Tuberculosis in captive Asian elephants (Elephas maximus) in Peninsular Malaysia

Abstract

SUMMARY A cross-sectional study was conducted from 10 January to 9 April 2012, to determine the seroprevalence of tuberculosis (TB) of all captive Asian elephants and their handlers in six locations in Peninsular Malaysia. In addition, trunk-wash samples were examined for tubercle bacillus by culture and polymerase chain reaction (PCR). For 63 elephants and 149 elephant handlers, TB seroprevalence was estimated at 20·4% and 24·8%, respectively. From 151 trunk-wash samples, 24 acid-fast isolates were obtained, 23 of which were identified by hsp65-based sequencing as non-tuberculous mycobacteria. The Mycobacterium tuberculosis-specific PCR was positive in the trunk-wash samples from three elephants which were also seropositive. Conversely, the trunk wash from seven seropositive elephants were PCR negative. Hence, there was evidence of active and latent TB in the elephants and the high seroprevalence in the elephants and their handlers suggests frequent, close contact, two-way transmission between animals and humans within confined workplaces.

Full text

Tuberculosis in captive Asian elephants (Elephas maximus)in
Peninsular Malaysia
B. L. ONG
1
*, Y. F. NGEOW
2
*, M. F. A. ABDUL RAZAK
3
,Y.YAKUBU
1
,
Z. ZAKARIA
1
,A.R.MUTALIB
1
,L.HASSAN
1
,H.F.NG
2
AND K. VERASAHIB
4
1
Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor, Malaysia
2
Faculty of Medicine, Universiti Malaya, Kuala Lumpur, Malaysia
3
Department of Wildlife and National Parks, Peninsular Malaysia, Kuala Lumpur, Malaysia
4
Ministry of Health, Putrajaya, Malaysia
Received 29 October 2012; Final revision 8 January 2013; Accepted 22 January 2013
SUMMARY
A cross-sectional study was conducted from 10 January to 9 April 2012, to determine the
seroprevalence of tuberculosis (TB) of all captive Asian elephants and their handlers in six
locations in Peninsular Malaysia. In addition, trunk-wash samples were examined for tubercle
bacillus by culture and polymerase chain reaction (PCR). For 63 elephants and 149 elephant
handlers, TB seroprevalence was estimated at 20·4% and 24·8%, respectively. From 151 trunk-
wash samples, 24 acid-fast isolates were obtained, 23 of which were identified by hsp65-based
sequencing as non-tuberculous mycobacteria. The Mycobacterium tuberculosis-specific PCR was
positive in the trunk-wash samples from three elephants which were also seropositive. Conversely,
the trunk wash from seven seropositive elephants were PCR negative. Hence, there was evidence
of active and latent TB in the elephants and the high seroprevalence in the elephants and their
handlers suggests frequent, close contact, two-way transmission between animals and humans
within confined workplaces.
Key words: Captive Asian elephants, elephant handlers, Mycobacterium tuberculosis, non-tuberculous
mycobacteria (NTM), Peninsular Malaysia.
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 [1–5].
Transmission between human and captive animals
has occurred following close and frequent contact [6].
TB has been recognized as a disease of elephants for
over 2000 years [7,8]. However, naturally occurring
TB has not been reported in wild elephants, suggesting
that captive elephants could most likely have con-
tracted the disease via contact with infected humans
[1]. In captive elephants, the disease is primarily
caused by Mycobacterium tuberculosis, although
infection with Mycobacterium bovis has been recorded
[9]. Asian elephants (Elephas maximus) are more fre-
quently infected with TB compared to African ele-
phants (Loxodonta africana)[10]. The difference in
prevalence may reflect a closer association of Asian
* Author for correspondence: Dr B. L. Ong, Department of
Veterinary Clinical Study, Faculty of Veterinary Medicine,
Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia.
(Email: ong_beelee@putra.upm.edu.my) [B. L. Ong]
(Email: yunngeow@yahoo.com) [Y. F. Ngeow]
Epidemiol. Infect., Page 1 of 7. © Cambridge University Press 2013
doi:10.1017/S0950268813000265

