title: Novel methods for the molecular discrimination of Fasciola spp. on the basis of nuclear protein-coding genes.
abstract: Fasciolosis is an economically important disease of livestock caused by Fasciola hepatica, Fasciola gigantica, and aspermic Fasciola flukes. The aspermic Fasciola flukes have been discriminated morphologically from the two other species by the absence of sperm in their seminal vesicles. To date, the molecular discrimination of F. hepatica and F. gigantica has relied on the nucleotide sequences of the internal transcribed spacer 1 (ITS1) region. However, ITS1 genotypes of aspermic Fasciola flukes cannot be clearly differentiated from those of F. hepatica and F. gigantica. Therefore, more precise and robust methods are required to discriminate Fasciola spp. In this study, we developed PCR restriction fragment length polymorphism and multiplex PCR methods to discriminate F. hepatica, F. gigantica, and aspermic Fasciola flukes on the basis of the nuclear protein-coding genes, phosphoenolpyruvate carboxykinase and DNA polymerase delta, which are single locus genes in most eukaryotes. All aspermic Fasciola flukes used in this study had mixed fragment pattern of F. hepatica and F. gigantica for both of these genes, suggesting that the flukes are descended through hybridization between the two species. These molecular methods will facilitate the identification of F. hepatica, F. gigantica, and aspermic Fasciola flukes, and will also prove useful in etiological studies of fasciolosis.
authors.affiliation: Laboratory of Veterinary Parasitology, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan; Department of Pathogenetic Veterinary Science, United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Shoriki
authors.affiliation: Laboratory of Veterinary Parasitology, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan; Department of Pathogenetic Veterinary Science, United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan. Electronic address: madoka@iwate-u.ac.jp.
authors.forename: Madoka
authors.initials: M
authors.lastname: Ichikawa-Seki
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, 2-11 Nishi, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Suganuma
authors.affiliation: Laboratory of Veterinary Microbiology, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan.
authors.forename: Ikunori
authors.initials: I
authors.lastname: Naito
authors.affiliation: Laboratory of Veterinary Parasitology, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan; Department of Pathogenetic Veterinary Science, United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Kei
authors.initials: K
authors.lastname: Hayashi
authors.affiliation: Department of Parasitology, Asahikawa Medical University, Asahikawa, Hokkaido 078-8510, Japan.
authors.forename: Minoru
authors.initials: M
authors.lastname: Nakao
authors.affiliation: Laboratory of Veterinary Parasitology, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan; Department of Pathogenetic Veterinary Science, United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Junya
authors.initials: J
authors.lastname: Aita
authors.affiliation: Laboratory of Veterinary Parasitology, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan; Department of Pathogenetic Veterinary Science, United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Uday Kumar
authors.initials: UK
authors.lastname: Mohanta
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, 2-11 Nishi, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
authors.forename: Noboru
authors.initials: N
authors.lastname: Inoue
authors.affiliation: Laboratory of Veterinary Microbiology, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Murakami
authors.affiliation: Laboratory of Veterinary Parasitology, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, Iwate 020-8550, Japan; Department of Pathogenetic Veterinary Science, United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Itagaki
mesh_terms: D000818:Animals; D004258:DNA Polymerase III / Q000235:genetics; D005209:Fasciola / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D005211:Fascioliasis / Q000469:parasitology*; D005838:Genotype; D010730:Phosphoenolpyruvate Carboxylase / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D004258:DNA Polymerase III; D010730:Phosphoenolpyruvate Carboxylase
keywords: Fasciola spp.; Multiplex PCR; PCR–RFLP; pepck; pold