Molecular identification of Fasciola spp. (Digenea: Fasciolidae) in Egypt

A total of 134 Egyptian liver flukes were collected from different definitive hosts (cattle, sheep, and buffaloes) to identify them via the use of PCR-RFLP and sequence analysis of the first nuclear ribosomal internal transcribed spacer (ITS1). Specimens of F. hepatica from France, as well as F. gigantica from Cameroon were included in the study for comparison. PCR products of ITS1 were subjected for digestion by RsaI restriction enzyme and visualized on agarose gel. According to RFLP pattern, Egyptian flukes were allocated into two categories. The first was identical to that of French hepatica flukes to have a pattern of 360, 100, and 60 (bp) band size, whereas the second resembled to that of Cameroonian gigantica worms to have a profile of 360, 170, and 60 bp in size. Results of RFLP analysis were confirmed by sequence analysis of representative ITS1 amplicons. No hybrid forms were detected in the present study. Taken together, this study concluded that both species of Fasciola are present in Egypt, whereas the hybrid form may be not very common.

T he species of genus Fasciola (Platyhelmintha: Digenea) are the common liver flukes of a wide range of animals (Mas-Coma et al., 2005). Severe negative economic impact due to fascioliasis has been estimated in ruminants (Urquhart et al., 1996;Spithill & Dalton, 1998). In addition, human fascioliasis represents a significant health problem, as the estimated number of infected people is between 2.4 and 17 million and the number of those at the risk is more than 180 million throughout the world (World Health Organization, 1995;Mas-Coma et al., 1999;Ishii et al., 2002;Mas-Coma et al., 2009). Although, liver flukes have a global geographical distribution, the disease probably exerts most of its impact on developing countries. In Africa, Fasciola infection has been recognized as a major constraint for public health and animal farm industry (Mekroud et al., 2006;Pfukenyi et al., 2006;Phiri et al., 2007). In Egypt, fascioliasis has serious veterinary and medical impacts, as it provokes the mortality of livestock and affects people at all ages (Haseeb et al., 2002;Curtale et al., 2003;Esteban et al., 2003) (Periago et al., 2008) and molecularly based on ribosomal and mitochondrial gene markers (Amer et al., 2011). Latter findings suggested a possible hybridization between these two species of Fasciola, as demonstrated in Japanese, Korean, Chinese and Vietnamese flukes (Itagaki & Tsutsumi, 1998;Agatsuma et al., 2000;Lin et al., 2007;Itagaki et al., 2009). Such hybridization of both fluke species may result in messing up chromosomal numbers with the appearance of diploid and triploid Research note Parasite, 2012, 19, 177-182 forms with spermic and aspermic (with no or a few spermatozoa in the seminal vesicle) features. The present study aimed to spot light on speciation of Fasciola population in Egypt. Fasciola spp. collected from different hosts and localities in Egypt were identified by PCR-RFLP based on ITS1 fragment using RsaI restriction endonuclease. Results of RFLP analysis were confirmed by sequence analysis of representative ITS1 amplicons.

MATERIALS AND METHODS
A total of 134 adult Fasciola spp. (Table I)

DNA EXTRACTION AND PCR-RFLP ANALYSIS
Genomic DNA was extracted from the adult flukes using QIAamp DNA Mini Kits (Qiagen, USA) following manufacturer's recommendations. The ITS1 fragment was amplified by PCR (Itagaki et al., 2005), using a set of 5'-TTGCGCTGATTACGTCCCTG-3' and 5'-TTG-GCTGCGCTCTTCATCGAC-3' as forward and reverse primers, respectively. The reaction was done in a total volume of 25 μl containing of 12.5 μl Qiagen Multiplex, 1 μl of each primer (0.3 μM), 1 μl genomic DNA, and 9.5 μl H 2 O. Reaction cycles consisted of an initial denaturation step at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 90 sec, annealing at 53 °C for 30 sec and extension at 72 °C for 60 sec, followed by a final extension step at 72 °C for 10 min. RFLP reaction was performed using RsaI restriction enzyme as described by Ichikawa and Itagaki (2010).
The digested fragments were electrophoresed on 2 % agarose gel and visualized by ethidium bromide using GELSMART 7.0 software (Clara Vision). The size of each band was determined by a 100-bp plus ladder molecular weight marker.

ITS1 SEQUENCING
For confirmation of RFLP reaction, ITS1 amplicons of representative samples of Egyptian as well as French and Cameroonian flukes were subjected to sequence analysis. PCR products of ITS1 were purified using rapid PCR purification systems (Marligen Bioscience, Inc., USA) according to manufacturer's instructions. The sequencing reaction was performed utilizing the same PCR primers using ABI Big Dye kit on 3130xl genetic analyzer (Applied Biosystems, France). Accuracy of data was confirmed by two-directional sequencing. Representative sequences were deposited in the GenBank under the accession numbers of JF294998, JF294999, JF295000, and JF295001.

