Available online at www.sciencedirect.com International Journal for Parasitology 38 (2008) 725–730 www.elsevier.com/locate/ijpara Human fascioliasis and the presence of hybrid/introgressed forms of Fasciola hepatica and Fasciola gigantica in Vietnam Thanh Hoa Le a, Nguyen Van De b, Takeshi Agatsuma c, Thanh Giang Thi Nguyen d, Quoc Doanh Nguyen d, Donald P. McManus e, David Blair f,* a Department of Immunology, Institute of Biotechnology, Hanoi, Viet Nam b Department of Parasitology, Hanoi Medical University, Viet Nam c Department of Environmental Health Sciences, Kochi Medical School, Kochi, Japan d National Institute of Veterinary Research, Hanoi, Viet Nam e Queensland Institute of Medical Research, Brisbane, Qld 4006, Australia f School of Marine and Tropical Biology, James Cook University, Townsville, Qld 4811, Australia Received 19 June 2007; received in revised form 1 October 2007; accepted 2 October 2007 Abstract The two species common of liver ﬂuke, Fasciola hepatica and Fasciola gigantica, cause human fascioliasis. Hybrids between these spe- cies, and introgressed forms of Fasciola, are known from temperate and subtropical regions of eastern Asia. Here, we report the presence of hybrid and/or introgressed liver ﬂukes in Vietnam where it has recently been recognised that human fascioliasis is an important zoo- notic disease. Specimens examined came from domestic stock (cattle and buﬀalo) at slaughter and also from human patients. DNA sequences were obtained from the nuclear ribosomal second internal transcribed spacer (ITS-2) and from portions of two mitochondrial protein-coding genes. Mitochondrial sequences in every case were similar to those of Fasciola gigantica. Nuclear ITS-2 sequences belonged to one or other of the Fasciola species, or, sequences from both were found in the same individual worm. This study extends the known range of hybrids or introgressed forms of Fasciola into tropical regions of Asia. Ó 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Fasciola gigantica; Fasciola hepatica; Hybridisation; Internal transcribed spacer region; Introgression; Vietnam 1. Introduction et al., 2006), but the existence of individuals with intermedi- ate morphological characteristics can cause confusion (e.g. The commonest and most widespread liver ﬂukes of the Terasaki et al., 1982; Itagaki et al., 2005a) and has led to genus Fasciola are Fasciola hepatica Linnaeus, 1758 (mostly the increasing use of molecular methods (e.g. Marcilla in temperate regions) and Fasciola gigantica Cobbold, 1856 et al., 2002) or morphometric methods (e.g. Ashraﬁ et al., (mostly tropical in distribution). Adults of both species occur 2006) to distinguish between the species. It is desirable to in many domestic ruminants and in humans (Mas-Coma know which species is the agent of human or animal disease et al., 2005; Le et al., 2007) and can cause serious disease. in a given area. The two species diﬀer in pathological and epi- The two liver ﬂuke species appear to be sympatric in many demiological characteristics (Mas-Coma et al., 2005). subtropical and warm temperate areas, especially in Africa Diﬃculties in speciﬁc identiﬁcation have been most and Asia (Le et al., 2007). They can generally be distin- intensively studied in Japan and adjacent areas. Research guished on the basis of their morphology (e.g. Ashraﬁ there has revealed not only a confusing range of morpho- logical forms but also the presence of worms of diﬀerent ploidies (diploid, triploid and ‘‘mixoploid’’), all of which * Corresponding author. Tel.: +61 7 4781 4322; fax: +61 7 4725 1570. are parthenogenetic and do not produce normal sperm E-mail address: firstname.lastname@example.org (D. Blair). (Terasaki et al., 1982, 2000). Genetic studies on Japanese 0020-7519/$34.00 Ó 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2007.10.003 726 