INTRODUCTION
Tuberculosis (TB) is a serious chronic infection in humans and animals throughout the world. It affects a large variety of animal hosts including non-human primates, marine mammals like seals and sea lions, psittacine and other birds, domestic, captive and wild animals like cats, rats, cattle, sheep, goats, swine, deer, fox, badgers, moles and elephants [Reference Montali, Mikota and Cheng1–Reference Millan5]. Transmission between human and captive animals has occurred following close and frequent contact [Reference Michalak6].
TB has been recognized as a disease of elephants for over 2000 years [Reference Iyer7, Reference McGaughley8]. However, naturally occurring TB has not been reported in wild elephants, suggesting that captive elephants could most likely have contracted the disease via contact with infected humans [Reference Montali, Mikota and Cheng1]. In captive elephants, the disease is primarily caused by Mycobacterium tuberculosis, although infection with Mycobacterium bovis has been recorded [Reference Payeur9]. Asian elephants (Elephas maximus) are more frequently infected with TB compared to African elephants (Loxodonta africana) [Reference Isaza, Fowler and Miller10]. The difference in prevalence may reflect a closer association of Asian elephants with humans [Reference Montali, Mikota and Cheng1]. Rothschild & Laub [Reference Rothschild and Laub11] identified tuberculous lesions in 52% (59/113) of mastodon (Mammut americanum) skeletons, and implied that pandemic TB may be one of the probable causes of the mastodons’ extinction. This discovery has alerted us to the need to protect living elephants from TB.
Transmission of TB from captive Asian elephants to other animals and humans has been described in several outbreaks [Reference Oh12–Reference Lewerin14]. Clinical signs in infected elephants are usually absent or only shown in the terminal stages of the chronic disease. Transmission of M. tuberculosis occurs by aerosolization of infectious respiratory droplets when the animals cough, trumpet or trunk spray, and is affected by the bacterial load, droplet size, duration of exposure, proximity to infected animals and the immune status of the exposed individual. TB can only be transmitted from elephants with active pulmonary disease following primary infection or after reactivation of latent infection [Reference Michalak6].
The interest in elephant TB has been increasing over the past years due to its public health threat as well as increased concern for the healthcare and conservation of elephants. According to the World Conservation Union (IUCN red list 2006), the Asian elephant is an endangered species. There are probably about 1100–1200 Asian elephants in the wild and slightly more than 60 captive Asian elephants in Peninsular Malaysia. Although there are a number of recent reports of TB in captive Asian elephants from the USA [Reference Payeur9], Sweden [Reference Lewerin14], Thailand [Reference Angkawanish15], Nepal [Reference Mikota and Maslow16] and Sri Lanka [Reference Dangolla and Silva17], very little is known about its prevalence in Peninsular Malaysia. The aim of this study is to determine the prevalence of TB in captive Asian elephants in zoos and conservation centres in Peninsula Malaysia and to assess its potential risk of transmission between elephants and to elephant handlers. TB surveillance data would provide important information to determine the need for a strategy to prevent and control TB in elephants as well as a specific occupational health programme for elephant handlers and caregivers.
METHODS
Animal and human samples
We conducted a cross-sectional study between 10 January and 9 April 2012 to estimate the seroprevalence of M. tuberculosis infection in all 63 captive elephants in six locations (zoos and elephant conservation centres) across Peninsular Malaysia. Only 58 elephants that were approachable and allowed handling, without imposing significant risk to both elephants and handlers were sampled. At the same time, blood samples from elephant handlers (zoo keepers and workers, veterinary staff and mahouts) who gave informed consent for participation in the study were collected to estimate the extent of human exposure to TB. Only those with direct contact with elephants were screened. The Animal Care and Use Committee at the University Putra Malaysia and Department of Wildlife and National Parks Malaysia (DWNP) granted approval for the study on captive Asian elephants while the Medical Ethical Committee from the University Malaya Medical Centre granted approval for the study on elephant handlers.
Blood sampling and testing in elephants
We collected blood from the auricular vein behind the elephant's ear, using a 21-gauge needle butterfly catheter and a 10-ml syringe to draw blood into a plain tube. Blood was allowed to clot at room temperature and samples were shipped in an ice chest to the laboratory within 3–12 hours. Sera were collected by centrifuging the blood tubes at 600 g for 10 min at ambient temperature (25–30°C) and then used in the ElephantTB Stat-Pak assay (ChemBio, USA) which contains a cocktail of recombinant antigens for rapid detection of antibodies to the M. tuberculosis complex in elephants [Reference Lyashchenko18].
