Journal of Threatened Taxa |
www.threatenedtaxa.org | 26 June 2021 | 13(7): 18713–18718
ISSN 0974-7907 (Online) | ISSN 0974-7893
(Print)
https://doi.org/10.11609/jott.5502.13.7.18713-18718
#5502 | Received 28 October 2019 | Final
received 10 April 2021 | Finally accepted 27 April 2021
Sero-diagnosis of
tuberculosis in elephants in Maharashtra, India
Utkarsh Rajhans
1 , Gayatri Wankhede 2,
Balaji Ambore 3, Sandeep
Chaudhari 4, Navnath Nighot 5,
Vitthal Dhaygude
6 & Chhaya
Sonekar 7
1,2,3 Department of Veterinary Clinical
Medicine Ethics and Jurisprudence, 6 Department of Veterinary
Pathology,
Krantisinh Nana Patil
College of Veterinary Science (KNPCVS), Shirwal, Satara District, Maharashtra 412801, India.
4,7 Department of Veterinary Public
Health and Epidemiology, Nagpur Veterinary College, Maharashtra Animal and
Fisheries Sciences UniveristY, Maharashtra 440006,
India.
5 Rajiv Gandhi Zoological Park and
Wildlife Research Centre, Katraj, Pune, Maharashtra
411046, India.
1 utkarshrajhans01@gmail.com
(corresponding author), 2 gayatriwd87@gmail.com, 3 balajiavet@gmail.com,
4 vphsandeep@gmail.com,
5 drnknighot@rediffmail.com, 6
drvitthalp@gmail.com, 7 chhayasonekar178@gmail.com
Editor: Bahar S. Baviskar,
Wild-CER, Nagpur, India. Date of publication:
26 June 2021 (online & print)
Citation: Rajhans,
U., G. Wankhede, B. Ambore, S. Chaudhari, N. Nighot, V. Dhaygude & C. Sonekar (2021). Sero-diagnosis of tuberculosis in elephants in
Maharashtra, India. Journal of Threatened Taxa 13(7): 18713–18718. https://doi.org/10.11609/jott.5502.13.7.18713-18718
Copyright: © Rajhans
et al. 2021. Creative Commons Attribution
4.0 International License. JoTT allows unrestricted use, reproduction, and
distribution of this article in any medium by providing adequate credit to the
author(s) and the source of publication.
Funding: (1) Department
of Veterinary Public Health and
Epidemiology, Nagpur Veterinary
College, Maharashtra Animal
and Fisheries Sciences Univeristy, India; (2) Department
of Veterinary Medicine, Ethic and Jurisprudence,
Department of Veterinary Pathology, Krantisinh Nana Patil College of Veterinary Science, Shirwal, Satara District, Maharashtra, Inida [Project Number:
10(14)/2014-Ep & Hs].
Competing interests: The authors
declare no competing interests.
Author details: Utkarsh Rajhans is currently working as Veterinarian and is
Managing director at Fauna Healthcare Veterinary Clinic, Pune, Maharashtra. He
has also worked as Veterinarian and CT scan consultant at Cessna Lifeline
Veterinary Hospital, Bangalore, Karnataka for 2 years. Gayatri
Wankhede is currently
working as Assistant Professor. Author is young veterinarian & academician
having experience of 8yrs of teaching, research & extension activities. She
has published research more than 10 articles in reputed national journals. Balaji Ambore is currently working as Assistant Professor
& Head. Author has total 24 years of experience in teaching, research,
extension and Clinician in the field of Veterinary Science. He has published
more than 30 research articles in national and international journals of
repute. Dr.
Sandeep Chaudhari is currently working as Professor & Head, having
experience of 21 years in teaching, research and extension activities has
published more than 80 research articles in reputed international and national
journals. Dr. Navnath Keshav Nighot,
MVSc (Vet. Medicine), Formarly
Deupty Director, Rajiv Gandhi Zoological Park and
Wildlife Research Center, Pune. Having more 16yrs experience of wild animal
medicine and therapeutics. Vitthal Dyaygude is
currently working as Assistant Professor and Head. Author has 13 years of
experience as an academician and He has published more than 30 research papers
and articles in journal of national and international reputes. Dr. Chhyaa P. Sonekar, PhD
scholar in the Department of Veterinary Public Health, Nagpur Veterinary
College, Nagpur. Currently working on tuberculosis in livestock animals, in and
around the Nagpur region along with the zoonotic aspect in humans.
