Development of high-resolution melting (HRM) analysis for population studies of Fascioloides magna (Trematoda: Fasciolidae), the giant liver fluke of ruminants
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The high-resolution melting (HRM) technique was successfully optimized as fast and effective method for population study of digenetic fluke, Fascioloides magna (Trematoda: Fasciolidae), originally North American liver parasite of free-living and domestic ruminants. Previously selected variable region (439 bp) of mitochondrial cytochrome c oxidase subunit I (cox1) of 249 fluke individuals from enzootic European and North American regions were sequenced and mutually compared. The sequence analysis of partial cox1 revealed presence of seven structurally different haplotypes. Based on the sequence structure and alignments of six of them (Ha1–Ha6), three internal probes were designed and applied in HRM-based haplotype determination of all F. magna specimens. HRM analysis, performed with three designed probes, resulted in classification of samples into the seven haplogroups, equally with their assortment according to the sequence analysis. The representative of the haplotype, which was not involved in probe design (Ha7), was characterized by a unique melting curve shape as well. This provided an evidence of optimally settled conditions in HRM assay and indicated a probability of successful discrimination of novel haplotypes in future population studies on F. magna. The successful optimization of HRM method stands for an opportunity of detection of genetically unknown North American variants of F. magna and promises its application as fast and cheap screening technique for phylogeography studies of the giant liver fluke on its original continent.
KeywordsPolymorphic Site Mitochondrial Haplotype Single Strand Conformational Polymorphism Internal Probe Cox1 Haplotype
The authors would like to acknowledge Dr. Marta Špakulová (Parasitological Institute SAS, Košice, Slovakia) for critical readings of the manuscript and valuable comments. We are sincerely thankful to the following scientists for donation of parasitic material: Prof. Luca Rossi (Faculty of Veterinary Medicine, University of Turin, Italy), Assoc. Prof. Dušan Rajský (Faculty of Forestry, Technical University in Zvolen, Zvolen, Slovakia), Dr. Martin Kašný (Department of Parasitology, Faculty of Science, Charles University, Prague, Czech Republic), Prof. Břetislav Koudela and Dr. Adam Novobilský (Department of Parasitology, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic), Prof. Albert Marinculić and Dr. Relja Beck (Department of Parasitology, Veterinary Faculty, University of Zagreb, Zagreb, Croatia), Prof. Egri Borisz (University of West Hungary, Institute of Animal Science, Department of Animal Health, Mosonmagyaróvár, Hungary), Dr. Margo Pybus (Alberta Fish and Wildlife Division, Edmonton, Alberta, Canada), and Dr. Kevin M. Keel (Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA).
This work was supported by the Slovak Research and Development Agency under contract APVV-51-062205 and project of the Grant Agency of the Slovak Republic (VEGA 1/0602/08). The publication has been created within realization of the project “Centre of Excellence for Parasitology” (Code ITMS: 26220120022), based on the support of Operational Programme “Research & Development” funded from the European Regional Development Fund (rate 0.1).
- Andriantsoanirina V, Lascombes V, Ratsimbasoa A, Bouchier C, Hoffman J, Tichit M, Rabarijaona LP, Durand R, Ménard D (2009) Rapid detection of point mutations in Plasmodium falciparum genes associated with antimalarial drugs resistance by using high-resolution melting analysis. J Microbiol Meth 78:165–170CrossRefGoogle Scholar
- Areekit S, Kanjanavas P, Pakpitchareon A, Khawsak P, Khuchareontaworn S, Sriyaphai T, Chansiri K (2009) High resolution melting real-time PCR for rapid discrimination between Brugia malayi and Brugia pahangi. J Med Assoc Thai 3:24–28Google Scholar
- Bassi R (1875) Sulla cachessia ittero-vermicosa, o marciaia dei Cervi, causata dal Distomum magnum. II Medico Veterinario 4:497–515Google Scholar
- Bazsalovicsová E, Králová-Hromadová I, Špakulová M, Reblánová M, Oberhauserová K (2010) Determination of ribosomal internal transcribed spacer 2 (ITS2) interspecific markers in Fasciola hepatica, Fascioloides magna, Dicrocoelium dendriticum and Paramphistomum cervi (Trematoda), parasites of wild and domestic ruminants. Helminthologia 47:76–82CrossRefGoogle Scholar
- Erhardová-Kotrlá B (1971) The occurrence of Fascioloides magna (Bassi, 1875) in Czechoslovakia. Czechoslovak Academy of Sciences, PragueGoogle Scholar
- Králová-Hromadová I, Špakulová M, Horáčková E, Turčeková L, Novobilský A, Beck R, Koudela B, Marinculić A, Rajský D, Pybus M (2008) Sequence analysis of ribosomal and mitochondrial genes of the giant liver fluke Fascioloides magna (Trematoda: Fasciolidae): intraspecific variation and differentiation from Fasciola hepatica. J Parasitol 94:58–67CrossRefPubMedGoogle Scholar
- Majoros G, Sztojkov V (1994) Appearance of the large American liver fluke Fascioloides magna (Bassi, 1875) (Trematoda: Fasciolata) in Hungary. Parasitol Hung 27:27–38Google Scholar
- Marinculić A, Džakula N, Janicky Z, Lućinger S, Živićniak T (2002) Appearance of American liver fluke (Fascioloides magna Bassi, 1875) in Croatia—a case report. Vet Archiv 72:319–325Google Scholar
- Rajský D, Patus A, Bukovjan K (1994) Prvý nález Fascioloides magna (Bassi, 1875) na Slovensku. Slov Vet Čas 19:29–30Google Scholar
- Špakulová M, Rajský D, Sokol J, Vodňanský M (2003) Giant liver fluke (Fascioloides magna), an important liver parasite of ruminants. PaRPRESS, BratislavaGoogle Scholar
- Ullrich K (1930) Über das Vorkommen von seltenen oder wening bekannten Parasiten der Säugetiere und Vögel in Böhmen und Mähren. Prager Arch Tiermed 10:19–43Google Scholar
- Winkelmayer R, Prosl H (2001) Riesenleberegel—jetzt auch bei uns? Österreichs Weidwerk 3:42–44Google Scholar