Introduction

Digenean trematodes of the genus Fasciola (Platyhelminthes: Trematoda: Digenea) are the common liver flukes of a range of animals with a global geographical distribution (Spithill and Dalton 1998). Fasciolosis caused by Fasciola spp. is a significant animal health problem, which causes substantial economic losses estimated at US$2 billion per annum worldwide (Spithill and Dalton 1998). Human infection with Fasciola spp. has been reported in a number of countries, millions of people are estimated to be infected, and hundreds of millions of people are at risk throughout the world (Mas-Coma et al. 1999, 2005; Haseeb et al. 2002; Ishii et al. 2002).

Several species have been described within the genus Fasciola, but only two species, Fasciola hepatica and Fasciola gigantica, are commonly recognized as taxonomically valid occurring in animals and humans (Yamaguti 1958; Mas-Coma et al. 2005), with F. hepatica mainly occurring in temperate areas, F. gigantica in tropical zones, and both overlapping in subtropical areas (Krämer and Schnieder 1998; Mas-Coma et al. 2005). In addition to F. hepatica and F. gigantica, several recent studies using the first and/or second internal transcribed spacers (ITS-1 and ITS-2) of ribosomal DNA (rDNA) as genetic markers have identified a so-called “intermediate Fasciola” between F. hepatica and F. gigantica from Japan, Korea, and China (Itagaki and Tsutsumi 1998; Agatsuma et al. 2000; Huang et al. 2004; Itagaki et al. 2005a, b; Lin et al. 2007).

In the Iberian Peninsula, F. hepatica has been found parasitizing domestic animals and wild animals, including European rabbit (Oryctolagus cuniculus), hare (Lepus granatensis), donkey (Equus asinus), horse (Equus caballus), pig and wild boar (Sus scrofa), fallow deer (Dama dama), cattle (Bos taurus), domestic sheep (Ovis aries), goat (Capra hircus), Iberian ibex (Capra pyrenaica), and man (Cordero del Campillo et al. 1994; Lavín et al. 1995; Pérez et al. 2006). In addition to F. hepatica, F. gigantica was reported to infect sheep in central Spain and Portugal (Cordero del Campillo et al. 1994).

However, before the present study, there had been no reports characterizing Fasciola from Spain using well-defined DNA sequences. Therefore, the objective of the present study was to characterize Fasciola samples from Spain from different host animals and geographical localities by sequences of the ITS-1 and ITS-2 rDNA because these sequences have been shown to provide specific markers for the identification of F. hepatica, F. gigantica and the “intermediate Fasciola”.

Materials and methods

Parasites

Adult trematodes were collected from the livers of infected hosts (all of them were local animals) at necropsy from nine host animal species and 19 geographical locations in Spain between 2002 and 2006. Individual worms were washed extensively in physiological saline, identified morphologically as Fasciola according to existing keys and descriptions (Yamaguti 1958), and fixed in 70% ethanol until extraction of genomic DNA. Their codes, host species, and geographical origins are listed in Table 1 and Fig. 1.

Table 1 Geographical locations in Spain and host origins of Fasciola samples used in this study
Full size table
Fig. 1
figure 1

Spain map showing approximate sites for sample collection. Codes next to the sites represent the sample codes in Table 1

Full size image

Extraction and purification of genomic DNA

Genomic DNA was extracted from a portion of individual adult trematodes by treatment with sodium dodecyl sulphate/proteinase K (Zhu et al. 2002), column-purified using Wizard™ DNA clean-up system (Promega) and then eluted into 65 μl H2O according to the manufacturer’s recommendations. DNA was also isolated from livers from cattle, buffalo or goat using the same method as for trematode samples. All the DNA samples were stored at −20°C until further use.

Enzymatic amplification of the ITS-1, 5.8S rDNA, and ITS-2

The DNA region comprising ITS-1, 5.8S rDNA, and ITS-2 plus primer flanking sequences (ITS+) was amplified by polymerase chain reaction (PCR) from trematode DNA using primers BD1 (forward; 5′-GTCGTAACAAGGTTTCCGTA-3′) and BD2 (reverse; 5′-TATGCTTAAATTCAGCGGGT-3′; Luton et al. 1992). PCR reactions (25 μl) were performed in 10 mM Tris–HCl, pH 8.4, 50 mM KCl, 2 mM MgCl2, 200 μM each of dNTP, 50 pmol of each primer and 0.25 U Taq polymerase (TaKaRa) in a thermocycler (Biometra) under the following conditions: 95°C for 5 min (initial denaturation) followed by 30 cycles of 95°C, 30 s (denaturation), 55°C, 30 s (annealing), 72°C, 30 s (extension), and a final extension of 72°C for 7 min. One microliter of genomic DNA was added to each PCR reaction. Samples with host DNA or without genomic DNA were included in each amplification run as “negative” controls. An aliquot (5 μl) of each amplicon was examined on 1% agarose-TBE (65 mM Tris–HCl, 22.5 mM boric acid, and 1.25 mM ethylenediamine tetraacetic acid, pH 9.0) gels, stained with ethidium bromide, and photographed using a gel documentation system (UVITEC). The DNA size marker DL2000 was used to estimate the length of the ITS+ amplicons.

