{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,5,31]],"date-time":"2025-05-31T09:43:31Z","timestamp":1748684611140,"version":"3.37.3"},"reference-count":47,"publisher":"Public Library of Science (PLoS)","issue":"4","license":[{"start":{"date-parts":[[2024,4,29]],"date-time":"2024-04-29T00:00:00Z","timestamp":1714348800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004253","name":"Quaid-i-Azam University","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100004253","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100010221","name":"Higher Education Commision, Pakistan","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100010221","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p><jats:italic>Dicrocoelium<\/jats:italic> lancet flukes cause significant production loss in ruminant livestock. Although co-infection with multiple <jats:italic>Dicrocoelium<\/jats:italic> species within a host is common, techniques for studying the composition of these complex parasite communities are lacking. The pathogenicity, epidemiology, and therapeutic susceptibility of different helminth species vary, and little is known about the interactions that take place between co-infecting species and their hosts. Here, we describe the first applicationof metabarcoding deep amplicon sequencing method to studythe <jats:italic>Dicrocoelium<\/jats:italic> species in sheep and goats. First, rDNA ITS-2 sequences of four <jats:italic>Dicrocoelium<\/jats:italic> species (<jats:italic>Dicrocoelium dendriticum<\/jats:italic>, <jats:italic>Dicrocoelium hospes<\/jats:italic>, <jats:italic>Dicrocoelium orientalis<\/jats:italic>, and <jats:italic>Dicrocoelium chinensis<\/jats:italic>) were extracted from the NCBI public database. Phylogenetic analysis revealed separate clades of <jats:italic>Dicrocoelium<\/jats:italic> species; hence, molecular differentiation between each species is possible in co-infections. Second, 202 flukes belonging to seventeen host populations (morphologically verified as belonging to the <jats:italic>Dicrocoelium<\/jats:italic> genus) were evaluated to determine the deep amplicon sequencing read threshold of an individual fluke for each of the four species. The accuracy of the method in proportional quantification of samples collected from single hosts was further assessed. Overall, 198 (98.01%) flukes were confirmed as <jats:italic>D<\/jats:italic>. <jats:italic>dendriticum<\/jats:italic> and 1.98% produced no reads. The comparison of genetic distances between rDNA ITS-2 revealed 86% to 98% identity between the <jats:italic>Dicrocoelium<\/jats:italic> species. Phylogenetic analysis demonstrated a distinct clustering of species, apart from <jats:italic>D<\/jats:italic>. <jats:italic>orientalis<\/jats:italic> and <jats:italic>D<\/jats:italic>. <jats:italic>chinensis<\/jats:italic>, which sit very close to each other in a single large clade whereas <jats:italic>D<\/jats:italic>. <jats:italic>hospes<\/jats:italic> and <jats:italic>D<\/jats:italic>. <jats:italic>dendriticum<\/jats:italic> are separated into their own clade. In conclusion each sample was identified as <jats:italic>D<\/jats:italic>. <jats:italic>dendriticum<\/jats:italic> based on the proportion of MiSeq reads and validated the presence of this group of parasites in the Gilgit Baltistan and Khyber Pakhtunkhwa provinces of Pakistan. The metabarcoding deep amplicon sequencing technology and bioinformatics pathway have several potential applications, including species interactions during co-infections, identifying the host and geographical distribution of <jats:italic>Dicrocoelium<\/jats:italic> in livestock, drug therapy response evaluation and understanding of the emergence and spread of drug resistance.