elephants with humans [1]. Rothschild & Laub [11]
identified tuberculous lesions in 52% (59/113) of mas-
todon (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 [12–14]. Clinical signs in infected ele-
phants are usually absent or only shown in the term-
inal 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 infec-
ted 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 [6].
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 [9], Sweden [14], Thailand [15], Nepal
[16] and Sri Lanka [17], very little is known about
its prevalence in Peninsular Malaysia. The aim of
this study is to determine the prevalence of TB in cap-
tive 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 impor-
tant 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 seropreva-
lence 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 mah-
outs) 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 temp-
erature 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 gfor 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 [18].
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’[19]. 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
2 B. L. Ong and others

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 decontami-
nated using the modified Petroff method [20]. Briefly,
the samples were centr ifuged at 13 000 gfor 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 cen-
trifuged at 13 000 gfor 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 inocu-
lated 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 ex-
clusively found in the M. tuberculosis complex, was
conducted for all acid-fast positive isolates. One loop-
ful 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 con-
sidered 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 sus-
pended in 0·2 ml sterile distilled water. The resulting
suspension was boiled at 100 °C for 30 min and then
centrifuged at 16 100 gfor 2 min. The resulting super-
natant was used for the subsequent hsp65-based PCR
analysis, as described by McNabb et al.[21].
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 se-
quence similarity with reference strains and the expec-
tation value (E value).
Blood sampling and testing of elephant handlers
Venous blood samples from the elephant handlers,
caregivers and veterinary staff were collected by medi-
cal 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)-γfrom TB-specific T lym-
phocytes [22]. 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 mito-
gen 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 incu-
bated at 37 °C, within 16 h of collection. After 16–24 h
incubation, the tubes were centrifuged and the super-
natant containing IFN-γreleased from the anti-
gen-stimulated T lymphocytes was harvested for
testing by QuantiFERON-TB Gold ELISA. As rec-
ommended by the manufacturer, optical density read-
ings >0·35 IU/ml were interpreted as indicative of
latent or active TB infection, depending on clinical
presentation.
TB in captive Asian elephants in Malaysia 3

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 Pvalue
<0·05 was considered statistically significant.
RESULTS
Fifty-eight serum samples were obtained from 63 ele-
phants 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 con-
tact 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 signifi-
cant association with duration of employment (P>
0·9999 for duration ∼1 year and P> 0·9999 for dur-
ation ∼5 years).
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 seronega-
tives 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.
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 amplifi-
cation and sequence alignment with reference NTM
species. M. arupense and M. colombiense made up
50% of the NTM species recovered (Table 4).
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 [15]. The
Chembio TB Stat-Pak assay used in this study is
Table 1. TB Stat-Pak test results for captive Asian elephants by location
Location
No. of
elephants
No. of sera
collected
No. of sera
tested
No. of Elephant TB
Stat-Pak positives
Seroprevalence
(%)
A 27 27 27 7 25·9
B 8 8 8 2 25·0
C 8 6 5 0 0·0
D 9 9 6 1 16·7
E 3 3 2 0 0·0
F 8 3 1 0 0·0
Total 63 58 49 10 20·4
Table 2. QuantiFERON test results of elephant
handlers by location
Location
No. of human
samples tested
No. of
QuantiFERON
test positives
Prevalence
(%)
A 70 13 18·6
B 21 5 23·8
C 20 5 25·0
D 12 6 50·0
E 18 6 33·3
F 8 2 25·0
Total 149 37 24·8
4 B. L. Ong and others