T.H. Le et al. / International Journal for Parasitology 38 (2008) 725–730 and Korean worm populations have detected individuals Adult worms of Fasciola sp. of Vietnamese origin, col- that have both nuclear and mitochondrial sequences typi- lected during 2001–2005 from human patients and animals cal of F. hepatica and others that appear on the same (cattle and buﬀaloes), were preserved in 70% ethanol and grounds to be F. gigantica. Individuals also occur that kept at À20 °C until used for extraction of DNA. Of 21 resemble one species in their nuclear DNA (usually assayed Vietnamese samples, 12 were of human origin, of which using ribosomal RNA gene or spacer sequences) but have a two (FspN-VN and FspQB-VN) were from the cases mitochondrial genotype typical of the other species (Agat- involving unusual cutaneous migration of worms reported suma et al., 2000; Itagaki et al., 2005a,b). Individuals have in Le et al. (2007). Specimens from eight human cases were also been found that have, in their tandem array of nuclear obtained surgically. In two further cases, eggs morpholog- ribosomal genes, copies of genes apparently derived from ically identiﬁed as being of a Fasciola sp. were recovered both liver ﬂuke species (Agatsuma et al., 2000 in Korea; from faeces of human patients serologically positive for Huang et al., 2004 in north eastern China; Itagaki et al., fascioliasis. These eggs were allowed to develop and hatch 2005a,b in Japan and Korea; Lin et al., 2007 in China). in water (1–2 weeks) and 10–20 miracidia from each patient An obvious conclusion is that hybridisation and introgres- were collected for subsequent DNA extraction. sion (deﬁnitions in Dowling and Secor, 1997) are common in Fasciola populations in this part of Asia, processes that 2.2. Genetic markers can lead to production of polyploid and parthenogenetic individuals (Dowling and Secor, 1997). Such individuals Genetic markers including mitochondrial genes (cox1, are often aspermic. There have been no convincing demon- nad1) and the nuclear second internal ribosomal spacer strations of hybrid/introgressed liver ﬂuke populations out- (ITS-2) sequences were obtained. ITS-2 is a useful marker side Asia, although aspermic triploid individuals of ‘‘pure’’ for distinguishing between F. gigantica and F. hepatica. It F. hepatica are now known from Britain (Fletcher et al., is rather conserved, especially in F. hepatica. There are 2004). Examples of triploid Fasciola sp. are also known seven sites at which the two species typically diﬀer. One from Assam and Hawaii (reviewed in Terasaki et al., of these is a deletion in F. gigantica relative to F. hepatica 2000) and Vietnam (Itagaki et al., 2005a). In addition to that appears to be a diagnostic diﬀerence between the spe- Japan and Korea, aspermic Fasciola spp. of unknown cies (e.g. Adlard et al., 1993; Semyenova et al., 2005). There ploidy are present (but generally uncommon) in the Philip- is some variability, especially in F. gigantica, that can be pines, Vietnam, Thailand, Taiwan, India, Nepal and confusing (Le et al., 2007). Sequence data from mitochon- Hawaii (Terasaki et al., 1982). drial genes are more variable than is the case for ITS-2, but In Vietnam, fascioliasis (caused by morphologically also provide unambiguous recognition of the two species identiﬁed F. gigantica) is very common in cattle and water (Le et al., 2007). buﬀaloes, with prevalences of more than 50% in the Red River and Mekong deltas, as well as in other coastal 2.3. DNA extraction, PCR and sequencing regions (Bui et al., 2003). In recent years, an extraordinary number of human cases of fascioliasis has been reported, to Total genomic DNA was extracted from adult worms such an extent that this zoonotic infection has become a and miracidia using the commercial QIAamp DNA extrac- major public health concern in Vietnam. More