<\/jats:p>","DOI":"10.1371\/journal.pone.0302455","type":"journal-article","created":{"date-parts":[[2024,4,29]],"date-time":"2024-04-29T17:27:47Z","timestamp":1714411667000},"page":"e0302455","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":2,"title":["Validation of deep amplicon sequencing of Dicrocoelium in small ruminants from Northern regions of Pakistan"],"prefix":"10.1371","volume":"19","author":[{"given":"Muhammad Asim","family":"Khan","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3979-7606","authenticated-orcid":true,"given":"Kiran","family":"Afshan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sabika","family":"Firasat","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4345-9121","authenticated-orcid":true,"given":"Muhammad","family":"Abbas","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Neil D.","family":"Sargison","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Martha","family":"Betson","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Umer","family":"Chaudhry","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2024,4,29]]},"reference":[{"issue":"4","key":"pone.0302455.ref001","doi-asserted-by":"crossref","first-page":"1179","DOI":"10.1007\/s00436-019-06270-5","article-title":"New insight into genetic variation and haplotype diversity of Fasciola hepatica from Algeria","volume":"118","author":"L Chougar","year":"2019","journal-title":"Parasitology research"},{"issue":"4","key":"pone.0302455.ref002","doi-asserted-by":"crossref","first-page":"1589","DOI":"10.1007\/s00436-013-3313-7","article-title":"A tool for diagnosis of Dicrocoelium dendriticum infection: hatching eggs and molecular identification of the miracidium","volume":"112","author":"H Sandoval","year":"2013","journal-title":"Parasitol Res"},{"issue":"10","key":"pone.0302455.ref003","doi-asserted-by":"crossref","first-page":"1297","DOI":"10.1017\/S0031182015000621","article-title":"Comparative recruitment, morphology and reproduction of a generalist trematode, Dicrocoelium dendriticum, in three species of host","volume":"142","author":"MA Beck","year":"2015","journal-title":"Parasitology"},{"issue":"1","key":"pone.0302455.ref004","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1016\/S1471-4922(02)00009-0","article-title":"Dicrocoeliosis of ruminants: a little known fluke disease","volume":"19","author":"D Otranto","year":"2003","journal-title":"Trends Parasitol"},{"issue":"13","key":"pone.0302455.ref005","doi-asserted-by":"crossref","first-page":"845","DOI":"10.1016\/j.ijpara.2017.04.006","article-title":"Population genetic analysis informs the invasion history of the emerging trematode Dicrocoelium dendriticum into Canada","volume":"47","author":"BJ van Paridon","year":"2017","journal-title":"Int J Parasitol"},{"key":"pone.0302455.ref006","doi-asserted-by":"crossref","first-page":"102276","DOI":"10.1016\/j.parint.2020.102276","article-title":"Molecular confirmation of Dicrocoelium dendriticum in the Himalayan ranges of Pakistan","volume":"81","author":"MA Khan","year":"2021","journal-title":"Parasitology International"},{"issue":"1","key":"pone.0302455.ref007","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1007\/s11686-022-00634-1","article-title":"Spatial Distribution of Dicrocoelium in the Himalayan Ranges: Potential Impacts of Ecological Niches and Climatic Variables.","volume":"68","author":"MA Khan","year":"2023","journal-title":"Acta Parasitol."},{"key":"pone.0302455.ref008","first-page":"344","article-title":"Coprological examination of domestic livestock for intestinal parasites in Village Bahlola, District Charsaddah (Pakistan).","volume":"33","author":"A Shah","year":"2001","journal-title":"Pakistan J. Zool"},{"issue":"2\u20133","key":"pone.0302455.ref009","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1016\/j.actatropica.2007.07.008","article-title":"Morphological and molecular differentiation between Dicrocoelium dendriticum (Rudolphi, 1819) and Dicrocoelium chinensis (Sudarikov and Ryjikov, 1951) Tang and Tang, 1978 (Platyhelminthes: Digenea).","volume":"104","author":"D Otranto","year":"2007","journal-title":"Acta Trop"},{"issue":"6","key":"pone.0302455.ref010","doi-asserted-by":"crossref","first-page":"1170","DOI":"10.1645\/GE-2828.1","article-title":"Human infections with Dicrocoelium dendriticum in Kyrgyzstan: the tip of the iceberg?","volume":"97","author":"A Jeandron","year":"2011","journal-title":"J