Author
contributions: Dr. Utkarsh Rajhans, designed and
conducted study on Sero-diagnosis of tuberculosis in
elephants in Maharashtra, India. Dr. Gayatri Wankhede and Dr. Balaji Ambore helped in coordinated and guided in the research and
manuscript writeup. Dr. Sandeep Chaudhari, Dr. Vitthal Dhaygude
and Dr. Chhaya Sonekar designed, performed and analyzed
the diagnostic procedures and data. The manuscript was written by Dr. Utkarsh Rajhans and commented
by all authors.
Acknowledgements: This research work was possible due to the
permission granted by central zoo authority (CZA), New Delhi and principal
chief conservator of forests (PCCF), Maharashtra. Dr. G.D. Wankhede, assistant
professor and Dr. B.N. Ambore, assistant professor and
sectional head, Department of Veterinary Clinical of Medicine, Ethics and
Jurisprudence, Krantisinh Nana Patil
College of Veterinary Science, Shirwal or her
valuable guidance moral support and constant encouragement during the entire
course of studies. Dr. S.P. Chaudhari
Professor and Head, Department of Veterinary Public Health and Epidemiology,
Nagpur Veterinary College, Nagpur for his expertise to formulate the research
methodology in particular and for providing chance to work at his departmental
laboratory facility. I wish to record
special thanks to Dr. N.K. Nighot, Deputy Director,
Rajiv Gandhi Zoological Park and Wildlife Research Centre, Pune whose
proficiency was invaluable in formulating the research topic and his timely
guidance. This work would not have been possible without the support of my
committee member Dr. V.S. Dhaygude.
Abstract: Tuberculosis is a highly
contagious zoonotic disease caused by Mycobacterium spp. A study was conducted to detect the presence
of Mycobacterium in captive elephants.
A total of 15 captive elephants were screened from various regions in
Maharashtra. The blood and serum samples
collected were subjected to rapid test kit, BacT/ALERT
3D system, Ziehl-Neelsen (ZN) staining and PCR. All the samples were found seronegative using
rapid test kit and whole blood PCR.
Whereas, all samples were signalled culture positive in BacT/ALERT 3D system which were further subjected to PCR,
only one amplicon was produced of 176bp of RD4 gene (Mycobacterium bovis) and no acid-fast organism was detected upon
ZN. Due to the atypical nature of this
organism, diagnosis of this disease in elephants using various tests is
complicated unlike the diagnostic tests that are validated in domestic
animals. Therefore, many tests have
sub-optimal sensitivity and specificity in elephants. As TB is a zoonotic disease, transmission can
occur between human-livestock-elephants interface. Therefore, the zoos and state forest
authority should inculcate a protocol of periodic TB screening for Mahouts and
elephants in captivity along with protocol of elephant-visitor interaction,
thus helping in conservation of this endangered species in India.
Keywords: Elephants, mycobacterium, serodiagnosis, Tuberculosis.
Introduction
Elephants are the largest
terrestrial mammals on the earth.
Elephants belong to the family Elephantidae in
animal kingdom. Two genera Elephas
and Loxodonta and three species are present
today – the Asian Elephant Elephas maximus, the African Bush Elephant Loxodonta africana,
and the African Forest Elephant Loxodonta cyclotis.
Currently, a population of 27,312
elephants has been estimated from 23 states in India (Project Elephant
Division, Government of India, 2017). In
past decades, the population of elephants has drastically been reduced and
since 1986, the Asian Elephant has been listed as ‘Endangered’ species on
the IUCN Red List, as the wild population has declined by at least
50% (Choudhury et al. 2008). The Asian
Elephant is placed in Schedule I and Part I of Indian Wildlife Protection Act
(1972); conferring it the highest level of protection.
Tuberculosis is a highly
contagious zoonotic disease in animals as well as humans. It is caused by highly pathogenic bacteria of
Mycobacterium tuberculosis complex (MTBC) which are M. tuberculosis,
M. bovis, and M. canetti.
The M. tuberculosis and M. bovis are
most pathogenic. Tuberculosis (TB) in
elephants was first observed more than 2,000 years ago by ancient Ayurvedic
physicians in Ceylon (Iyer 1937; McGaughey
1961). Transmission between human and
captive animals has occurred following close and frequent contact (Kathleen et
al. 2002). More frequent reporting of
this disease occurs in Asian Elephants than in African Elephants may be due to
closer human contact related to their use for performances, rides and in temple
rituals. Mycobacterium tuberculosis is the predominating disease-causing
agent in elephants, although TB cases have been caused by M. bovis (Mikota 2008). The reservoirs for M. tuberculosis and
M. bovis are infected human and cattle (Hirsch
2004).
