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Development of high-resolution melting (HRM) analysis for population studies of Fascioloides magna (Trematoda: Fasciolidae), the giant liver fluke of ruminants

Abstract

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.

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Acknowledgements

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).

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Correspondence to Eva Bazsalovicsová.

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Nucleotide sequence data reported in this paper are available in the GenBank™, EMBL, and DDBJ databases under the accession numbers GU599860–72.

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Radvánský, J., Bazsalovicsová, E., Králová-Hromadová, I. et al. Development of high-resolution melting (HRM) analysis for population studies of Fascioloides magna (Trematoda: Fasciolidae), the giant liver fluke of ruminants. Parasitol Res 108, 201–209 (2011). https://doi.org/10.1007/s00436-010-2057-x

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  • DOI: https://doi.org/10.1007/s00436-010-2057-x

Keywords

  • Polymorphic Site
  • Mitochondrial Haplotype
  • Single Strand Conformational Polymorphism
  • Internal Probe
  • Cox1 Haplotype