Parasitol"},{"key":"pone.0302455.ref011","doi-asserted-by":"crossref","first-page":"159","DOI":"10.4103\/0256-4947.60525","article-title":"Human infection with Dicrocoelium dendriticum in Turkey.","volume":"30","author":"Z.T. Cengiz","year":"2010","journal-title":"Ann Saudi Med"},{"issue":"3\u20134","key":"pone.0302455.ref012","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1016\/j.vetpar.2007.08.002","article-title":"Assessment of climatic and orographic conditions on the infection by Calicophoron daubneyi and Dicrocoelium dendriticum in grazing beef cattle (NW Spain).","volume":"149","author":"P D\u00edaz","year":"2007","journal-title":"Vet Parasitol"},{"key":"pone.0302455.ref013","doi-asserted-by":"crossref","first-page":"104736","DOI":"10.1016\/j.prevetmed.2019.104736","article-title":"Predicting environmental suitability and geographical distribution of Dicrocoelium dendriticum at littoral of Caspian Sea: An ecological niche-based modeling.","volume":"170","author":"B Meshgi","year":"2019","journal-title":"Preventive veterinary medicine."},{"key":"pone.0302455.ref014","doi-asserted-by":"crossref","first-page":"109975","DOI":"10.1016\/j.vetpar.2023.109975","article-title":"Histopathology and antibody responses describe the seasonal pattern of dicrocoeliosis in small ruminants in the Himalayan ranges of Pakistan","volume":"320","author":"MA Khan","year":"2023","journal-title":"Vet Parasitol"},{"issue":"1","key":"pone.0302455.ref015","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1645\/18-124","article-title":"Spatiotemporal Patterns of Infection for Emerging Larval Liver Fluke (Dicrocoelium dendriticum) in Three Species of Land Snail in Southern Alberta, Canada.","volume":"105","author":"ZW Dempsey","year":"2019","journal-title":"J Parasitol"},{"issue":"1","key":"pone.0302455.ref016","doi-asserted-by":"crossref","first-page":"342","DOI":"10.1186\/s13071-019-3609-2","article-title":"Dicrocoeliosis in extensive sheep farms: a survey.","volume":"12","author":"A Scala","year":"2019","journal-title":"Parasit Vectors"},{"issue":"6","key":"pone.0302455.ref017","doi-asserted-by":"crossref","first-page":"101276","DOI":"10.1016\/j.ttbdis.2019.101276","article-title":"Development of a deep amplicon sequencing method to determine the species composition of piroplasm haemoprotozoa","volume":"10","author":"U Chaudhry","year":"2019","journal-title":"Ticks Tick Borne Dis"},{"key":"pone.0302455.ref018","doi-asserted-by":"crossref","first-page":"104305","DOI":"10.1016\/j.meegid.2020.104305","article-title":"A novel metabarcoded 18S ribosomal DNA sequencing tool for the detection of Plasmodium species in malaria positive patients","volume":"82","author":"A Wahab","year":"2020","journal-title":"Infect Genet Evol"},{"key":"pone.0302455.ref019","doi-asserted-by":"crossref","first-page":"106416","DOI":"10.1016\/j.actatropica.2022.106416","article-title":"A novel metabarcoded deep amplicon sequencing tool for disease surveillance and determining the species composition of Trypanosoma in cattle and other farm animals","volume":"230","author":"G Yasein","year":"2022","journal-title":"Acta Trop"},{"issue":"10","key":"pone.0302455.ref020","doi-asserted-by":"crossref","first-page":"840","DOI":"10.1007\/s004360100451","article-title":"Description of the adult worms of a new Brazilian isolate of Echinostoma paraensei (Platyhelminthes:Digenea) from its natural vertebrate host Nectomys squamipes by light and scanning electron microscopy and molecular analysis.","volume":"87","author":"AJr Maldonado","year":"2001","journal-title":"Parasitol Res"},{"issue":"1","key":"pone.0302455.ref021","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1023\/A:1006358524045","article-title":"Molecular and morphological differentiation between species of the Plagiorchis vespertilionis group (Digenea, Plagiorchiidae) occurring in European bats, with a re-description of P. vespertilionis (M\u00fcller, 1780)","volume":"47","author":"VV Tkach","year":"2000","journal-title":"Syst Parasitol."