Sequencing of the ITS-1, 5.8S and ITS-2 rDNA and analysis

ITS+ products of 25 samples were sent to BioSune Biotechnology Company for sequencing using ABI 377 automated DNA sequencer (BioDye Terminator Chemistry) from both directions using the same primers as used in primary amplification. The 5′ and 3′ ends of the Fasciola ITS-1, 5.8S and ITS-2 sequences were determined by comparison with previously published Fasciola ITS-1, 5.8S and ITS-2 sequences (Itagaki and Tsutsumi 1998; Agatsuma et al. 2000; Huang et al. 2004; Itagaki et al. 2005a, b; Lin et al. 2007; also see GenBank™ accession numbers AJ557567, AJ557569, AJ557570, AJ557571, AJ628430, AJ628431, and AJ628043). The sequences were aligned and compared among themselves and with those of F. hepatica, F. gigantica and the “intermediate Fasciola”. Pairwise comparisons were made of the level of sequence differences (D) using the formula D = 1 − (M/L; Chilton et al. 1995), where M is the number of alignment positions at which the two sequences have a base in common and L is the total number of alignment positions over which the two sequences are compared.

Results and discussion

Genomic DNA was isolated from 25 individuals of Fasciola representing nine different host species and 19 geographical locations in Spain (Table 1, Fig. 1). The ITS+ fragment amplified from each sample using primers BD1 and BD2 was approximately 1,000 bp in length, and in no case was product amplified from no-DNA sample or host DNA control. Figure 2 shows the ITS+ fragment amplified from representative Fasciola samples.

Fig. 2
figure 2

Agarose gel electrophoresis of ITS+ PCR products of representative Fasciola samples from Spain. Lanes 1–9 represent Fasciola samples from Iberian ibex, Chamois, Roe deer, Fallow deer, Red deer, European mouflon, Horse, Ovine, and Bovine, respectively (cf. Table 1). Lanes 10 and 11 represent host (bovine) and no DNA control, respectively. M represents a DNA size marker (ordinate values in bp)

Full size image

Then, the 25 ITS+ PCR products were subjected to direct sequencing; 25 sequences of 946 bp were obtained and deposited in the GenBank™ (accession numbers AM709498-AM709500, AM707030, AM709609-AM709622, AM709643-AM709649; also see Table 2). The sequence was composed of the complete ITS-1 sequence of 422 bp, complete 5.8S sequence of 162 bp, and complete ITS-2 sequence of 362 bp. While there was no nucleotide variation in the ITS-1 and 5.8S rDNA among the 25 Fasciola samples, two different ITS-2 sequences were defined for the examined Spanish Fasciola samples, differing at one nucleotide (0.3%, 1/362) in the ITS-2 (sequence position 868, Table 2), indicating the existence of two genotypes among the examined Spanish Fasciola samples. However, it appears that this sequence variation was not related to particular host species and/or geographical origins of the samples because it occurred in the examined Fasciola samples from different host species and geographical locations in Spain (Tables 1 and 2).

Table 2 Comparison of the ITS-1 and ITS-2 sequences of Fasciola from different hosts and geographical locations in Spain with that of F. hepatica, F. gigantica and the “intermediate Fasciola” at variable sequence positions
Full size table

Comparison of the ITS sequences of the Spanish Fasciola samples examined in the present study with those of F. hepatica, F. gigantica and the “intermediate Fasciola” revealed that all Spanish Fasciola samples examined represent the single species of F. hepatica (see Table 2). It is interesting to find that the examined Spanish F. hepatica differed from Fasciola from elsewhere by two nucleotides in the ITS-2 (sequence positions 925 and 926, Table 2), which provided genetic marker for the differentiation of Spanish F. hepatica from Fasciola from other geographical localities.

In the present study, 25 Fasciola samples from nine host species and 19 geographical locations in Spain were sequenced, and no sequence variation was detected either in their ITS-1 or 5.8S rDNA, while their ITS-2 sequences differed only by one nucleotide (0.3%). The explanation for such a low level of intraspecific variation seems to be that repeated DNA sequences, such as rDNA, have been subject to concerted evolution, which tends to homogenize sequences among individuals and among populations (Dowling et al. 1990). This tendency increases the discriminating power of repeated sequences at the species level by reducing the incidence of intraspecific sequence divergence.

In conclusion, the present study demonstrated that the 25 liver fluke samples from nine host species and 19 geographical locations in Spain represented the single species of F. hepatica, and two different ITS-2 sequence types were defined and that F. hepatica from Spain differed from Fasciola from elsewhere by the two nucleotides in the ITS-2. Further studies using more variable genetic markers, such as the sequence-related amplification polymorphism (Li and Quiros 2001), are warranted to examine the genetic variability and population genetic structure within F. hepatica from different hosts and geographical locations in Spain.