Tuberculosis in elephants-a reemergent disease: diagnostic dilemmas, the natural history of infection, and new immunological tools.

Tuberculosis (TB) in elephants has been described since ancient times. However, it was not until 1996 when infection with Mycobacterium tuberculosis was identified in a herd of circus elephants that significant research into this disease began. The epidemiology and natural history of TB were unknown in elephants since there had been no comprehensive screening programs, and diagnostic techniques developed for cervidae and bovidae were of unknown value. And, while precepts of test and slaughter were the norm for cattle and deer, this was considered untenable for an endangered species. With no precedent for the treatment of TB in animals, treatment regimens for elephants were extrapolated from human protocols, which guided changes to the Guidelines for the Control of Tuberculosis in Elephants. In the absence of diagnostic testing to confirm cure in elephants, the efficacy of these treatment regimens is only beginning to be understood as treated elephants die and are examined postmortem. However, because of pressures arising from public relations related to elephant husbandry and the added considerations of TB infection in animals (whether real or imagined), sharing of information to aid in research and treatment has been problematic. Here we review the challenges and successes of the diagnosis of tuberculosis in elephants and discuss the natural history of the disease to put the work of Landolfi et al on the immunological response to tuberculosis in elephants in perspective.

Tuberculosis in Laos, who is at risk: the mahouts or their elephants?

SUMMARY Tuberculosis (TB) in elephants has the potential to infect humans and is an increasing public health concern. Lao PDR is one of the last countries where elephants are still used for timber extraction and where they live in close contact with their mahouts. There are 500 animals at work in the country, some interacting with wild herds. Although human TB prevalence is known to be high in Laos, studies on elephant TB had yet to be undertaken. From January to July 2012, screening was performed using the ElephantTB Stat-Pak assay on 80 elephants working around the Nam Pouy National Park in Sayaboury Province. This represents more than 18% of the total registered national working elephant population. Here we report that 36% of the elephants were seroreactive to the test. Of these, 31% had contacts with wild individuals, which suggests potential transmission of mycobacteria to the local wild herds. Clinical examination, chest X-rays, sputum microscopy and culture were performed on their 142 mahouts or owners. Despite high TB seroreactivity in elephants, no participant was smear- or culture-positive for Mycobacterium tuberculosis or M. bovis, although atypical mycobacteria were isolated from 4% of participants.

Population pharmacokinetics of pyrazinamide in elephants.

This study was undertaken to characterize the population pharmacokinetics (PK), therapeutic dose, and preferred route of administration for pyrazinamide (PZA) in elephants. Twenty-three African (Loxodonta africana) and Asian (Elephas maximus) elephants infected with or in contact with others culture positive for Mycobacterium tuberculosis were dosed under treatment conditions. PZA was dosed daily at 20-30 mg/kg via oral (fasting or nonfasting state) or rectal (enema or suppository) administration. Blood samples were collected 0-24 h postdose. Population PK was estimated using nonlinear mixed effect modeling. Drug absorption was rapid with T(max) at or before 2 h regardless of the method of drug administration. C(max) at a mean dose of 25.6 (+/-4.6) mg/kg was 19.6 (+/-9.5 microg/mL) for PZA given orally under fasting conditions. Under nonfasting conditions at a mean dose of 26.1 +/- 4.2 mg/kg, C(max) was 25% (4.87 +/- 4.89 microg/mL) and area under concentration curve (AUC) was 30% of the values observed under fasting conditions. Mean rectal dose of 32.6 +/- 15.2 mg/kg yielded C(max) of 12.3 +/- 6.3 microg/mL, but comparable AUC to PZA administered orally while fasting. Both oral and rectal administration of PZA appeared to be acceptable and oral dosing is preferred because of the higher C(max) and lower inter-subject variability. A starting dose of 30 mg/kg is recommended with drug monitoring between 1 and 2 h postdose. Higher doses may be required if the achieved C(max) values are below the recommended 20-50 microg/mL range.