than 500 tion kit (QIAGEN Inc.) according to the manufacturer’s human cases were recorded in the three years from 1997 instructions. In the case of adult worms, only a single spec- to 2000 based on serological tests (Tran et al., 2001). Prior imen was used in each DNA extraction. Genomic DNA to 1991, fascioliasis had been regarded as rare in Vietnam. was diluted to a working concentration of 50 ng/lL and For example, only two cases were reported in 1978 (Tran 2 lL of this was used as template in a PCR reaction of et al., 2001). Unusual cases of cutaneous fascioliasis have 50 lL. also been described (e.g. Xuan et al., 2005; Le et al., PCR was used to amplify the entire nuclear ITS-2 and 2007). Morphologically, the adult ﬂukes found in animals two mitochondrial genetic markers (a portion of each of and human patients in Vietnam fall into two categories, cox1 and nad1). Primers 3SF (forward) (5 0 GGTACCG one typical of F. gigantica and the other closely resembling GTGGATCACTCGGCTCGTG3 0 ) and BD2R (reverse) F. hepatica. Here we report data indicating that hybrid/int- (5 0 TATGCTTAAATTCAGCGGGT3 0 ) were used for rogressed populations of Fasciola occur in Vietnam and ampliﬁcation of ITS-2; JB3F (forward) (5 0 TTTTTTGG that these are implicated in human infection. GCATCCTGAGGTTTAT3 0 ) and JB4.5R (reverse) (5 0 TAAAGAAAGAACATAATGAAAATG3 0 ) for cox1 2. Materials and methods as previously published (Bowles and McManus, 1993; Bowles et al., 1995). Primers FND1F (forward) (5 0 TGG 2.1. Sources of Fasciola specimens GGTCTGTTGCAGAGATTTGC3 0 ) and FND1R (reverse) (5 0 ATCCAATGGAGTACGGTTACA3 0 ) for Parasite material and nucleotide sequences of Fasciola nad1 were designed for use in this study. species, their host and geographical origin used in this PCR ampliﬁcation was carried out in a ﬁnal volume of study are listed in Table 1. 50 lL, including 100 ng template, 10 pmol of each primer T.H. Le et al. / International Journal for Parasitology 38 (2008) 725–730 727 Table 1 List of Fasciola specimens and sequences used in this study, their host and geographical origins Species Country Code of samples DNA Extracted from Host Markers used Genbank no/Refs cox1 nad1 ITS-2 p p p F. sp G Vietnam FspBD-VN Adult Human EU260063 p p p F. sp G Vietnam FspPY-VN Adult Cattle EU260074 p p p F. spa H Vietnam FspH1-VN Adult Human EU260068 p p p F. spa H Vietnam FspH2-VN Adult Human EU260069 p p p F. spa H Vietnam FspCB1-VN Adult Cattle EU260064 p p p F. spa H Vietnam FspFG1-VN Adult Human EU260066 p p p F. spa H Vietnam FspFG2-VN Adult Human EU260067 p p p F. spa H Vietnam FspNA-VN Adult Cattle EU260071 p p p F. sp G Vietnam FspTH-VN Adult Cattle EU260076 p p p F. spb H Vietnam Fsp1-VN Adult Human EU260059 p p p F. spb H Vietnam Fsp2-VN Adult Human EU260060 p p p F. sp G Vietnam FspB3-VN Adult Cattle EU260061 p p p F. sp G Vietnam FspBDB-VN Adult Cattle EU260057 p p p F. sp ? Vietnam FspM-VN miracidia Human EU260070 p p p F. sp G Vietnam FspNB-VN Adult Cattle EU260072 p p p F. sp G Vietnam FspT4-VN Adult Buﬀalo EU260075 p p p F. sp ? Vietnam FspX-VN miracidia Human EU260077 p p p F. spa H Vietnam FspBD1-VN Adult Human EU260062 p p p F. spa H Vietnam FspCB2-VN Adult Buﬀalo EU260065 p p p F. spa H Vietnam FspN-VN Adult Humand EU260073 p p p F. sp H Vietnam FspQB-VN Adult Humand EU260078 p p p F. hepatica Australia Fh-AU Adult Cattle EU260058 p p p F. hepatica Uruguay Fh-UR Adult N/A AB010974 p F. hepatica Spain FhCa-SP N/A Cattle AJ272053 p F. hepatica France Fh-FR Adult Cattle AJ557567 p F. hepatica China Fh(Si)-CN Adult N/A AJ557568 p F. hepatica Ireland Fh-IR Adult N/A AB207148 p F. hepatica C. Asia FhSemyenovaH1 Adult N/A Semyenova et al., 2005 p F. hepatica Armenia FhSemyenovaH2 Adult N/A Semyenova et al., 2005 p F. hepatica Austria Fh_AT N/A N/A DQ683546 p F. gigantica C. Asia FgSemyenovaG5, G6, G7, G8 Adult N/A Semyenova