Trunk-wash collection in elephants
Trunk-wash samples were collected by the elephant handlers under the supervision of the veterinarians in zoos and conservation centtes, using the ‘triple sample method’ [Reference Isaza and Ketz19]. A series of three trunk-wash samples was collected on separate days within a 1-week period. Each elephant was carefully restrained by its mahouts and 60 ml of sterile normal saline was instilled into a nostril using a 60-ml syringe. The trunk was lifted up and then lowered to collect the fluid in a sterile plastic bag. The wash was later split into two 25-ml aliquots and transferred into a 50-ml sterile Falcon tube™ (BD Biosciences, USA). One set of samples was taken for liquid culture (BACTEC MGIT960 system, BD Biosciences) and polymerase chain reaction (PCR) at the University of Malaya (UM), and the other set for culture on Lowenstein–Jensen (LJ) agar (Oxoid, UK) at the University Putra Malaysia (UPM). A total of 154 trunk-wash samples were collected of which only 151 could be used.
Decontamination of trunk-wash samples
Prior to culture, trunk-wash samples were decontaminated using the modified Petroff method [Reference Cruickshank20]. Briefly, the samples were centrifuged at 13 000 g for 15–25 min at 4°C. The supernatant was discarded and 5 ml was retained in the tube to which 5 ml 2% NaOH was added. The mixture was incubated at 37°C for 15 min. After incubation, 40 ml phosphate buffered saline (1 × PBS) was added and the mixture was centrifuged at 13 000 g for a further 15–25 min. The supernatant was then discarded, 1 ml 1 × PBS was added to resuspend the pellet and the sample was transferred into a labelled sterile bijou bottle.
Culture and microscopic identification
A loopful of each decontaminated sample was inoculated onto a LJ slant and incubated at 37°C for up to 8 weeks. The cultures were examined daily for 7 days, then weekly for 7 weeks, during which time the growth rate and pigmentation of visible colonies were recorded. Ziehl–Neelsen staining was performed to look for acid-fast bacilli under a light microscope.
TB antigen rapid test (TiBilia™ TB, Genesis, China)
The TiBilia test, an immunochromatographic assay that detects the presence of MPB64 antigens exclusively found in the M. tuberculosis complex, was conducted for all acid-fast positive isolates. One loopful of each colony was suspended in 200 μl extraction buffer in a 1·5-ml tube. The mixture was vortexed and 100 μl was dropped into the sample well of the test device at room temperature. Results were read after 15 min and the presence of two red lines at the test and control areas indicated positive detection for M. tuberculosis, while a red line only at the control area was considered negative. Test results showing no red line or a red line at the test area only were considered invalid.
PCR detection of M. tuberculosis in trunk wash
DNA in the trunk wash was extracted and purified using DNA-sorb-B Nucleic Acid Extraction kit (AmpliSens Biotechnologies, Russia), according to the manufacturer's instructions. The purified DNA then served as a template for PCR amplification of the M. tuberculosis complex using the AmpliSens MBT-EPh PCR kit (AmpliSens Biotechnologies).
Identification of non-tuberculous mycobacterium (NTM) species
One loopful of an isolate on LJ medium was suspended in 0·2 ml sterile distilled water. The resulting suspension was boiled at 100°C for 30 min and then centrifuged at 16 100 g for 2 min. The resulting supernatant was used for the subsequent hsp65-based PCR analysis, as described by McNabb et al. [Reference McNabb21]. PCR amplicons were purified by the QIAquick PCR Purification kit (Qiagen, Germany) and sequenced by 1st BASE Laboratories (Malaysia). The resulting DNA sequences were aligned, using BLAST, against hsp65 locus sequences in a web-accessible database (http://msis.mycobacteria.info/). The most probable species of each isolate was identified based on the sequence similarity with reference strains and the expectation value (E value).
