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Journal of Threatened Taxa Building evidence for conservaton globally www.threatenedtaxa.org ISSN 0974-7907 (Online) | ISSN 0974-7893 (Print) Short Communication

Molecular detection of Murshidia linstowi in a free-ranging dead elephant calf

Sourabh Ranjan Hota, Sonali Sahoo, Manojita Dash, Avisek Pahari, Bijayendranath Mohanty & Niranjana Sahoo

26 February 2020 | Vol. 12 | No. 3 | Pages: 15359–15363 DOI: 10.11609/jot.4961.12.3.15359-15363

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Journal of Threatened Taxa | www.threatenedtaxa.org | 26 February 2020 | 12(3): 15359–15363 ISSN 0974-7907 (Online) | ISSN 0974-7893 (Print) PLATINUM OPEN ACCESS DOI: htps://doi.org/10.11609/jot.4961.12.3.15359-15363

#4961 | Received 24 March 2019 | Final received 31 December 2019 | Finally accepted 26 January 2020

S h o Molecular detecton of Murshidia linstowi r t in a free-ranging dead elephant calf C o Sourabh Ranjan Hota 1 , Sonali Sahoo 2 , Manojita Dash 3 , Avisek Pahari 4 , m 5 6 m Bijayendranath Mohanty & Niranjana Sahoo u n 1,3,4,6 Centre for Wildlife Health, College of Veterinary Science and Animal Husbandry, Bhubaneswar, Odisha 751003, India. i 2 c Department of Veterinary Epidemiology and Preventve Medicine, College of Veterinary Science and Animal Husbandry, Bhubaneswar, a Odisha 751003, India. t 5 Department of Veterinary Parasitology, College of Veterinary Science and Animal Husbandry, Bhubaneswar, Odisha 751003, India. i 1 [email protected], 2 [email protected], 3 [email protected], 4 [email protected], o 5 [email protected], 6 [email protected] (corresponding author) n

Abstract: Gastrointestnal helminths are ubiquitous in both Helminths are ubiquitous across vertebrate taxa. domestc and wild animals. Infectons are ofen sub-clinical except in They pose a threat to the welfare, management circumstances of destabilizaton of host-parasite equilibrium by innate or environmental factors. The present case deals with microscopic and and conservaton of captve as well as free-ranging molecular diagnosis of Murshidia linstowi recovered from an elephant. elephants. Strongyles of the genus Murshidia reside in A post-mortem examinaton of a free-ranging juvenile male elephant the alimentary canal of Indian and African elephants. calf that had died of electrocuton in Athagarh Wildlife Division revealed the presence of slender, whitsh nematodes in the stomach. Murshidia spp. afectng elephants include M. linstowi No gross lesions were notced either in the site of predilecton or any (Heinrich 2016; McLean et al. 2012), M. murshida other internal organs. The average length of the parasites was 3.8cm. These parasites were collected for further gross as well as microscopic (Ajitkumar et al. 2009; Chandra et al. 2018; Edwards et examinaton following routne parasitological techniques. Temporary al. 1978; Muraleedharan 2016), M. falcifera (Ajitkumar mounts prepared afer cleaning the nematodes in lactophenol were et al. 2009; Chandra et al. 2018; Edwards et al. 1978; observed under a microscope. Morphological features such as a well-developed mouth collar, large and globular buccal capsule with Matsuo and Supramah 1997),M. longicaudata (Heinrich fne tubercles, cone shaped oesophageal funnel, short bursa having 2016; McLean et al. 2012), M. indica (Ajitkumar et al. indistnctly divided lobes and closely apposed ventral rays and stout 2009; Muraleedharan 2016) and M. Africana (McLean et spicules with club shaped tps bent dorsally corroborated with that of M.linstowi (male). Amplifcaton of the rDNA from the internal al. 2012). Murshidiasis in elephants has been reported transcribed spacer (ITS) region using universal nematode primers from across the globe like Sri Lanka, Nigeria, Kenya, NC2 and NC5 revealed a product size of 870bp. The PCR product was Burma, Indonesia and India. The present case report subjected to sequencing followed by NCBI-BLAST which revealed 98% homology with M. linstowi. A phylogenetc study showed a maximum deals with molecular identfcaton of Murshidia linstowi similarity with M.linstowi recovered from elephants in Kenya. This recovered from a free-ranging elephant calf that died of partcular nematode species belonging to the family Strongylidae and electrocuton. sub-family Cyathostominae appears to be the frst documented report in India.