et al., 2005 p p p c F. gigantica Indonesia Fg(IndoA)-ID Adult N/A p p p F. gigantica Indonesia Fg-ID Adult N/A EU260080 p p p F. gigantica China Fg(GxB10)-CN Adult Buﬀalo EU260079 p F. gigantica China Fg(Gx)-CN Adult Buﬀalo AJ557569 p F. gigantica Indonesia Fg(IndoT)-ID Adult Cattle AB010977 p F. gigantica Thailand Fg-TL Adult Cattle AB207149 p F. gigantica Burkina Faso Fg(Bobo)-BF Adult Cattle AJ853848 p F. gigantica Zambia FgZamI-ZA Adult N/A AB010975 p F. gigantica Zambia FgZamII-ZA Adult N/A AB010976 p F. sp China Fsp(He1)-CN Adult Sheep AJ557570 p F. sp China Fsp(He2)-CN Adult Sheep AJ557571 p F. sp Japan FspI-JP Adult N/A AB010978 p F. sp Japan FspII-JP Adult N/A AB010979 p F. sp Japan FspKochi1-JP Adult Cattle AB207152 p F. sp Japan FspSaita-JP Adult Cattle AB207151 p F. spa Japan FspHokai-JP Adult Cattle AB207150 p p p F. spa Korea Fsp(Kor2)-KR Adult N/A c p p p c F. sp Korea Fsp(Kor4)-KR Adult N/A p p p c F. sp Korea Fsp(Kor5)-KR Adult N/A p p p c F. sp Japan FspKochi2-JP Adult N/A N/A: not available. a Indicates apparent hybrid/ introgressed specimens (see text). b Indicates two worms known to have ITS-2 sequences of both Fasciola hepatica and Fasciola gigantica type. For Vietnamese worms, G = F. gigantica – like and H = F. hepatica - like. c Indicates data from Agatsuma et al. (2000). d Indicates specimens that had undertaken cutaneous migration in humans as reported in Le et al. (2007). and a mix of the remaining PCR components (PCR Master for cox1 with initiation at 94 °C for 5 min, then 35 cycles Mix from Promega). The PCR reactions were carried out including denaturation at 94 °C for 1 min, annealing at in a MJ Thermal Cycler PTC-100 (MJ Research, USA) 37 °C for 1 min, extension at 72 °C for 2 min. For ITS-2 728 T.H. Le et al. / International Journal for Parasitology 38 (2008) 725–730 and nad1 the above conditions were used, except that which has many probable errors near the 5 0 end. Sequences annealing was at 50 °C for 1 min. Finally, all reactions were reported by Adlard et al. (1993) (F. gigantica from Indone- held for 10 min at 72 °C to complete the ampliﬁcation. sia and Malaysia; F. hepatica from Australia, New Zea- The PCR products were puriﬁed using QIAquick Puriﬁ- land, Hungary and Mexico; Fasciola sp. from Japan) are cation kit (QIAGEN). PCR products were initially sub- shorter than those presented here and have not been jected to direct sequencing using the PCR primers as included. Their inclusion would not have altered the con- sequencing primers. However, when traces with ambigui- clusions reached. All ITS-2 sequences reported by Huang ties were obtained (ITS-2), PCR products were cloned et al. (2004; AJ557567- AJ557571) include a pair of sites using a TA cloning kit (Invitrogen, USA) and recombinant near the 3 0 end that are inverted relative to all other avail- plasmid DNAs subjected to sequencing using Big Dye Ter- able sequences. However, Prof. Zhu Xing-Quan (personal minator Cycle Sequencing technology on an automated communication), one of the authors of that paper, has con- sequencer (ABI 3100 Avant Genetic Analyzer) using M13 ﬁrmed that this inversion is a sequencing error: their forward and reverse primers. sequences have been corrected as used in this paper (note that Alasaad et al. (2007), have used the uncorrected 2.4. Data analysis and phylogenetic construction sequences). Sequences for some haplotypes from Turkmen- istan and adjacent regions of West and Central Asia The sequences were edited in SeqEd v 1.03, aligned reported by Semyenova et al. (2005) have been recon- using AssemblyLIGN v 1.9c and analysed using the Mac- structed from information in that paper: none was avail- Vector 8.2 package (Accelrys) in a Macintosh computer able from any database. Immediately prior to submission system. Speciﬁc identiﬁcation was conﬁrmed by compari- of this