Population pharmacokinetics of isoniazid in the treatment of Mycobacterium tuberculosis among Asian and African elephants (Elephas maximus and Loxodonta africana).

We recently described the clinical presentation and treatment of 18 elephants from six herds infected with TB. Treatment protocols and methods varied between herds to include both oral and rectal dosing using multiple drug doses and formulations. In this paper we present information regarding the pharmacokinetics (PK) of isoniazid (INH) in elephants and provide suggestions regarding initial treatment regimens. Forty-one elephants received INH daily by either oral or rectal administration with different formulations. Population PK analysis was performed using Non-linear Mixed Effect Modeling (NONMEM). Results of oral administration indicated that compared with premixed INH solution, the drug exposure was highest with a suspension prepared freshly with INH powder. When INH was concomitantly given as an admixture over food, Tmax was delayed and variability in drug absorption was significantly increased. Compared with oral administration, similar drug exposures were found when INH was dosed rectally. The data generated suggest that a starting dose of 7.5 mg/kg of INH is appropriate for initial TB treatment in elephants when premixed solution is administered directly into the oropharynx or rectal vault and 4 mg/kg are when INH is administered following immediate suspension from powdered form.

Mycobacterium tuberculosis in zoo and wildlife species.

Tuberculosis caused by Mycobacterium tuberculosis and M. tuberculosis-like organisms has been identified in a wide range of species, including non-human primates, elephants and other exotic ungulates, carnivores, marine mammals and psittacine birds. Disease associated with M. tuberculosis has occurred mostly within captive settings and does not appear to occur naturally in free-living mammals. Mycobacterium tuberculosis probably originated as an infection of humans, but from the zoonotic standpoint, non-human primates, Asian elephants and psittacine birds have the potential to transmit this disease to humans. However, the overall prevalence of disease in these susceptible species is low and documented transmissions of M. tuberculosis between animals and humans are uncommon. Mycobacterium tuberculosis causes progressive pulmonary disease in mammals and a muco-cutaneous disease in parrots. In all cases, the disease can disseminate and be shed into the environment. Diagnosis in living animals is based on intradermal tuberculin testing in non-human primates, culturing trunk secretions in elephants, and biopsy and culture of external lesions in parrots. Ancillary testing with deoxyribonucleic acid probes and nucleic acid amplification, and enzyme-linked immunosorbent assays have been adapted to some of these species with promising results. Additionally, new guidelines for controlling tuberculosis in elephants in the United States of America, and programmes for tuberculosis prevention in animal handlers have been established.

Epidemiology and diagnosis of Mycobacterium tuberculosis in captive Asian elephants (Elephas maximus).

The deaths of two Asian elephants (Elephas 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.

Evaluation of a multiple-antigen enzyme-linked immunosorbent assay for detection of Mycobacterium tuberculosis infection in captive elephants.

Mycobacterium tuberculosis has become an important agent of disease in the captive elephant population of the United States, although current detection methods appear to be inadequate for effective disease management. This investigation sought to validate a multiple-antigen enzyme-linked immunosorbent assay (ELISA) for screening of M. tuberculosis infection in captive elephants and to document the elephant's serologic response over time using a cross-sectional observational study design. Serum samples were collected from 51 Asian elephants (Elephas maximus) and 26 African elephants (Loxodonta africana) from 16 zoos and circuses throughout the United States. Infection status of each animal was determined by mycobacterial culture of trunk washes. Reactivity of each serum sample against six antigens was determined, and the linear combination of antigens that accurately predicted the infection status of the greatest number of animals was determined by discriminant analysis. The resulting classification functions were used to calculate the percentage of animals that were correctly classified (i.e., specificity and sensitivity). Of the 77 elephants sampled, 47 fit the criteria for inclusion in discriminant analysis. Of these, seven Asian elephants were considered infected; 25 Asian elephants and 15 African elephants were considered noninfected. The remaining elephants had been exposed to one or more infected animals. The specificity and sensitivity of the multiple-antigen ELISA were both 100% (91.9-100% and 54.4-100%, respectively) with 95% confidence intervals. Mycobacterium bovis culture filtrate showed the highest individual antigen specificity (95%; 83.0-100%) and sensitivity (100%; 54.4-100%). Serum samples from 34 elephants were analyzed over time by the response to the culture filtrate antigen; four of these elephants were culture positive and had been used to calculate the discriminant function. Limitations such as sample size, compromised ability to ascertain each animal's true infection status, and absence of known-infected African elephants suggest that much additional research needs to be conducted regarding the use of this ELISA. However, the results indicate that this multiple-antigen ELISA would be a valuable screening test for detecting M. tuberculosis infection in elephant herds.

