Journal of Threatened Taxa |
www.threatenedtaxa.org | 26 February 2020 | 12(3): 15359–15363
ISSN 0974-7907 (Online) | ISSN 0974-7893
(Print)
doi: https://doi.org/10.11609/jott.4961.12.3.15359-15363
#4961 | Received 24 March 2019 | Final
received 31 December 2019 | Finally accepted 26 January 2020
Molecular detection of Murshidia linstowi in
a free-ranging dead elephant calf
Sourabh Ranjan Hota 1, Sonali Sahoo
2, Manojita Dash 3, Avisek Pahari 4, Bijayendranath
Mohanty 5 & Niranjana
Sahoo 6
1,3,4,6 Centre for Wildlife Health,
College of Veterinary Science and Animal Husbandry, Bhubaneswar, Odisha 751003,
India.
2 Department of Veterinary
Epidemiology and Preventive Medicine, College of Veterinary Science and Animal
Husbandry, Bhubaneswar, Odisha 751003, India.
5 Department of Veterinary
Parasitology, College of Veterinary Science and Animal Husbandry, Bhubaneswar,
Odisha 751003, India.
1 sourabhranjanhota@gmail.com, 2
sahoosonali75@gmail.com, 3 manojita.dash@gmail.com, 4 avishekpahari@gmail.com,
5 bijayendranath@gmail.com, 6 niranjanasahoo@hotmail.com
(corresponding author)
Abstract: Gastrointestinal helminths are
ubiquitous in both domestic and wild animals. Infections are often sub-clinical
except in circumstances of destabilization of host-parasite equilibrium by
innate or environmental factors. The present case deals with microscopic and
molecular diagnosis of Murshidia linstowi recovered from an elephant. A post-mortem
examination of a free-ranging juvenile male elephant calf that had died of
electrocution in Athagarh Wildlife Division revealed
the presence of slender, whitish nematodes in the stomach. No gross lesions
were noticed either in the site of predilection or any other internal organs.
The average length of the parasites was 3.8cm.
These parasites were collected for further gross as well as microscopic
examination following routine parasitological techniques. Temporary mounts
prepared after cleaning the nematodes in lactophenol were observed under a
microscope. Morphological features such as a well-developed mouth collar, large
and globular buccal capsule with fine tubercles, cone shaped oesophageal
funnel, short bursa having indistinctly divided lobes and closely apposed
ventral rays and stout spicules with club shaped tips bent dorsally
corroborated with that of M.linstowi (male).
Amplification of the rDNA from the internal transcribed spacer (ITS) region
using universal nematode primers NC2 and NC5 revealed a product size of 870bp.
The PCR product was subjected to sequencing followed by NCBI-BLAST which
revealed 98% homology with M. linstowi. A
phylogenetic study showed a maximum similarity with M.linstowi
recovered from elephants in Kenya. This particular nematode species belonging
to the family Strongylidae and sub-family Cyathostominae appears to be the first documented report in
India.
Keywords: Gastrointestinal helminths,
infection, nematode.
Editor: Mandar Paingankar, Government
Science College Gadchiroli, Maharashtra, India. Date of publication: 26 February 2020
(online & print)
Citation: Hota,
S.R., S. Sahoo, M. Dash, A. Pahari, B. Mohanty & N. Sahoo (2020). Molecular detection of Murshidia linstowi
in a free-ranging dead elephant calf. Journal of Threatened Taxa 12(3): 15359–15363. https://doi.org/10.11609/jott.4961.12.3.15359-15363
Copyright: © Hota
et al. 2020. 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: None.
Competing interests: The authors
declare no competing interests.
Acknowledgements: Authors are thankful to the
Divisional Forest Officer, Athagarh Forest Division,
Forest and Environment Department, Government of Odisha for providing all
necessary support towards this field investigation.
Helminths are ubiquitous across
vertebrate taxa. They pose a threat to the welfare, management and conservation
of captive as well as free-ranging elephants. Strongyles
of the genus Murshidia reside in the alimentary canal
of Indian and African elephants. Murshidia spp. affecting elephants include M. linstowi (Heinrich 2016; McLean et al. 2012), M. murshida (Ajitkumar et al. 2009;
Chandra et al. 2018; Edwards et al. 1978; Muraleedharan
2016), M. falcifera (Ajitkumar
et al. 2009; Chandra et al. 2018; Edwards et al. 1978; Matsuo and Supramah 1997),M. longicaudata
(Heinrich 2016; McLean et al. 2012), M. indica
(Ajitkumar et al. 2009; Muraleedharan
2016) and M. Africana (McLean et al. 2012). Murshidiasis in
elephants has been reported from across the globe like Sri Lanka, Nigeria,
Kenya, Burma, Indonesia and India. The present case report deals with molecular
identification of Murshidia linstowi recovered from a free-ranging elephant calf
that died of electrocution.
History
A free-ranging juvenile, male
elephant calf about 4½ years that had died of electrocution in Athagarh Wildlife Division (20.320N & 85.410E)
was referred for investigation. The body condition of the elephant was almost
normal. During post-mortem examination, two types of helminths (trematode and
nematode) were recovered from its stomach. No gross lesions were noticed either
in the site of predilection of the parasites or any other internal organs.