paper, a further sequence of F. gigantica, from son with known sequences of the corresponding species Meghalaya, India, appeared in the public databases. This in GenBank or by reference to our previous published data is EF027103, identical in sequence to the Indonesian refer- Le et al., 2001; Le and Nguyen, 2002. ence sequence of F. gigantica mentioned below. Sequences The multiple alignment of sequences was performed of F. hepatica from Bolivia (Mas-Coma et al., 2001) are using GeneDoc v2.5 (Nicholas and Nicholas, 1997. Gene- identical to those from Spain. Doc: A tool for editing and annotating multiple sequence For reference, F. gigantica from Indonesia (Fg_ID) and alignment. Distributed by author) and/or ClustalW incor- F. hepatica from Australia and Europe (Fh_AU) are porated into the MacVector 8.2 package. Modeltest v3.7 regarded as representing genetically pure forms of each (Posada and Crandall, 1998) was used to ﬁnd the best sub- species that have not experienced introgression or hybrid- stitution model. This model (HKY+G) was then speciﬁed isation (Agatsuma et al., 2000) (indicated by arrows on for likelihood analyses in PAUP* v4.0b10 (Swoﬀord, D. Fig. 1). L. 2000. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sinauer Associates, Sun- 3. Results derland, Massachusetts). PaupUP graphical interface (Cal- endini, F., Martin J.-F. 2005. PaupUP v18.104.22.168 A free Excluding ﬂanking regions in the 5.8S and 28S genes, graphical frontend for PAUP* Dos software. Available the length of the ITS-2 alignment was 362 bp. For the mito- from http://www.agro-montpellier.fr/sppe/Recherche/ chondrial markers, partial sequences were obtained from JFM/PaupUp/main.htm) was used to facilitate working cox1 (423 bp) and nad1 (435 bp). with PAUP*. The dataset was resampled 100 times using The main purpose of the phylogenetic analyses was to the bootstrap method. Phylogenetic analyses are presented compare species-of-origin of nuclear and mitochondrial only for the nuclear ITS-2 sequences. Outgroups sequences in a number of individual worms from Vietnam (sequences from Fascioloides magna DQ683545 and Fasci- and elsewhere. This is easily done by presenting a tree olopsis buski DQ341852) were used in some analyses to based on one marker on which can be indicated, for each conﬁrm monophyly of each Fasciola species. individual, species-of-origin of the other marker(s). Here As far as possible, all available published ITS-2, cox1 we present only the tree of the ITS-2 sequences (Fig. 1). and nad1 sequences have been included. Some sequences Given the small number of diﬀerences between the species have been omitted – this is especially so for ITS-2 in which in the ITS-2 region, and the slight variation within each there are relatively few variable sites. Sequences with species, it is not surprising that bootstrap values are rather ambiguous sites or incomplete sequences can bias the tree low. However, the two species are clearly distinguished dramatically. Available ITS-2 sequences not used were: (and remain so in analyses using other fasciolids as out- AB207153 (Hiroshima and Kagoshima, Japan), which groups). It is immediately apparent that the Vietnamese includes several ambiguous sites reﬂecting the fact that worms fall readily into the F. gigantica or the F. hepatica ITS-2 sequences of both Fasciola species are present (Itag- cluster based on ITS-2 data. However, all Vietnamese aki et al., 2005a); AB259058 (Kyoto, Japan), which is worms had mitochondrial sequences (both cox1 and incomplete; L07844, one of the earliest reported sequences nad1) typical of F. gigantica only. Mitochondrial genomes of F. hepatica, which has possible sequencing errors at the in animals are maternally inherited. Our data thus demon- 3 0 end, and DQ383512 from Egyptian F. gigantica and strate that hybridisation or introgression has