Keywords: Gastrointestnal helminths, infecton, nematode.

Editor: Mandar Paingankar, Government Science College Gadchiroli, Maharashtra, India. Date of publicaton: 26 February 2020 (online & print)

Citaton: Hota, S.R., S. Sahoo, M. Dash, A. Pahari, B. Mohanty & N. Sahoo (2020). Molecular detecton of Murshidia linstowi in a free-ranging dead elephant calf. Journal of Threatened Taxa 12(3): 15359–15363. htps://doi.org/10.11609/jot.4961.12.3.15359-15363

Copyright: © Hota et al. 2020. Creatve Commons Atributon 4.0 Internatonal License. JoTT allows unrestricted use, reproducton, and distributon of this artcle in any medium by providing adequate credit to the author(s) and the source of publicaton.

Funding: None.

Competng interests: The authors declare no competng interests.

Acknowledgements: Authors are thankful to the Divisional Forest Ofcer, Athagarh Forest Division, Forest and Environment Department, Government of Odisha for providing all necessary support towards this feld investgaton.

15359 J TT Murshidia linstowi in dead elephant calf Hota et al.

History 0.16µl DNA polymerase, 1.2µl of each primer(10mM) A free-ranging juvenile, male elephant calf about and 2.4µl of dNTP mixture (2pmol). Amplifcaton was 4½ years that had died of electrocuton in Athagarh preceded by a 10 minute polymerase actvaton step at Wildlife Division (20.320N & 85.410E) was referred for 95ºC followed by 40 cycles of 45 sec each at 95ºC, 55ºC investgaton. The body conditon of the elephant was and 72ºC. A 5-min extension step at 72ºC concluded the almost normal. During post-mortem examinaton, two reacton. The amplifcaton products were subjected types of helminths (trematode and nematode) were to electrophoresis on 1.5% agarose gel. The parasite recovered from its stomach. No gross lesions were sample was run in duplicates along with nuclease free notced either in the site of predilecton of the parasites water as negatve control. The purifed PCR products or any other internal organs. were subjected to sequencing for further identfcaton. The similarity of the sequence with Genbank database Materials and Methods submissions was carried out by using BLAST (htp://blast. These parasites thus collected (Image1) were ncbi.nlm.nih.gov) (Altschul et al. 1990). The sequence subjected to gross and microscopic examinaton was submited to Genbank for generaton of accession following routne parasitological techniques and number. Additonal 21 gene sequences were retrieved identfed based on their features (Singh 2003). from the NCBI GenBank database (htp://www.ncbi.nlm. nih.gov/). All the sequences were aligned and compared Molecular diagnosis using ClustalW (htp://www.ebi.ac.uk), with gaps and The internal transcribed spacer (ITS) region within missing data eliminated from the dataset (“complete the rDNA provides a reliable marker to diferentate deleton opton”).There were a total of 707 positons in between several strongyles. Genomic DNA of the the fnal dataset. Molecular phylogenetc analysis was parasite was extracted using commercially available performed using MEGA 6.05. The best ft model for DNA mini kit (QIAGEN, Germany). According to the nucleotde substtuton was selected from 24 models manufacturer’s instructons, 25mg of the parasite was using MEGA 6.05 (Tamura et al. 2013) based on the taken for the said purpose. The universal nematode minimum Bayesian Informaton Criterion (BIC) value (Nei primers NC2 (5’- TTAGTTTCTTTTCCTCCGCT-3’) and NC5 and Kumar 2000; Schwarz 1798). The best ft nucleotde (5’- GTAGGTGAACCTGCGGAAGGATCATT-3’) were used substtuton model was used for testng the phylogenetc for amplifcaton (McLean et al. 2012). PCR was carried hypothesis using maximum likelihood method based out in a 24µl reacton mixture containing2µl (640ng/µl)of on the Tamura-Nei model (Tamura and Nei 1993). The

genomic DNA, 2.4µl 10X PCR bufer, 2.4µl of 25mM MgCl2, branch support for the correct locaton of branches was assessed through 1,000 bootstrap replicates.

