Molecular Characterization of Fasciola spp. in Egypt on the Basis of certain rDNA fragments and Highly Repetitive DNA Sequences

the Egyptian Society for Biological Sciences ,Department of Entomology ,Faculty of Sciences Ain Shams University . Medical Entomology and Parasitology Journal publishes original research and review papers on all aspects of basic and applied medical entomology, parasitology and host-parasite relationships, including the latest discoveries in parasite biochemistry, molecular biology , genetics, ecology and epidemiology in the content of the biological, medical entomology and veterinary sciences


INTRODUCTION
Fasciola hepatica Linnaeus, 1758 and Fasciola gigantica Cobbold, 1855 (Platyhelminthes: Trematoda: Digenea) are two common species causing fascioliasis in domestic animals and humans.Several studies have revealed that F. hepatica is prevalent in temperate areas, while F. gigantica is widespread in tropical zones, and both species interfere in subtropical areas (Bargues and Mas-Coma, 2005;Mas-Coma et al., 2005;Ashrafi et al., 2006;Mas-Coma et al., 2009).This overlapping distribution of both species has even become the basis of an already long controversy on the taxonomic identity of Fasciola species occurring in Far East countries, especially Japan, Taiwan, the Philippines and Korea.In these countries a wide range of morphological types was detected (Mas-Coma and Bargues, 1997).
These different forms indicated the necessity of applying other techniques, in addition to the morphology as an approach to the taxonomy of Fasciola species.In several earlier studies, the two species and their intermediate forms have been discriminated by sequence analysis of the first (ITS1), the 5.8S, and second Internal Transcribed Spacers (ITS2) of the nuclear ribosomal DNA (rDNA), 28S ribosomal ribonucleic acid (rRNA) (Adlard et al., 1993;Itagaki and Tsutsumi, 1998;Marcilla et al., 2002;Itagaki et al., 2005a;Le et al., 2008;Ichikawa & Itagaki, 2010), 18S rRNA (Karimi, 2008), mitochondrial NADH dehydrogenase I (NDI) and Cytochrome C Oxidase I (COI) genes (Hashimoto et al., 1997;Itagaki et al., 2005b).Molecular analyses of the intermediate forms were mainly done in the Far East Asian countries such as China, Japan, Korea and Vietnam.
These studies have detected individuals that have nuclear DNA related to one species, while their mitochondrial DNA is typical of the other species.In addition, individuals might have nuclear ribosomal genes with divergent copies apparently derived from both Fasciola species (Bandelt et al., 1999;Itagaki et al., 2005;Ashrafi et al., 2006;Semyenovaet al., 2006;Ichikawa et al., 2010).
In Egypt, fascioliasis posed a public health problem as the infections dramatically increased among human populations (Farag et al., 1979;Hassan et al., 1995;El Shazly et al., 2009).It is not exactly known whether Fasciola spp. is belonging to a single or several species or it may be a hybrid of the two species, Lotfy et al. (2002) used morphological, morphoanatomical and morphometric analysis to identify the identity of Fasciola species in Egypt.They also used isoelectrofocusing assay to confirm their findings.However, further studies are needed to provide additional taxonomic criteria for the identification of Fasciola species in Egypt.Lotfy and Hillyer (2003) in their review of Fasciola species in Egypt recommended studying the molecular characterization of Fasciola species in Egypt.Recently, Periago et al. (2008) claimed the existence of F. hepatica/F.gigantica intermediate forms in Egypt utilizing morphometric criteria.Therefore, Amer et al. (2011) molecularly ascertain the nature of Fasciola population derived from different hosts and different geographic locations in Egypt.They used sequence analysis of ribosomal first and second internal transcribed spacer (ITS1 and ITS2) in addition to mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit I (NDI) and cytochrome C oxidase subunit I (COI) as target gene markers.They revealed profound genetic heterogeneity in the Fasciola sp.populations in Egypt and existence of hybrid form.
The present study aimed to use sequence analysis of the ITS2 region of rDNA and a highly repetitive DNA sequences to determine the identity and heterogeneity among Fasciola isolated from buffalo, cow and sheep hosts.

Parasites collection
Three isolates of adult worms of Fasciola spp.were obtained from bile ducts of three Egyptian natural hosts: Buffaloes, cows and sheep.The worms were washed several times in 0.85% NaCl to remove adherent host cells and empty the intestinal caeca, and then stored at -70 °C until used.