occurred T.H. Le et al. / International Journal for Parasitology 38 (2008) 725–730 729 had probably not experienced introgression/hybridisation, were placed where expected on the tree (Fig. 1). However, for many of these worms, ITS-2 only was available and nothing was known about their mitochondrial genome. The exceptional sequence was from a specimen of F. gigan- tica from Zambia (AB010975). This sequence had the sin- gle-base deletion characteristic of F. gigantica, but at most other variable sites it possessed the base typical of F. hepatica. We are uncertain how to interpret this. Individual worms from human patients were distributed in both clades of the tree (Fig. 1). 4. Discussion It is clear that hybridisation and/or introgression has occurred in Vietnam involving both of the common species of Fasciola. In every case in that country, the mitochon- drial genome was typical of F. gigantica, whereas the nuclear genome could be from either species. Distinguish- ing between hybridisation and introgression requires more data than we have available. We regard hybrids as the F1 oﬀspring of a mating between the two species. In such a case, all oﬀspring will carry the mitochondrial genome of the maternal parent. However, their nuclear ribosomal Fig. 1. Tree inferred from ITS-2 sequences. Specimen codes are those used RNA cluster will contain copies from both parents. in Table 1. GenBank accession numbers are included where available. Sequences from human cases are shown in bold-italic type. Arrows point Back-crossing of hybrids and of subsequent generations to reference sequences from ‘‘pure’’ Fasciola gigantica or Fasciola hepatica. with one parent species (=introgression) should homoge- With the few exceptions discussed in the text, sequences fall into two nise the ribosomal array so that sequences of the other spe- groups corresponding to F. gigantica and F. hepatica. Sequences are cies will eventually disappear. However, the non- labeled according to the species name or code given to them in the relevant recombining nature of the mitochondrial genome means original publications: Fg = F. gigantica, Fh = F. hepatica and Fsp for those not assigned to a species. Specimens from Vietnam are indicated by that it will be passed on largely unchanged from generation the letters ‘‘VN’’ after the specimen code. Other country codes: AR, to generation. If back-crossing of hybrids is with the pater- Armenia; AT, Austria; AU, Australia; BF, Burkina Faso; BV, Bolivia; nal species, then the mitochondrial genome of the maternal CA, Turkmenistan and adjacent regions of Central Asia; CN, China; FR, species will introgress into the paternal species (reviewed in France; ID, Indonesia; IN, India; IR, Ireland; JP, Japan; Kr, Korea; RU, Blair, 2005). All this assumes that the organisms involved Russia, and neighbouring countries; SP, Spain; TL, Thailand; UR, Uruguay; ZA, Zambia. Specimens known to have F. gigantica-like reproduce sexually and undergo normal meiosis. For many mitochondrial sequences (this study or from the literature) are indicated eastern Asian populations of Fasciola species, this assump- by *. Similarly, specimens with F. hepatica-like mitochondrial sequences tion seems to be false. If hybrids are parthenogenetic, then are indicated with # after the specimen code. Neither symbol appears if worms should carry ribosomal repeats from both parents, mitochondrial data are not available for that individual. The two as well as the mitochondrial genome of the maternal par- sequences marked with Ù came from the same worm specimen in north eastern China (Huang et al., 2004). The symbol à indicates worms from ent, for as many generations as the clonal lineage persists. Vietnam for which direct-sequencing traces indicated the presence of both We do not yet know the ploidy of any of the Vietnamese F. hepatica and