Materials and Methods
These parasites thus collected
(Image1) were subjected to gross and microscopic examination following routine
parasitological techniques and identified based on their features (Singh 2003).
Molecular diagnosis
The internal transcribed spacer
(ITS) region within the rDNA provides a reliable marker to differentiate
between several strongyles. Genomic DNA of the
parasite was extracted using commercially available DNA mini kit (QIAGEN,
Germany). According to the manufacturer’s instructions, 25mg of the parasite
was taken for the said purpose. The universal nematode primers NC2 (5’-
TTAGTTTCTTTTCCTCCGCT-3’) and NC5 (5’- GTAGGTGAACCTGCGGAAGGATCATT-3’) were used
for amplification (McLean et al. 2012). PCR was carried out in a 24µl reaction
mixture containing2µl (640ng/µl)of genomic DNA, 2.4µl 10X PCR buffer, 2.4µl of
25mM MgCl2, 0.16µl DNA polymerase, 1.2µl of each primer(10mM) and
2.4µl of dNTP mixture (2pmol). Amplification was preceded by a 10 minute
polymerase activation step at 95ºC followed by 40 cycles of 45 sec each at
95ºC, 55ºC and 72ºC. A 5-min extension step at 72ºC concluded the reaction. The
amplification products were subjected to electrophoresis on 1.5% agarose gel.
The parasite sample was run in duplicates along with nuclease free water as
negative control. The purified PCR products were subjected to sequencing for
further identification. The similarity of the sequence with Genbank
database submissions was carried out by using BLAST (http://blast.ncbi.nlm.nih.gov)
(Altschul et al. 1990). The sequence was submitted to
Genbank for generation of accession number.
Additional 21 gene sequences were retrieved from the NCBI GenBank database (http://www.ncbi.nlm.nih.gov/).
All the sequences were aligned and compared using ClustalW
(http://www.ebi.ac.uk), with gaps and missing data eliminated from the dataset
(“complete deletion option”).There were a total of 707 positions in the final
dataset. Molecular phylogenetic analysis was performed using MEGA 6.05. The best fit model for nucleotide
substitution was selected from 24 models using MEGA 6.05 (Tamura et al. 2013)
based on the minimum Bayesian Information Criterion (BIC) value (Nei and Kumar 2000; Schwarz 1798). The best fit nucleotide
substitution model was used for testing the phylogenetic hypothesis using maximum
likelihood method based on the Tamura-Nei model
(Tamura and Nei 1993). The branch support for the
correct location of branches was assessed through 1,000 bootstrap replicates.
Results and
Discussion
Microsopic examination of the anterior end
of the slender whitish nematode measuring about 3.8cm revealed the presence of
a well-developed mouth collar, large and globular buccal capsule having fine
tubercles and cone shaped oesophageal funnel (Image2). The posterior end
consisted of a short bursa having indistinctly divided lobes and closely
apposed ventral rays. Spicules were stout, straight with club shaped tips bent
dorsally (Image3). Such morphological features corroborated with those of the
male M. linstowi
(Singh 2003). Molecular analysis showed a product size of 870bp (Image4). The
sequencing results were compared with reference sequences of NCBI database
using BLAST and 98% similarity was found with M. linstowi
recovered from elephants in Kenya. The sequence was submitted to GenBank,
with the accession number MK968095. Nucleotide substitution model with
invariant sites (T92+I, BIC=3284.19, InL= -1282.61, I
= 0.69) was chosen as the best nucleotide substitution model (Tamura 1992).
Nucleotide sequence of the sample and 21 reference sequences were used for the
construction of a maximum likelihood phylogenetic tree (Figure 1). The
bootstrap values shown in the nodes of the branches within the different
clusters of Murshidia are relatively
high. Therefore, the sample is likely to be M. linstowi.
Based on gross and microscopic
examinations, the trematode was identified as Pseudodiscus
hawkesii (Singh 2003). P.hawkesi
measuring approximately 3.6–11mm in length and 2–6mm in breadth possessed
the salient features like ventral mouth opening with oral suckers, well
developed esophageal muscular bulb, lobed testes,
sub-median ovary and coiled uterus.
Like other members of the
subfamily Cyathostominae, M.linstowi
probably has a direct life cycle. Eggs passed in the faeces hatch on the ground
to release the first stage larva which subsequently develops into the third
stage. These strongyles are inadvertently ingested by
their hosts as infective third-stage larvae on vegetation (Newton-Fisher et al.
2006). Helminthic infections in many wild animals are often sub-clinical except
in circumstances where the host-parasite equilibrium is being destabilized by
stressors like concurrent infections, pregnancy, lactation and changes in
climatic conditions. Clinical signs such as reduction in feed intake, edematous swelling on dependent parts of body, debility and
reduction in body weight have been recorded in elephants suffering from murshidiasis (Tripathy et al.
1991). However, in the present case, no such clinical signs were evident. A
single dose of fenbendazole at the rate of 5mg/kg body weight has been found to
be successful against murshidiasis in elephants (Nei and Kumar 2000).
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