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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 For Focus, Scope, Aims, Policies, and Guidelines visit htps://threatenedtaxa.org/index.php/JoTT/about/editorialPolicies#custom-0 For Artcle Submission Guidelines, visit htps://threatenedtaxa.org/index.php/JoTT/about/submissions#onlineSubmissions For Policies against Scientfc Misconduct, visit htps://threatenedtaxa.org/index.php/JoTT/about/editorialPolicies#custom-2 For reprints, contact <[email protected]> The opinions expressed by the authors do not refect the views of the Journal of Threatened Taxa, Wildlife Informaton Liaison Development Society, Zoo Outreach Organizaton, or any of the partners. 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Member Threatened Taxa 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.