Elephants with tuberculosis
infection show clinical signs like weight loss, wasting and weakness, coughing
or dyspnoea have been reported but appear to be uncommon. Exercise intolerance may be observed in
working elephants (Mikota 2008). In some cases, ventral oedema has been
reported, but other pathologic factors could be the initiating cause (Seneviratna et al. 1966). Majority of times elephants infected with TB
do not have any clinical signs. In some
cases, elephants manifest symptoms only in advance stage of disease or may not
be diagnosed until necropsy (Paudel & Tsubota 2016).
The study presents the clinical,
serological, and culture data from 15 elephants present in captivity thus
helping to diagnose and decrease TB risk to these wild animals.
Materials
and Methods
Study animals and sample
collection
Blood and serum samples were collected
from the 15 elephants in captivity of Forest Camp areas of Gadchiroli
(19.4290° N, 80.0563° E), Pune Zoo (18.4520N, 73.8650E),
Mumbai Zoo (18.9780N, 72.8350E), Shegaon
temple (20.7890N, 76.7010E) in Maharashtra. The elephants were included in the study
irrespective of their health status, age, sex or habitat.
Serological testing
The Wild TB alert kit is a
lateral flow chromatographic immunoassay for the detection of antibodies of
mycobacterium tuberculosis complex antigenserum,
plasma and whole blood of elephants.
This kit contents a unique cocktail of tuberculosis specific recombinant
proteins (ESAT-6, CFP-10, MPB83, MPB70) and crude protein impregnated on
nitrocellulose membrane housed in a disposable plastic cassette. After adding sample to the well followed by
addition of diluent they travel through the membrane by capillary action. If antibodies are present, they bind to the
antigen and a red colour band is observed in test
area.
BacT/ALERT 3D system
BacT/ALERT 3D system is an automated
microbial detection system which offers microbiological
culture of blood. This
mycobacteria detection systems utilize a colorimetric sensor and reflected
light to monitor the presence and production of carbon dioxide (CO2)
dissolved in the culture medium. BacT/ALERT MB are disposable culture bottles with a
removable closure contain 10 ml of media and an internal sensor that detects
carbon dioxide as an indicator of microbial growth. The media formulation consists of: Middlebrook
7H9 Broth (0.47% w/v), Pancreatic Digest of Casein (0.1% w/v), Bovine Serum
Albumin (1.0% w/v), Catalyse (48 µ/ml), in purified
water. Bottle reflectance is monitored
and recorded by the instrument every 10 minutes. The growth curve enters lag phase then the
bottle is flagged positive. At the time
of detection, approximate colony forming units (CFUs) are 106–107 per ml.
Ziehl-Neelsen/Acid Fast staining
Bacterial culture smear was
prepared from samples indicated positive in BacT/ALERT
3D system on clean and grease free slide, using standard protocol of Ziehl-Neelsen staining kit (Hi-Media Pvt. Ltd, India).
PCR detection of mycobacterium
DNA was extracted from blood
samples and samples signaled positive in BacT/ALERT
3D system of 15 elephants using the extraction protocol described by Samrook et al. 1989 and Tissue Genomic DNA Extraction Mini
Kit (FAVORGEN Biotech Corp, Taiwan). The
extracted DNA was subjected to PCR by using the standard primer RD4 F
5’-AATGGTTTGGTCATGACGCCTTC-3’; R 5’-CCCGTAGCGTTACTGAGAAATTGC-3’ and RD1 F
5’-CCCTTTCTCGTGTTTATAGTTTGA-3’ R 5’-GCCATATCGTCCGGAGCTT-3’ which was amplified
176 and 110 bp of Mycobacterium tuberculosis
and Mycobacterium bovis. The PCR reaction was carried out at 94°C for
10 minutes followed by 35 cycles of denaturation at 94°C for 1 minute,
annealing at 60°C for 30 seconds and extension at 72°C for 1 minute, with final
extension at 72°C for 10 minutes. The
PCR products were analysed by electrophoresis in 1.5%
agarose gel at100 V for 45 minutes and documented. Amplicon of size 176bp and 110bp is specific
for Mycobacterium genus.