F. gigantica ITS-2 sequences. Clean sequence for only one worms, nor do we know if any are parthenogenetic. How- type of ITS-2 was subsequently obtained from cloned PCR products, ever, in at least some cases, individual worms carried ribo- which is why the worms are included in only one clade. Bootstrap values somal sequences from both parent species, suggesting (as percentage support for each branch) are shown. either that they were F1 hybrids, had undergone recent involving the two Fasciola species and that F. gigantica was introgression, or that these worms were from parthenoge- the maternal parent in each case. netic lineages of unknown age. Direct sequencing of PCR products from some of the Not all human infections are due to hybrid/introgressed Vietnamese worms yielded traces with ambiguities indicat- worms. Some were apparently due to ‘‘pure’’ F. gigantica ing the presence of both F. hepatica and F. gigantica ITS-2 infection. In addition, worms from human patients were sequences (data not shown). Clean sequence for only one scattered in both clades in the tree (Fig. 1), suggesting that type of ITS-2 was subsequently obtained from cloned multiple genotypes of Fasciola in Vietnam are capable of PCR products, which is why each of these worms is infecting humans. We have no information on whether included in only one clade in Fig. 1. the mixing of genetic material of the two Fasciola species All ITS-2 sequences (except one) from populations of in Vietnam is a recent phenomenon. Nor do we know if either species that, because of their geographical origins, ‘‘pure’’ F. hepatica is present in the country. 730 T.H. Le et al. / International Journal for Parasitology 38 (2008) 725–730 In conclusion, we have demonstrated the presence in Fletcher, H.L., Hoey, E.M., Orr, N., Trudgett, A., Fairweather, I., Vietnam of hybrid and/or introgressed populations of liver Robinson, M.W., 2004. The occurrence and signiﬁcance of triploidy in the liver ﬂuke, Fasciola hepatica. Parasitology 128, 69–72. ﬂukes bearing genetic material from both F. hepatica and Huang, W.Y., He, B., Wang, C.R., Zhu, X.Q., 2004. Characterisation of F. gigantica. Some of these worms were from human Fasciola species from mainland China by ITS-2 ribosomal DNA patients. This appears to be the ﬁrst demonstration from sequence. Vet. Parasitol. 120, 75–83. a tropical country of the presence of liver ﬂukes containing Itagaki, T., Kikawa, M., Sakaguchi, K., Shimo, J., Terasaki, K., genetic material from both common species of Fasciola. Shibahara, T., Fukuda, K., 2005a. Genetic characterization of parthenogenetic Fasciola sp. in Japan on the basis of the sequences of ribosomal and mitochondrial DNA. Parasitology 131, 679–685. Acknowledgments Itagaki, T., Kikawa, M., Terasaki, K., Shibahara, T., Fukuda, K., 2005b. Molecular characterization of parthenogenic Fasciola sp. in Korea on This investigation received ﬁnancial support from IC- the basis of DNA sequences of ribosomal ITS1 and mitochondrial GEB (Project No. CRP/VIE05-02 awarded to Thanh NDI gene. J. Vet. Med. Sci. 67, 1115–1118. Le, T.H., Blair, D., McManus, D.P., 2001. Complete DNA sequence and Hoa Le) and the Wellcome Trust (Grant No. 068762 gene organization of the mitochondrial genome of the liverﬂuke, awarded to Le, McManus and Blair). We thank our collab- Fasciola hepatica L. (Platyhelminthes; Trematoda). Parasitology 123, orators for their kind provision of materials used in this 609–621. study and staﬀ in Dr Le’s Laboratory (Institute of Biotech- Le, T.H., De, N.V., Agatsuma, T., Blair, D., Vercruysse, J., Dorny, P., nology, Hanoi, Vietnam) for their laboratory work. Nguyen, T.G.T., McManus, D.P., 2007. Molecular conﬁrmation that Fasciola gigantica can undertake aberrant migrations in human hosts. J. Clin. Microbiol. 45, 648–650. 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