Endoscopic ovariohysterectomy in two lions (Panthera leo).

Endoscopic techniques were used to ovariohysterectomize two hybrid Asian lions (Panthera leo) in order to reduce the risk of postoperative wound complications associated with standard surgical techniques. One of the lions was aged, overweight, and considered an anesthetic risk. The animals were anesthetized, intubated, catheterized intravenously, and placed in dorsal recumbency with the head lower (Trendelenburg position). Ventilation was assisted mechanically. Following abdominal insufflation, a surgical trocar was placed in the abdominal cavity. Two additional 12-mm surgical trocars were placed under direct visualization using a videoscope. The ovaries and uterus were removed endoscopically, and the abdominal cavity was inspected for hemorrhage under decreased insufflation pressure before closure. The surgery was complicated by obesity, by uterine enlargement from cystic endometrial hyperplasia and endometrial polyps, and by ovarian enlargement and fragility because of bilateral cystic rete ovarii. The procedure and anesthetic recovery were uneventful. Postsurgical recovery time and convalescence lasted less than 3 days, and the animals were reintroduced to an exhibit mate and placed on exhibit within 8 days. The technique is appropriate for use in lions, even those with pathologic reproductive changes, in zoos.

Management protocols for animals in captive propagation and reintroduction programmes.

Species Survival Plans in North America--and comparable programmes in other parts of the world--address the management issues related to maintaining populations of endangered species in captivity. The need has been recognized for universal methods of assessing, evaluating, monitoring and preventing the transmission of disease to naive wildlife by reintroduced species. Standardized protocols are presented for evaluation of the health status of captive animals intended for release.

Epizootic of Mycobacterium bovis in a zoologic park.

An epizootic of Mycobacterium bovis in a zoologic park resulted in the death of 4 southern white rhinoceroses and 2 colobus monkeys. Zoo personnel were detected that had positive intradermal tuberculin skin test results after exposure to mycobacterial-infected animals. On the basis of DNA fingerprinting, all 3 mycobacterial isolates (from 1 rhinoceros and 2 monkeys) were determined to be genetically similar and probably originated from the same source. The 3 animals (1 rhinoceros and 2 colobus monkeys) that had confirmed infections lived in separate, but adjacent, areas. Aerosolization of bacteria during routine cleaning was believed to have contributed to the unusual distance between infected animals. Tuberculosis has reemerged as a major disease problem in human and veterinary medicine.

Isoflurane anesthesia in a rhinoceros.

During surgical exploration of the reproductive tract of a 15-year-old female white rhinoceros, anesthesia was maintained by administration of isoflurane in oxygen. Anesthesia was induced with IM administration of etorphine, and, to facilitate endotracheal intubation, muscle relaxation was achieved by IV administration of additional etorphine, acepromazine maleate, guaifenesin, and thiamylal sodium. Minor complications encountered during the anesthetic monitoring period included hypoventilation and ventilation/perfusion inequality, which contributed to hypoxemia. Recovery was not evaluated because the rhinoceros was euthanatized at the end of the surgical procedure.