},{"issue":"4","key":"pone.0302455.ref022","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1016\/j.biotechadv.2008.03.003","article-title":"Toward practical, DNA-based diagnostic methods for parasitic nematodes of livestock\u2014bionomic and biotechnological implications","volume":"26","author":"RB Gasser","year":"2008","journal-title":"Biotechnol Adv"},{"issue":"4","key":"pone.0302455.ref023","doi-asserted-by":"crossref","first-page":"375","DOI":"10.1292\/jvms.68.375","article-title":"Morphometry on lancet flukes found in Japanese sika deer (Cervus nippon centralis) captured in Iwate Prefecture, Japan.","volume":"68","author":"K Taira","year":"2006","journal-title":"J Vet Med Sci"},{"issue":"4","key":"pone.0302455.ref024","doi-asserted-by":"crossref","first-page":"e20180436","DOI":"10.1590\/0001-3765201920180436","article-title":"Morphological and morphometric variations of Dicrocoelium rileyi (Digenea: Dicrocoelidae) parasitizing Tadarida brasiliensis (Chiroptera: Molosiidae) in Mexico.","volume":"91","author":"J Falc\u00f3n-Ordaz","year":"2019","journal-title":"An Acad Bras Cienc"},{"key":"pone.0302455.ref025","doi-asserted-by":"crossref","first-page":"102071","DOI":"10.1016\/j.parint.2020.102071","article-title":"Genetic diversity and multiplicity of infection in Fasciola gigantica isolates of Pakistani livestock","volume":"76","author":"ZU Rehman","year":"2020","journal-title":"Parasitol Int"},{"key":"pone.0302455.ref026","doi-asserted-by":"crossref","first-page":"105012","DOI":"10.1016\/j.meegid.2021.105012","article-title":"High-throughput sequencing of Fasciola spp. shows co-infection and intermediate forms in Balochistan, but only Fasciola gigantica in the Punjab province of Pakistan","volume":"94","author":"ZU Rehman","year":"2021","journal-title":"Infect Genet Evol"},{"issue":"17","key":"pone.0302455.ref027","doi-asserted-by":"crossref","first-page":"5112","DOI":"10.1128\/AEM.01043-13","article-title":"Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform","volume":"79","author":"JJ Kozich","year":"2013","journal-title":"Applied and Environmental Microbiology"},{"issue":"23","key":"pone.0302455.ref028","doi-asserted-by":"crossref","first-page":"7537","DOI":"10.1128\/AEM.01541-09","article-title":"Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities","volume":"75","author":"PD Schloss","year":"2009","journal-title":"Applied and Environmental Microbiology"},{"key":"pone.0302455.ref029","article-title":"Bio-Strings: A Relational Database Data-Type for Dealing with Large Biosequences.","volume":"11","author":"S Lifschitz","year":"2022","journal-title":"BioTech (Basel)."},{"issue":"6","key":"pone.0302455.ref030","doi-asserted-by":"crossref","first-page":"1547","DOI":"10.1093\/molbev\/msy096","article-title":"MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms","volume":"35","author":"S Kumar","year":"2018","journal-title":"Molecular biology and evolution"},{"issue":"4","key":"pone.0302455.ref031","doi-asserted-by":"crossref","first-page":"1237","DOI":"10.1093\/molbev\/msz312","article-title":"Molecular Evolutionary Genetics Analysis (MEGA) for macOS.","volume":"37","author":"G Stecher","year":"2020","journal-title":"Molecular biology and evolution"},{"issue":"2","key":"pone.0302455.ref032","doi-asserted-by":"crossref","first-page":"150","DOI":"10.1016\/j.actatropica.2009.04.005","article-title":"Fluke egg characteristics for the diagnosis of human and animal fascioliasis by Fasciola hepatica and F. gigantica","volume":"111","author":"MA Valero","year":"2009","journal-title":"Acta Tropica"},{"key":"pone.0302455.ref033","article-title":"Molecular and Morphological Characterization of Fasciola spp. Isolated from Different Host Species in a Newly Emerging Focus of Human Fascioliasis in Iran","author":"R Shafiei","year":"2014","journal-title":"Veterinary Medicine International."