Blood sampling and testing of elephant handlers
Venous blood samples from the elephant handlers, caregivers and veterinary staff were collected by medical staff from the Ministry of Health, Malaysia, for testing with the QuantiFERON®-TB Gold In-Tube test (Cellestis Inc., Australia), which detects the release of interferon (IFN)-γ from TB-specific T lymphocytes [Reference Mori22]. Three millilitres of blood was drawn directly into three blood collection tubes, i.e. 1 ml each into a nil control tube with saline, TB antigen tube with a mixture of synthetic peptides representing ESAT-6, CFP-10 and TB7·7 test antigens, and a mitogen control tube containing phytohaemagglutinin. To ensure complete mixing of the blood with the tube contents, the tubes were shaken immediately after blood collection and again just before they were incubated at 37°C, within 16 h of collection. After 16–24 h incubation, the tubes were centrifuged and the supernatant containing IFN-γ released from the antigen-stimulated T lymphocytes was harvested for testing by QuantiFERON-TB Gold ELISA. As recommended by the manufacturer, optical density readings >0·35 IU/ml were interpreted as indicative of latent or active TB infection, depending on clinical presentation.
Data analysis
TB seroprevalence was estimated by the number of seropositives divided by the total number tested, and reported as a percentage (%). Statistical analysis was performed using Fisher's exact test from GraphPad InStat version 3 (GraphPad Inc., USA). A P value <0·05 was considered statistically significant.
RESULTS
Fifty-eight serum samples were obtained from 63 elephants in captivity, of which only 49 could be tested as the rest were haemolysed. Using the rapid Stat-Pak assay, 10/49 (20·4%) elephants tested were seropositive (Table 1). Herd prevalence ranged from 0% to 25·9% in the six study locations. Of 149 staff who had contact with elephants, the overall seroprevalence by QuantiFERON test was 24·8% (range 18·6–50%) (Table 2). Besides Malaysians, there were 19 foreign nationals among the elephant handlers. There was no significant difference in seropositivity (P = 0·2537) between Malaysian (23·18%, 30/130) and foreign (36·8%, 7/19) workers. Neither was there any significant association with duration of employment (P > 0·9999 for duration ∼1 year and P > 0·9999 for duration ∼5 years).
TB Stat-Pak test results for captive Asian elephants by location
QuantiFERON test results of elephant handlers by location
The TB detection results for trunk-wash samples are given in Table 3. Samples were only collected from five locations as the elephants in location F were not approachable. TB PCR was performed on all 10 seropositive elephants and 12 of the seronegatives animals. Of these 22 elephants, three (13·6%) were positive in both tests and therefore considered to have laboratory evidence of active TB. Eight elephants (with negative serology and PCR) were probably not infected. Seven (seropositive but PCR-negative) were considered to have latent infection and the remaining four (seronegative but PCR-positive) could be false-positive PCR or false-negative serology results.
Culture and TB PCR results of elephant trunk washes by location
PCR, Polymerase chain reaction.
* From 55 elephants.
† From 22 elephants tested.
All trunk-wash samples were cultured; none grew M. tuberculosis. Most of the liquid cultures were heavily contaminated by non acid-fast bacteria and fungi, despite prior decontamination and the incorporation of antibiotics (BBL, MGIT PANTA; BD Biosciences) into the MGIT culture medium. However, on LJ slants it was possible to obtain pure subcultures of acid-fast bacteria, but 23/24 positive cultures turned out to be NTM species, identified by a negative TiBilia test followed by hsp65gene amplification and sequence alignment with reference NTM species. M. arupense and M. colombiense made up 50% of the NTM species recovered (Table 4).
Non-tuberculous mycobacteria (NTM) identification by hsp65 sequencing
* Of 24 acid-fast bacilli-positive cultures, 23 were NTM species. The remaining isolate was identified as Nocardia nova.
DISCUSSION
Many techniques have been used for the detection of TB in elephants but few have been reported to be entirely satisfactory when used alone. A combination of diagnostic assays is often required [Reference Angkawanish15]. The Chembio TB Stat-Pak assay used in this study is licensed by the U.S. Department of Agriculture as a screening test for TB in elephants. The sensitivity and specificity of this test for the detection of anti-M. tuberculosis complex antibodies have been reported to be 100% and 95%, respectively [Reference Greewald23]. However, other workers have noted an inadequacy of the Stat-Pak assay for the unequivocal identification of TB-infected animals [Reference Payeur9, Reference Angkawanish15]. Our results also showed poor correlation between serology and trunk-wash culture and PCR. In humans, serological results are affected by the phase of TB infection and the immune competence of the host. A positive serological result in the absence of clinical features and M. tuberculosis detection is usually interpreted as indicative of latent infection, while negative serology in the presence of positive TB culture or PCR can be explained by immunological anergy. It is not known whether the same interpretations are applicable in elephants. With trunk-wash tests, sensitivity has been reported to be poor [Reference Lyashchenko18, Reference Greewald23] and affected by collection and processing methods as well as the degree of contamination in the samples collected. The PCR assay we used is designed for a wide range of human specimen types but has not been adequately evaluated for elephant respiratory samples. Hence, although the combined use of both the Stat-Pak assay and TB PCR did provide some evidence for active and latent TB infection in our captive elephants, continued monitoring of the health of elephants, particularly those seropositive, and repeated examination of trunk washes are required to confirm TB infection in elephants. Nevertheless, there is sufficient indication that there exists a sizable reservoir of silent infection in the elephants that would maintain continued transmission if not controlled.