P CR amplification of ITS1 resulted in a fragment
size of 680 bp (Fig. 1). The products were subsequently subjected to digestion by RsaI restriction enzyme. Electrophoresis of the digested products revealed to two different bands patterns (Fig.  2), regardless of their geographical origins. The first pattern composed of three bands of 360, 100, and 60 bp in size, whereas the second was 360, 170, and 60 bp in size. Alignment of obtained sequences revealed that the members of the first pattern including French flukes belonged to F. hepatica type, whereas those of the second pattern including Cameroonian flukes was belonging to F. gigantica type (Fig. 3)

DISCUSSION
P CR-RFLP based on ITS1 is a reliable tool to differentiate F. hepatica from F. gigantica (Ichikawa & Itagaki, 2010;Rokni et al., 2010). In the present study, results showed the presence of two different ITS1-RFLP patterns corresponding to F. hepatica (360, 100, and 60 bp band size) and F. gigantica (360, 170, and 60 bp band size) as confirmed by PCR-sequence analysis. These results come in full agreement with those described by Ichikawa & Itagaki (2010) for Fasciola from several geographical regions. Although nucleotide composition reveals five (hepatica) and four (gigantica) restriction sites in the affiliated sequences, only three bands in each case were recognized. The band of 60 bp was thought to include the three fragments of 68 bp (only F. hepatica), 59 and 54 bp, and the small fragment of 28 bp was not detected on agarose gels (Ichikawa & Itagaki, 2010). Although the intermediate form of Fasciola sp.  Lanes 1-5 denote to those of Fasciola gigantica isolated from cattle at Yaoundé, Cameron (1) and Cairo, Egypt (2, 3); as well as from sheep at Cairo, Egypt (4, 5), respectively. Lanes 6-10 denote to those of Fasciola hepatica isolated from sheep and cattle (6, 7) at Cairo, Egypt, and sheep and cattle (8,9) at Tanta, Egypt, respectively, as well as from cattle at Limoges, France (10). 680 bp ➝ 360 bp ➝ 170 bp ➝ 100 bp ➝ 60 bp ➝ has been reported in Egypt (Amer et al., 2011), the present study did not detect the discriminating pattern (360, 170, 100, and 60 bp band size as described by Ichikawa and Itagaki, 2010) by RFLP reaction nor by sequence analysis. This may indicate that the hybrid form of Fasciola is not very common in Egypt and could be detected on sporadic bases. Of note, several PCR-RFLP techniques were developed to differentiate between F. hepatica and F. gigantica as described by El-Gozamy and Shoukry (2009) using 18S rRNA, Marcilla et al. (2002) using 28S rRNA and Rokni et al. (2010 using ITS1 utilizing different array of endonucleases. Although nucleotide sequence analysis was consistent with the results of RFLP reaction, Egyptian F. gigantica proved heterogeneity at position 116 compared to Cameroonian gigantica flukes. Such heterogeneity was reported in Egyptian flukes (Amer et al., 2011). Moreover, nucleotide variability was reported in Fasciola flukes (Vara-Del Río et al., 2007, Walker et al., 2011 based on sequence of different genetic markers. The percentage of F. hepatica (69.4 %) noted among the slaughtered animals in the present study gave an idea about the adaptation of this fluke to the local environment in Egypt. Occurrence of this digenean, which may have be introduced into this country from Europe through imported infected animals (Lofty et al., 2002;Mas-Coma et al., 2005), was supported by a recent report suggesting the role of Radix natalensis (the principal snail host of F. gigantica) as a potential intermediate host for F. hepatica (Dar et al., 2010). The other fasciolid, F. gigantica present in 30.7 % in the analyzed samples, was considered as an indigenous species of Fasciola found in Egypt (Farag, 1998), as it was recorded in nearly all governorates, especially those of the Nile Delta in Lower Egypt (Lotfy & Hillyer, 2003;Dar et al., 2005). The restriction sites of RsaI endonuclease are underlined and shaded in gray. The nucleotide differences between the two Fasciola species are denoted in bold. Dots indicate that the nucleotides are identical to those in the upper line. 'Y' represents a nucleotide indicating the presence of a double peak, C and T. (Irl), GenBank accession number: AB514850 for Fasciola hepatica from Ireland. (Zam), GenBank accession number: AB514855 for Fasciola gigantica from Zambia. Underline denote to primer position.