Results and Discussion Microsopic examinaton of the anterior end of the slender whitsh nematode measuring about 3.8cm revealed the presence of a well-developed mouth collar, large and globular buccal capsule having fne tubercles and cone shaped oesophageal funnel (Image2). The posterior end consisted of a short bursa having indistnctly divided lobes and closely apposed ventral rays. Spicules were stout, straight with club shaped tps 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 submited to GenBank, with the accession number MK968095. © Sonali Sahoo Nucleotde substtuton model with invariant sites Image 1. Helminths recovered from stomach of elephant. (T92+I, BIC=3284.19, InL= -1282.61, I = 0.69) was chosen

15360 Journal of Threatened Taxa | www.threatenedtaxa.org | 26 February 2020 | 12(3): 15359–15363 J TT Murshidia linstowi in dead elephant calf Hota et al.

Image 2. Anterior end of Murshidia linstowi (male). © Sonali Sahoo

Image 4. Lane a—negatve control | Lanes b & c—samples in duplicate (870bp) |Lane d—100bp DNA ladder. © Sonali Sahoo

(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 frst stage larva which subsequently develops into the third stage. Image 3. Posterior end of Murshidia linstowi (male). © Sonali Sahoo These strongyles are inadvertently ingested by their hosts as infectve third-stage larvae on vegetaton (Newton- Fisher et al. 2006). Helminthic infectons in many wild as the best nucleotde substtuton model (Tamura animals are ofen sub-clinical except in circumstances 1992). Nucleotde sequence of the sample and 21 where the host-parasite equilibrium is being destabilized reference sequences were used for the constructon of by stressors like concurrent infectons, pregnancy, a maximum likelihood phylogenetc tree (Figure 1). The lactaton and changes in climatc conditons. Clinical signs bootstrap values shown in the nodes of the branches such as reducton in feed intake, edematous swelling within the diferent clusters of Murshidia are relatvely on dependent parts of body, debility and reducton in high. Therefore, the sample is likely to be M. linstowi. body weight have been recorded in elephants sufering Based on gross and microscopic examinatons, the from murshidiasis (Tripathy et al. 1991). However, in the trematode was identfed as Pseudodiscus hawkesii present case, no such clinical signs were evident. A single

Journal of Threatened Taxa | www.threatenedtaxa.org | 26 February 2020 | 12(3): 15359–15363 15361 J TT Murshidia linstowi in dead elephant calf Hota et al.

Figure 1. Evolutonary analysis of M. linstowi (DKAT-18) using 16s rDNA sequencing.