The QuantiFERON test has been well established for the diagnosis of latent TB in humans and is often used as a supplementary test to aid the diagnosis of active TB. None of the 37 staff who tested positive in this study had TB-like symptoms. All 13 seropositive individuals from location A were examined by a chest physician and found to have no physical or radiological signs. Only one gave a history of recent contact with a known case of TB. The 24·8% seroprevalence is probably entirely due to latent infection but it is substantially higher than the seroprevalence previously obtained (authors’ unpublished data) for asymptomatic university lecturers and students (4%), general laboratory staff (6%) and staff working in a TB diagnostic laboratory (12·5%). The overall 20·4% seroprevalence in our elephants is comparable to reports of 20% from Nepal [Reference Mikota and Maslow16] and 12–25% from India (25% of elephants in temples vs. 12–15% in other elephant groups) [Reference Mikota and Maslow16]. This finding is rather unexpected as the human TB incidence in Malaysia (3-year average of 83/100 000 population from 2008 to 2010) is substantially lower than incidences in Nepal (163/100 000 population) and India (190/100 000 population) [24]. Hence, the high seroprevalence in elephants and their handlers in this study could be the result of frequent, close contact, two-way transmission between animals and humans within confined workplaces.
The isolation of NTM from trunk wash illustrates the ubiquitous presence of these environmental bacteria. Many of the species recovered are potential animal and human pathogens. M. avium has been reported to be the most frequently isolated mycobacterial species from trunk washes in the USA [Reference Payeur9]. Similarly, in this study, among the most frequently isolated NTM were two members of the M. avium complex, M. colombiense (a new sequevar of M. avium [Reference Murcia25]) and M. intracellulare. M. arupense, the other common isolate, is associated with human respiratory infections and has been isolated from various domestic and wild animals [Reference Neonakis26]. The role of these NTM species as elephant pathogens requires further investigation.
There is a paucity of information of TB in elephants particularly in Asia. To the best of our knowledge, this study is the first to look at the extent of TB infection in captive Asian elephants in Peninsular Malaysia. Our findings could contribute to the development of a long-term surveillance and healthcare programme for this endangered species. From the conservation point of view, the implication of potential transmission and spread to wild elephant populations cannot be underestimated, e.g. if TB-infected elephants are used during rescue and release operations [Reference Mikota27]. Just as important is the prevention of elephant–human transmission in zoos and conservation centres. Elephant handlers need to be aware of the risk of TB acquisition from infected animals and be educated with regard to infection control measures. On the other hand, as elephants become infected by humans with open TB, there must be workplace policies (e.g. pre-employment screening and annual chest X-rays) to ensure that elephant handlers are free from active TB. Hence, a One Health approach [Reference Mikota and Maslow16] involving both animal and human health sectors should be undertaken to develop a comprehensive prevention, treatment and prophylaxis control strategy to protect the elephants and their caregivers from TB.
ACKNOWLEDGEMENTS
We thank all the staff in zoos and conservation centres for their assistance in the field in the collection of elephant blood samples and trunk washes. We also thank the medical staff at the Ministry of Health Malaysia, as well as Mr J. B. Chook and Ms, Vishala Sivapalan from the University of Malaya for their assistance in the collection of blood samples from the elephant handlers, and Ms. Krishnammah Kuppusamy from the University Putra Malaysia and Ms. Jennifer Chong from the University Malaya for technical assistance in the laboratory. This study was funded by University Putra Malaysia Research Grant (UPM/700-1/2/RUGS/05-02-12-1833RU); and University of Malaya HIR grant (UM.C/625/1/HIR/004).
DECLARATION OF INTEREST
None.