},{"issue":"9\u201310","key":"pone.0302455.ref034","doi-asserted-by":"crossref","first-page":"774","DOI":"10.1515\/znc-2005-9-1018","article-title":"Understanding genetic diversity of the liver fluke Fasciola hepatica","volume":"60","author":"M Dosay-Akbulut","year":"2005","journal-title":"Z Naturforsch C J Biosci"},{"key":"pone.0302455.ref035","doi-asserted-by":"crossref","first-page":"100086","DOI":"10.1016\/j.crpvbd.2022.100086","article-title":"Current methods for the detection of Plasmodium parasite species infecting humans.","volume":"2","author":"L Slater","year":"2022","journal-title":"Curr Res Parasitol Vector Borne Dis"},{"key":"pone.0302455.ref036","article-title":"A 4 year observation of gastrointestinal nematode egg counts, nemabiomes and the benzimidazole resistance genotypes of Teladorsagia circumcincta on a Scottish sheep farm","author":"MJ Evans","year":"2021","journal-title":"Int J Parasitol"},{"key":"pone.0302455.ref037","doi-asserted-by":"crossref","first-page":"109339","DOI":"10.1016\/j.vetpar.2020.109339","article-title":"Nemabiome metabarcoding reveals differences between gastrointestinal nematode species infecting co-grazed sheep and goats","volume":"289","author":"LM Costa-Junior","year":"2021","journal-title":"Vet Parasitol"},{"issue":"12","key":"pone.0302455.ref038","doi-asserted-by":"crossref","first-page":"e0143559","DOI":"10.1371\/journal.pone.0143559","article-title":"Exploring the gastrointestinal \u201cnemabiome\u201d: deep amplicon sequencing to quantify the species composition of parasitic nematode communities.","volume":"10","author":"RW Avramenko","year":"2015","journal-title":"PLoS One"},{"key":"pone.0302455.ref039","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/j.vetpar.2019.02.007","article-title":"A high throughput deep amplicon sequencing method to show the emergence and spread of Calicophoron daubneyi rumen fluke infection in United Kingdom cattle herds","volume":"268","author":"ND Sargison","year":"2019","journal-title":"Vet Parasitol"},{"issue":"14","key":"pone.0302455.ref040","doi-asserted-by":"crossref","first-page":"1916","DOI":"10.1017\/S0031182011001375","article-title":"Detection of Dicrocoelium dendriticum larval stages in mollusc and ant intermediate hosts by PCR, using mitochondrial and ribosomal internal transcribed spacer (ITS-2) sequences.","volume":"138","author":"AM Martinez-Ibeas","year":"2011","journal-title":"Parasitology"},{"issue":"6","key":"pone.0302455.ref041","doi-asserted-by":"crossref","first-page":"683","DOI":"10.3109\/19401736.2013.772168","article-title":"Genetic variability among Dicrocoelium dendriticum isolates from different regions in Shaanxi Province, China revealed by sequences of three mitochondrial genes","volume":"24","author":"GH Zhao","year":"2013","journal-title":"Mitochondrial 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counterimmunoelectrophoresis and passive haemagglutination tests for the diagnosis of Dicrocoelium dendriticum infection in sheep and goats","volume":"61","author":"KP Jithendran","year":"1996","journal-title":"Vet Parasitol"},{"issue":"9","key":"pone.0302455.ref045","doi-asserted-by":"crossref","first-page":"2605","DOI":"10.1007\/s00436-017-5557-0","article-title":"Phylogenetic relationships between Dicrocoelium chinensis populations in Japan and China based on mitochondrial nad1 gene sequences","volume":"116","author":"K Hayashi","year":"2017","journal-title":"Parasitol Res"},{"issue":"7)","key":"pone.0302455.ref046","article-title":"Rumen and Liver Fluke Infections in Sheep and Goats in Northern and Southern Germany.","volume":"12","author":"U Alstedt","year":"2022","journal-title":"Animals (Basel)."},{"issue":"1\u20132","key":"pone.0302455.ref047","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/S0167-5877(02)00215-5","article-title":"Serum antibodies to Dicrocoelium dendriticum in sheep from Sardinia (Italy).","volume":"57","author":"R S\u00e1nchez-Andrade","year":"2003","journal-title":"Preventive veterinary medicine."}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0302455","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,4,29]],"date-time":"2024-04-29T17:28:17Z","timestamp":1714411697000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0302455"}},"subtitle":[],"editor":[{"given":"Hudson 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