dose of fenbendazole at the rate of 5mg/kg body weight Heinrich, L. (2016). Prevalence and molecular identfcaton of helminthes in wild and captve Sri Lankan Elephants, has been found to be successful against murshidiasis in Elephasmaximus. Research project-Royal Veterinary College. elephants (Nei and Kumar 2000). University of London. Matsuo, K.&H. Supramah (1997). Some parasites from Sumatran elephants in Indonesia. Journal of the Helminthological Society of References Washington 64: 298–299. McLean, E.R., J.M. Kinsella, P. Chiyo, V.Obanda, C. Moss & E.A. Ajitkumar, G., K.S. Anil & P.C. Alex (2009). Healthcare management of Archie (2012). Genetc identfcaton of fve Strongyle nematode Captve AsianElephants. Elephant Study Centre of Kerala Agricultural parasites in wild African Elephants(Loxondata Africana). Journal of University, 94pp. Wildlife Diseases 48: 707–716. htps://doi.org/10.7589/0090-3558- Altschul, S.F., W. Gish, W. Miller, E.W. Myers&D.J. Lipman (1990). Basic 48.3.707 local alignment search tool. Journal of Molecular Biology 215: 403– Muraleedharan, K. (2016).Endoparasites of Wildlife(Herbivores, 410. htps://doi.org/10.1016/S0022-2836(05)80360-2 Primates and Reptles) of Karnataka state, India - an overview. Chandra, K., D. Gupta, K.C. Gopi, B.Tripathy&V. Kumar (2018).Faunal Veterinary Research Internatonal 4: 89–98. Diversity of Indian Himalaya. Zoological Survey of India, Kolkata, Nei, M. &S. Kumar(2000). Molecular Evoluton and 105pp. Phylogenetcs. Oxford University Press, New York, 333pp. Edwards, S.J., M.W. Hood, J.H. Shaw, J.D. Rayburn, M.D. Kirby, Newton-Fisher, N.E., H. Notman, J.D. Paterson & V. Reynolds (2006). D.T.Hanfman& J.A. Zidar (1978). Index-catalogue of medical and Primates of Western Uganda. Springer, New York, 516pp. https:// veterinary zoology, parasite-subject catalogue, parasites: Nematoda doi.org/10.1007/978-0-387-33505-6 and Acanthocephala, Supplement 21, Part 4: 118. Schwarz, G. (1978). Estmatng the dimension of a model. Annals of

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Statstcs 6: 461–464. Singh, K.R.S. (2003). Veterinary Helminthology. Indian Council of agricultural Research, New Delhi, 347–348. Tamura, K. (1992). Estmaton of the number of nucleotde substtutons when there are strong transiton-transversion and G+C content biases. Molecular Biology and Evoluton 9(4): 678–687. htps://doi.org/10.1093/oxfordjournals.molbev.a040752 Tamura, K. & M.Nei (1993).Estmaton of the number of nucleotde substtutons in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evoluton 10(3):512–526. htps://doi.org/10.1093/oxfordjournals.molbev.a040023 Tamura, K., G. Stecher, D. Peterson, A.Filipski& S. Kumar (2013). MEGA6: Molecular Evolutonary Genetcs Analysis version 6.0. Molecular Biology and Evoluton 30: 2725–2729. Tripathy,S.B., L.N.M. Acharjyo&N.K. Padhi (1991). Use of Fenbendazole against murshidiasis in zoo elephant.Internatonal Seminar on Veterinary Medicine in Wild and Captve Animals, 29pp.

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Journal of Threatened Taxa | www.threatenedtaxa.org | 26 February 2020 | 12(3): 15359–15363 15363 PLATINUM The Journal of Threatened Taxa (JoTT) is dedicated to building evidence for conservaton globally by publishing peer-reviewed artcles online every month at a reasonably rapid rate at www.threatenedtaxa.org. OPEN ACCESS All artcles published in JoTT are registered under Creatve Commons Atributon 4.0 Internatonal License unless otherwise mentoned. JoTT allows allows unrestricted use, reproducton, and distributon of artcles in any medium by providing adequate credit to the author(s) and the source of publicaton.

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February 2020 | Vol. 12 | No. 3 | Pages: 15279–15406 Date of Publicaton: 26 February 2020 (Online & Print) www.threatenedtaxa.org DOI: 10.11609/jot.2020.12.3.15279-15406

Review New record of Tulostoma squamosum (Agaricales: Basidiomycota) from India based on morphological features and phylogenetc analysis Ramifcatons of reproductve diseases on the recovery of the – Arun Kumar Duta, Soumitra Paloi & Krishnendu Acharya , Pp. 15375– Sumatran Rhinoceros Dicerorhinus sumatrensis (Mammalia: 15381 Perissodactyla: Rhinocerotdae) – Nan E. Schafer, Muhammad Agil & Zainal Z. Zainuddin, Pp. 15279–15288 Notes

