1997. Elephant Al had died on the premises of herd D in August 1996, and elephant Cl (a culture-positive animal) had lived in this group. Herd D consisted of four female Asian elephants ranging in age from 29 to 32 yr (D1-D4). Three elephants (D5-D7) had been sent to other institutions for breeding. One, a 13-yr-old female Asian elephant (D5), left the herd in April 1996 and returned in September 1997. Elephants D6 and D7 left in February 1995 and returned in April 1998. Housing at facility D was contained within an 18.3-m X 18.3-m concrete block structure with a well-drained concrete floor Within that structure was one large (12 m X 12 m) and one smaller (6 m X 9 m) housing area. There was no shared air space connecting the two housing areas. Each area, when in use, was cleaned and sanitized daily. Each housing area had large doors on two sides that were left open throughout the day, affording cross ven- tilation and exposure to sunlight while the elephants were in one of two separate, 0.2-ha exercise corrals. The interior of one of the exercise corrals was ar- ranged so the elephants could commingle or be sep- arated from one another. The elephants were housed outdoors during daylight hours throughout the year. Herd D was first tested in May 1997. Mycobac- terium tuberculosis was isolated from elephants Dl and D2. Treatment was initiated in June 1997. HerdE Herd E consisted of one 10-yr-old male Asian elephant (El), three female Asian elephants ages 24?40 yr (E2-E4), four male African elephants ages 15?19 yr, (E5-E8), and two female African elephants ages 15 and 20 yr (E9 and ElO, respec- tively). The Asian and African herds were housed outside in large paddocks, 24 hr/day, for 7 mo of the year. The paddocks were separate but shared a common fence. For 5 mo, the herds were housed in two barns at night, again separated by species. The Asian elephant bull (El) was housed in a sep- arate barn and paddock with one of the Asian ele- JOURNAL OF ZOO AND WILDLIFE MEDICINE phant females. In previous years, some of the Asian elephant females had been housed with the African elephants. In June 1998, trunk washes were performed on the 10 elephants. Mycobacterium tuberculosis was isolated from the male Asian elephant (El); ele- phants E2-E10 were culture negative. Treatment of elephant El was initiated in October 1998. Sero- logie studies were conducted in October 1998 and March 1999. Cultures were obtained weekly on el- ephant El and every other month on elephants E2? ElO. Herd F Herd F consisted of four groups of elephants (FB, PR, FTl, and FT2). The first, FB, was a breed- ing group comprised of 23 female Asian elephants ranging in age from 2 to 57 yr and eight male Asian elephants ranging in age from 1 mo to 32 yr (FB 1? FB31). Group FR was a retirement group and in- cluded 12 female Asian elephants 31-57 yr, three male Asian elephants 12?54 yr, and one 52-yr-old female African elephant (FRl?FR16). Group FTl included traveling elephants comprised of 14 fe- male Asian elephants ages 15?47 yr (FTl?FT14), and FT2 was a traveling group of 12 female Asian elephants 29?49 yr and one 4-yr-old male Asian elephant (FT1-FT13). Housing at the breeding facility consisted of one large barn (1,579 m^), several smaller barns, and a series of large outdoor paddocks. Housing at the retirement facility was similar. The two facilities were located in the same state but were separated geographically. The two traveling elephant groups were generally housed at separate fairgrounds fa- cilities for 2 mo of the year. Screening for TB be- gan in 1997. Mycobacterium tuberculosis was iso- lated from elephants FBI and FB2 and elephants FR3 and FR4. Contact between herds There was extensive contact between five of the six herds. Elephant Al died on the premises of fa- cility D in August 1996. After her death, the barn used for treatment and necropsy was thoroughly cleaned with a mycobactericidal disinfectant. There had been no direct contact between elephant Al and herd D elephants. Elephant Bl resided in facility D from October 1991 through April 1994, when she moved to herd B. Elephants B3 and B4 had also been housed tem- porarily with herd D from fall 1993 through fall 1994. Otherwise, elephants B2, B3, and B4 were long-term members (>10 yr) of herd B. Elephant Cl had an extensive travel history. She joined herd D in October 1991, moved to facility B in April 1994, was returned to facility D in November 1996, and then finally moved to facility C in March 1997. Elephant El, born in 1988, was the son of elephant FBI. He was housed at facility D from September 1993 to February 1994 and again from March to May 1996. He was with herd A in early February 1996 and joined herd E in May 1996. There was no contact between herd F and the other five herds. At the time of testing, the four groups of herd F did not intermingle. However, in previous years, there was extensive commingling among the four groups. Elephants of herd F pri- marily moved between the retirement group FR and group FT2, although movements also occurred be- tween the breeding facility and both traveling groups. Seven of the elephants in the breeding group arrived from a separate facility in 1996. The FTl and FT2 groups each included three elephants that were owned by another facility. The histories of the six herds and the 12 M. to- ?)ercM/o5W?positive elephants are summarized in Tables 1 and 2. METHODS Cultures Cultures were processed at six laboratories: the National Veterinary Services Laboratories (NVSL; Ames, Iowa 50011, USA; herds A, C, D, E, and F); the County of Orange Public Health Laboratory (Santa Ana, California 92706, USA; herds B, C, D, and P); the San Bernardino County Laboratory (San Bernardino, California 92412, USA; herd B); the San Francisco City and County Public Health Lab- oratory (San Francisco, California 94110, USA; herd C); the Arkansas Department of Health, Di- vision of Public Health Laboratories (Little Rock, Arkansas 72205-3867, USA; herd E); and the Iowa State University (Ames, Iowa 50011, USA; herd F). Specimens consisted of trunk washes, trunk swabs, oral swabs, and feces. Drug susceptibilities of isolates of M. tubercu- losis were determined by the National Jewish Med- ical and Research Center (Denver, Colorado 80206, USA; elephant Bl), the San Bernardino County Laboratory (elephant Al), the San Francisco City and County Public Health Laboratory (elephant Cl), or the County of Orange Public Health Lab- oratory (elephants D2, FBI, FB2, and FR4). Sus- ceptibility testing for isolates from elephants A2, A3, El, and FR3 was not performed. Trunk washes were obtained by one of two meth- ods. The first method consisted of inserting a 14 MIKOTA ET AL.?ELEPHANT TUBERCULOSIS Table 1. Mycobacterial 1 culture results from six elephant herds." No. samples with atypi- cal, unidenti- fied myco- No. ele- No. ele- bacteria or No. No. No. Age phants phants No. No. sam- with Nocar- Herd elephants Asian African range M.tb+i' M.tb- samples ples M.tb+ dia A 21 19 2 3-55 3 18 380 4 4 B 5 3 2 12-39 1 4 62 1 14 C 4 2 2 29-37 1 3 160 7 18 D 7 7 0 13-32 2 5 411 4 93 E 10 4 6 10-40 1 9 199 2 117 F 75 74 1