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 re-
ported to be 100% and 95%, respectively [23].
However, other workers have noted an inadequacy
of the Stat-Pak assay for the unequivocal identifi-
cation of TB-infected animals [9,15]. 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 sero-
logical 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 applic-
able in elephants. With trunk-wash tests, sensitivity
has been reported to be poor [18,23] and af-
fected by collection and processing methods as well
as the degree of contamination in the samples col-
lected. 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 evi-
dence for active and latent TB infection in our cap-
tive 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 sero-
positive 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% sero-
prevalence 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 [16] and 12–25% from
India (25% of elephants in temples vs. 12–15% in
Table 3. Culture and TB PCR results of elephant trunk washes by location
Location
No. of
elephants
No. of trunk
washes collected
No. of acid-fast
positive cultures
No. of elephants
TB PCR positive
A27 78 13 0
B824 4 2
C818 2 0
D927 4 3
E3 4 1 2
F8 0 0 0
Total 63 151 24* 7†
PCR, Polymerase chain reaction.
* From 55 elephants.
†From 22 elephants tested.
Table 4. Non-tuberculous mycobacteria (NTM)
identification by hsp65 sequencing
Most probable species No. of isolates
M. arupense 7
M. colombiense 5
M. intracellulare 2
M. asiaticum 2
M. mantenii 2
M. fortuitum 2
M. gilvum 1
M. hiberniae 1
M. kumamotonense 1
Total 23*
* Of 24 acid-fast bacilli-positive cultures, 23 were NTM
species. The remaining isolate was identified as Nocardia
nova.
TB in captive Asian elephants in Malaysia 5

other elephant groups) [16]. 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 popul ation) [24]. Hence, the high seropreva-
lence 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 bac-
teria. Many of the species recovered are potential ani-
mal and human pathogens. M. avium has been
reported to be the most frequently isolated mycobac-
terial species from trunk washes in the USA [9].
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 [25]) and M. intracellulare. M. arupense,
the other common isolate, is associated with human
respiratory infections and has been isolated from var-
ious domestic and wild animals [26]. 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 infec-
tion in captive Asian elephants in Peninsular
Malaysia. Our findings could contribute to the devel-
opment of a long-term surveillance and healthcare
programme for this endangered species. From the
conservation point of view, the implication of poten-
tial transmission and spread to wild elephant popu-
lations cannot be underestimated, e.g. if TB-infected
elephants are used during rescue and release oper-
ations [27]. Just as important is the prevention of ele-
phant–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 pol-
icies (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
[16] involving both animal and human health sectors
should be undertaken to develop a comprehensive
prevention, treatment and prophylaxis control strat-
egy 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 ele-
phant 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.
REFERENCES
1. Montali RJ, Mikota SK, Cheng LI. Mycobacterium
tuberculosis in zoo and wildlife species. Revue Scien-
tific et Technique de l’Office International des Epizooties
2001; 20: 291–303.
2. Cousins DV, et al.Tuberculosis in seals caused by a
novel member of the Mycobacterium tuberculosis com-
plex: Mycobacterium pinnipedii sp. nov. International
Journal of Systematic and Evolutionary Microbiology
2003; 53: 1305–1314.
3. Washko RM, et al.Mycobacterium tuberculosis infec-
tion in a green-winged macaw (Ara chloroptera):
Report with public health implications. Journal of
Clinical Microbiology 1998; 36: 1101–1102.
4. Kremer K, et al.Mycobacterium microti: more wide-
spread than previously thought. Journal of Clinical
Microbiology 1998; 36: 2793.
5. Millan J, et al.Disseminated bovine tuberculosis in a
wild red fox (Vulpes vulpes) in Southern Spain.
Journal of Wildlife Diseases 2008; 44: 701–706.
6. Michalak K, et al.Mycobacterium tuberculosis infection
as a zoonotic disease: transmission between humans
and elephants. Emerging Infectious Disease 1998; 4:
283–287.
7. Iyer AK. Veterinary science in India, ancient and mod-
ern with special reference to tuberculosis. Agriculture
Livestock India 1937; 7: 718–724.
8. McGaughley CA. Diseases of elephants: part III. Ceylon
Veterinary Journal 1961; 9:94–98.
9. Payeur JB, et al.Mycobacterial isolation in captive ele-
phants in the United States. Annals of the New York
Academy of Sciences 2002; 969: 256–258.
6 B. L. Ong and others