Results
The Table 1 shows the results of
various diagnostic tests used for diagnosis of mycobacterium in elephants. The serum samples collected from the 15
elephants were seronegative by the rapid test kit as no coloured
band was observed in the test area of the rapid test kit (Image 1). All the 15 samples were detected positive by
the BacT/ALERT 3D system in 6 mean days. These samples were further subjected to ZN
staining, no sample detected the presence of acid fast bacilli (Amer et al. 2016; Bapat et al. 2017) (Image 2). Isolates of DNA extracted from the blood
samples of these 15 elephants were subjected to PCR which did not produce
specific amplicon of 176bp and 110bp RD4 and RD1 gene. Similarly, the DNA isolates from the BacT/ALERT culture system did not produce amplicon of 176
and 110 bp but one isolate produced amplicon of 176bp
of RD4 of targeted gene indicating presence of Mycobacterium bovis (BCG) (Bapat et al. 2017) as illustrated in Image
3 and 4.
Discussion
Tuberculosis is a highly
contagious zoonotic disease with high incidence and prevalence in human,
domestic and wild animals of developing countries. Tuberculosis infection in captive elephants
is ongoing and complex problem with respect to their conservation. Due to atypical nature of the mycobacteria
that causes diseases, the diagnosis is rather complicated, apart from the fact
that many diagnostic tests are developed for domestic species however, those
are not validated for wild animals.
Therefore, many tests have sub-optimal specificity and sensitivity.
The major problem to designate a
perfect test among available tests for diagnosis of tuberculosis, which are
most accurate for elephants, giving veterinarians a standardized method, which
will allow them to make preventive measures and treatment protocols; thus,
helping in conservation of endangered species like elephant.
These samples were subjected to
diagnostic tests like BacT/ALERT 3D system, ZN
staining, PCR, Rapid TB test kit. All 15
samples were signalled positive by BacT/ALERT 3D system. This test is not yet used and
validated in animals, like in humans.
This was the first time when the test was used in detection of TB in
wild animals. Therefore, the specificity
still remains a question. On the other hand, other tests like ZN staining,
Rapid TB test kit and blood PCR did not detect any mycobacteria in the samples.
Molecular detection (duplex PCR)
of the samples that signalled positive in BacT/ALERT 3D system was carried out using RD4 and RD1 gene
primer with amplicon size of 176bp and 110bp respectively as described by Bapat
et al. (2017). Only one sample was
positive detecting the presence of M. bovis
(BCG) at 176bp of RD4 gene.
During the study it was not
possible to calculate the specificity of various diagnostic tests used. Development
and use of new and more species specific diagnostic methods are needed at the
moment, as it will help in early and accurate diagnosis that might permit early
application of preventive measures and will ensure safety of endangered species
as well as human staff involved.
Moreover, this mycobacterial disease requires long term surveillance
plans in order to be effective, as this organism has prolonged incubation and
latency.
Although, the reported case of TB
in elephant in present study was caused by M. bovis
(BCG) which is vaccine strain, its species predilection is still
unidentified. Moreover, this animal
should be screened multiple times over the period of time to confirm the
disease. Cultural isolation of
mycobacterium is currently the only gold standard test for TB diagnosis in
elephants, but ancillary tests like PCR, BacT/Alert
3D system, rapid TB test kit etc. may be useful. The molecular method (PCR) used in diagnosis
of mycobacterium in present study is not a confirmatory test due to its
possibility of cross contamination (false positive) and inability to determine
the pathogenicity of the organism. As
this is a zoonotic disease, transmission of TB can occur between humans,
livestock and elephants. Elephants are
at risk of contracting TB from infected human (Mahouts). Therefore, Mahouts (handlers) and elephants
should undergo periodic TB screening to minimize the risk of animals’
health. Zoos and forest elephant camp
areas should be encouraged to incorporate protocol for elephant-visitor
interactions and periodic screening of animals for tuberculosis.
This study highlights the
potential usefulness and efficacy of ante-mortem diagnostic methods. Use of
multiple tests helps to achieve high possibility (sensitivity) of tuberculosis
detection in elephants rather than using single test; however, it is important
to evaluate and validate the test regime and will require addition of more
animals in to the study; expectantly allowing in better understanding of
tuberculosis in elephants, thus contributing to undertake control measures by
state forest department and zoo authorities for conservation of this endangered
species.