Communicatons An account of a frst record of the Common Goldeneye Bucephala clangula Linnaeus, 1758 (Aves: Anseriformes: Anatdae) Diet ecology of tgers and leopards in Chhatsgarh, central India in Bhutan – Krishnendu Basak, Moiz Ahmed, M. Suraj, B.V. Reddy, O.P. Yadav & – Sangay Nidup, Gyeltshen & Tshering Tobgay, Pp. 15382–15384 Krishnendu Mondal, Pp. 15289–15300 First record of the hawkmoth Theretra lycetus (Cramer, 1775) Building walls around open wells prevent Asiatc Lion Panthera leo persica (Sphingidae: Macroglossinae) from Bhutan (Mammalia: Carnivora: Felidae) mortality in the Gir Lion Landscape, – Sangay Nidup & Jatshwor Singh Irungbam, Pp. 15385–15386 Gujarat, India – Tithi Kagathara & Erach Bharucha, Pp. 15301–15310 Occurrence and associaton of the Scarce Lilacfork Lethe dura gammiei (Moore, [1892]) (Lepidoptera: Nymphalidae: Taxonomic and ecological notes on some poorly known bats Satyrinae) with Woolly-leaved Oak Quercus lanata Smith, 1819 (Mammalia: Chiroptera) from Meghalaya, India (Fabaceae) forest in the Kumaon region of the Indian Himalaya – Utam Saikia, AdoraThabah & Manuel Ruedi, Pp. 15311–15325 – Arun P. Singh & Tribhuwan Singh, Pp. 15387–15390

Angiosperm diversity in Bhadrak region of Odisha, India Additons to the Odonata (Insecta) fauna of Asansol-Durgapur – Taranisen Panda, Bikram Kumar Pradhan, Rabindra Kumar Mishra, Industrial Area, West Bengal, India Srust Dhar Rout & Raj Ballav Mohanty, Pp. 15326–15354 – Amar Kumar Nayak, Pp. 15391–15394

Gynochthodes cochinchinensis (DC.) Razafm. & B. Bremer (Morindeae: Short Communicatons Rubioideae: Rubiaceae): an additon to the woody climbers of India – Pradeep Kumar Kamila, Prabhat Kumar Das, Madhusmita Mallia, Sightng of Petaurista petaurista (Pallas, 1766) (Mammalia: Rodenta: Chinnamadasamy Kalidass, Jagayandat Pat & Pratap Chandra Panda, Sciuridae) on limestone hills in Merapoh, Malaysia Pp. 15395–15399 – Priscillia Miard, Mohd Nur Arifuddin, Izereen Mukri, Sit Syuhada Sapno, Hafz Yazid, Nadine Ruppert & Jayaraj Vijaya Kumaran, Pp. 15355–15358 Record of Oldenlandia hygrophila Bremek. (Spermacoceae: Rubiaceae), a lesser known herb from Palghat Gap of Western Ghats, Kerala, India Molecular detecton of Murshidia linstowi in a free-ranging dead – Vadakkeveedu Jagadesh Aswani, Vasudevan Ambat Rekha, Pathiyil elephant calf Arabhi, Manjakulam Khadhersha Jabeena, Kunnamkumarath Jisha & – Sourabh Ranjan Hota, Sonali Sahoo, Manojita Dash, Avisek Pahari, Maya Chandrashekaran Nair, Pp. 15400–15404 Bijayendranath Mohanty & Niranjana Sahoo, Pp. 15359–15363

Parasite commonality at Swamp Deer (Mammalia: Artodactyla: Book Review Cervidae: Rucervus duvaucelii duvaucelii) and livestock interface – Animesh Talukdar, Bivash Pandav & Parag Nigam, Pp. 15364–15369 The State of Wildlife and Protected Areas in Maharashtra: News and Informaton from the Protected Area Update 1996-2015 Prevalence and seasonal variaton of gastrointestnal parasites – Reviewed by L.A.K. Singh, Pp. 15405–15406 among captve Northern Pig-tailed Macaque Macaca leonina (Mammalia: Primates: Cercopithecidae) – Phoebe Lalremruat & G.S. Solanki, Pp. 15370–15374 Publisher & Host

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