10. Isaza R. Tuberculosis in all taxa. In: Fowler ME, Miller
RE eds. Zoo and Wild Animal Medicine. Pennsylvania:
Saunders, 2003, pp. 689–696.
11. Rothschild BM, Laub R. Hyperdisease in the
late Pleistocene: validation of an early 20th century
hypothesis. Naturewurwissenschaften 2006; 93: 557–
564.
12. Oh P, et al.Human exposure following Mycobacterium
tuberculosis infection of multiple animal species in a
Metropolitan Zoo. Emerging Infectious Disease 2002;
8: 1290–1293.
13. Murphree R, et al.Elephant-to-human transmission of
tuberculosis. Emerging Infectious Disease 2011; 17:
366–370.
14. Lewerin SS, et al.Outbreak of Mycobacterium tubercu-
losis infection among captive Asian elephants in a
Swedish zoo. Veterinary Record 2005; 156: 171–175.
15. Angkawanish T, et al.Mycobacterium tuberculosis infec-
tion of domesticated Asian elephants, Thailand. Emerg-
ing Infectious Disease 2010; 16: 1290–1293.
16. Mikota S, Maslow JN. Tuberculosis at the human-
animal interface: an emerging disease of elephants.
Tuberculosis 2011; 91: 208–211.
17. Dangolla A, Silva I. The status and veterinary problems
in captive elephants in Sri Lanka. In Proceedings of the
54th Annual Convention of the Sri Lanka Veterinary
Association. Gannoruwa, Sri Lanka: Plant Genetic
Resource Centre, 3–4 May 2002, pp. 19 (http://www.
aserc.org/index.php?option=com_content&task=view&
id=37). Accessed 7 January 2013.
18. Lyashchenko KP, et al.Tuberculosis in elephants: anti-
body responses to defined antigens of Mycobacterium
tuberculosis, potential for early diagnosis, and monitor-
ing of treatment. Clinical and Vaccine Immunology
2006; 13: 722–732.
19. Isaza R, Ketz CJ. A trunk wash technique for the diag-
nosis of tuberculosis in elephants. Verhandlungsbericht
des Internationalen Symposiums uber die Erkrankungen
der Zootiere Vienna 1999; 39: 121–124.
20. Cruickshank R, et al.Medical Microbiology, 12th edn.
Edinburgh: Churchill Livingstone, 1975, pp. 390.
21. McNabb A, et al.Assessment of partial sequencing of
the 65-kilodalton heat shock protein gene (hsp65) for
routine identification of Mycobacterium species isolated
from clinical sources. Journal of Clinical Microbiology
2004; 42: 3000–3011.
22. Mori T, et al.Specific detection of tuberculosis infection
with an interferon-gamma based assay using new anti-
gens. American Journal of Respiratory and Critical
Care Medicine 2004; 170:59–64.
23. Greewald R, et al.Highly accurate antibody assays for
early and rapid detection of tuberculosis in African
and Asian elephants. Clinical and Vaccine Immunology
2009; 16: 605–612.
24. World Health Organization. Global tuberculosis con-
trol report 2011 (http://whqlibdoc.who.int/publications/
2011/9789241564380_eng.pdf), pp. 216–231 Accessed 7
January 2013.
25. Murcia MI, et al.Mycobacterium colombiense sp.nov.,
a novel member of the Mycobacterium avium complex
and description of MAC-X as a new ITS genetic vari-
ant. International Journal of Systematic and Evolu-
tionary Microbiology 2006; 56: 2049–2054.
26. Neonakis IK, et al.Mycobacterium arupense pulmonary
infection: antibiotic resistance and restriction fragment
length polymorphism analysis. Indian Journal of Medi-
cal Microbiology 2010; 28: 173–176.
27. Mikota SK. Review of tuberculosis in captive elephants
and implications for wild population. Gajah 2008; 28:
8–18.
TB in captive Asian elephants in Malaysia 7