Table 1. Overall results of test
applied (n= 15).
Elephant No. |
BacT/ALERT |
ZN Staining |
Blood PCR |
BacT/ALERT + ve PCR |
Rapid test |
(E1) |
Positive |
Negative |
Negative |
Negative |
Negative |
(E2) |
Positive |
Negative |
Negative |
Negative |
Negative |
(E3) |
Positive |
Negative |
Negative |
Negative |
Negative |
(E4) |
Positive |
Negative |
Negative |
Negative |
Negative |
(E5) |
Positive |
Negative |
Negative |
Negative |
Negative |
(E6) |
Positive |
Negative |
Negative |
Negative |
Negative |
(E7) |
Positive |
Negative |
Negative |
Negative |
Negative |
(E8) |
Positive |
Negative |
Negative |
Positive |
Negative |
(E9) |
Positive |
Negative |
Negative |
Negative |
Negative |
(E10) |
Positive |
Negative |
Negative |
Negative |
Negative |
(E11) |
Positive |
Negative |
Negative |
Negative |
Negative |
(E12) |
Positive |
Negative |
Negative |
Negative |
Negative |
(E13) |
Positive |
Negative |
Negative |
Negative |
Negative |
(E14) |
Positive |
Negative |
Negative |
Negative |
Negative |
(E15) |
Positive |
Negative |
Negative |
Negative |
Negative |
References
Bapat, P.R.,
A.S. Satav, S.D. Shekhawat,
S.D. Manke, A.A. Husain, A.R. Nayak, A.P. Kawle, L.R. Singh, H.F. Daginawala
& R.S. Kashyap (2017a). Molecular diagnosis of zoonotic Mycobacterium bovis
infection in Melghat, India. Journal of Zoonotic
Diseases 2(2): 2–16.
Bapat, P.R.,
R.S. Dubey, S.D. Shekhawat, A.A. Husain, A.R. Nayak,
A.P. Kawle, H.F. Daginawala,
L.R. Singh & R.S. Kashyap (2017b). Prevalence of zoonotic
tuberculosis and associated risk factors in Central Indian populations. Journal
of Epidemiology and Global Health 7: 277–283.
Choudhury,
L., D.K. Desai, A. Duckworth, J.W. Easa, P.S. Johnsingh, A.J.T. Fernando, P. Hedges, S. Gunawardena, M.
Kurt, F. Karanth, U. Lister, A. Menon, V. Riddle, H.
Rubel & A.E. Wikaranayake (2008). Elephas maximus. The ICUN Red List of Threatened
Species [online] https://doi.org/10.2305/IUCN.UK.2008.RLTS.T7140A12828813.en
[viewed 6 March 2019].
Hirsch, D.C.,
N.. MacLachian & R.L. Walker (eds.) (2004). Veterinary Microbiology.
2nd edition. Blackwell Publishing Ltd., USA, 223pp.
Iyer, A.K. (1937). Veterinary science in India,
Ancient and modern with special reference to tuberculosis. Agriculture
Livestock India 7: 718–724.
Kathleen,
A.A., E. Pleydell, M.C. Williams, E.P. Lane, J.F.C. Nyange & A.L. Michel (2002). Mycobacterium tuberculosis: an
emerging disease of free-ranging wildlife. Emerging Infectious Diseases
8(6): 598–601.
Mikota, S.K. (ed.) (2008). Zoo and Wild Animal Medicine
- 6th Edition. Elsevier Saunders Inc., USA, 355pp.
Mc Gaughey C.A. (1961). Diseases of elephants - Part 3. Ceylon
Veterinary Journal 9: 94–98.
Paudel, S. & T. Tsubota
(2016). Tuberculosis
in elephants: a zoonotic disease at the human-elephant interface. Japanese
Journal of Zoo and Wildlife Medicine 21(3): 65–69.
Project
Elephant Division Goverment
of India (2017). [online] http://www.moef.nic.in/division/project-elephant/
Seneviratna, P., S.G. Wttimuny
& D. Senevirtna (1966).Fatal tuberculosis pneumonia in
an elephant. Veterinary Medicine, Small Animal Clinician 60: 129–132.
Samrook J., E.F. Fritsch & T. Maniatis (1989). Molecular cloning: alaboratory manual. Cold Spring Harbor Laboratory
Press, New York. Brazilian Journal of Biology 62(3): 387–408.