DNA Isolation
Genomic DNA was extracted from flukes by the CTAB precipitation technique as described by Yap and Thompson (1987) and Mostafa et al. (2002).Briefly, the adult worms were homogenized individually in 1.5 ml of lysis buffer (8% Triton X-100, 0.25 M sucrose, 50mM Tris-HCl, 50 mM EDTA, pH 7.5).After homogenization, freshly prepared proteinase K (1mg/ml) was added.The homogenate was incubated at 65ºC in water bath for 2 hrs.To precipitate the genomic DNA, 1.0 ml of a sterile 2% CTAB solution was added to the homogenate and centrifuged at 1500 rpm.The supernatant was discarded and the precipitate was dissolved in 0.5 ml of 2.5 M NaCl, 10mM EDTA, pH 7.7 and diluted with 1.0 ml of 40 mM Tris-HCl, 2 mM EDTA, pH 7.7.Two volumes of chloroform-isoamyl was added to the mixture and centrifuged at 12000 rpm for 10 min.The aqueous phase was isolated, and the DNA was precipitated by two volumes of cold absolute ethanol.The DNA pellet was dissolved in 30 µl of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0).

PCR amplification
Fragments spanning 5.8S, ITS-2, and 28S in the rRNA gene region were amplified by PCR using the forward primer, 5-TGTGTCGATGAAGAGCGCAG-3 and the reverse primer, 5-TGGTTAGTTTCTTTTCCTCCGC-3.Amplification reactions were performed in a final volume of 50 l containing 10mM HCl buffer, pH8.3, 25 pmol of each primer, 200M each of dATP, dCTP, dTTP.and dGTP, 2.5 units of Taq DNA polymerase, 100ng Fasciola DNA, 1.5mM-MgCl  and 50 mM-KCl.The PCR protocol required 94C for 0.5 min initially, then for 1.5 min at the start of each cycle, 53C for 2 min, 72C for 2 min and 10 min at the end of all 30 cycles.PCR amplification of highly repetitive sequence PCR reaction was carried out in a total volume of 50µl containing 5µl of 15 mM MgCl2, 1 µl of 10 mM dNTPs, 2.5 µl of 50 pm of the two specific primers (Forward 5´-ATTCACCCATTTCTGTTAGTCC-3´; reverse 5´-ACTAGGCTTAAAGGCGTCC-3´).The design of the primers is based on 124 base-pair (bp) highly repeated sequence of DNA specific for Fasciola spp.(Kaplan et al., 1995 andKramer andSchnieder, 1998).The PCR reaction was performed in DNA thermal cycler (T-personal, Biometra) under the following conditions: 1x94ºc for 3 min.,35x (94ºc for 1 min., denaturing, 65ºc for 1 min.annealing, 72ºc for 1 min.extension); followed by a single incubation at 72ºc for 10 min.

ITS-2 Sequencing
The PCR products were purified using an ultra clean DNA purification kit (Qiagen) according to the manufacturer's protocol.Sequencing reactions were carried out with Big Dye 3.3 terminator cycle sequencing kit (Applied Biosystems, Foster City, A) using the PCR conditions recommended by the manufacturer.Samples were then analysed on 6% polyacrylamide gel on an applied Biosystems model 3100 Automated DNA Sequencing S-ystem at Genetic Engineering research Center, Ain Shams University-Cairo-Egypt.
Beside ITS region, the smallest band of the highly repetitive sequence of amplified products (approximately 124bp) were sequenced using a Big Dye terminator cycle sequencing kit (Applied Bio System, Foster City, A).Sequencing products were purified by using Centri-Sep Spin columns (Princeton separations, Adelphia, NJ) and were resolved on an applied Biosystems model 3100 automated DNA sequencing system at Vacsera Authority.Phylogenetic Analysis NCBI (National Center for Biotechnology Information) databases of Fasciola spp.Collected from various localities were used for sequence similarity analysis.Sequence of Fasciolopsis buski obtained from Gen Bank data base was used as outgroup.
The maximum likelihood tree was built using RAxML version 7.2.8 algorithm (Stamatakis et al., 2005) as proposed on the Black Box portal (http://phylobench.vitalit.ch/raxml-bb/)using the GTR+Г+I model.Model parameters were estimated in RAxML over the duration of the tree search.Two simultaneous MCMC chains were performed, and 500,000 generations.The generations were added until the standard deviation of split frequencies fell below 0.01 according to the manual of Mr Bayes 3.1.For every 100 th generation.The tree with the best likelihood score was saved, resulting in 5000 trees.The burn in value was set to 25%.Trees were viewed using Fig Tree (a program distributed as part of the BEAST package (http://tree.bio.ed.ac.uk/software/figtree/).
The divergences between sequences were calculated using the program BioEdit software (Hall, 1999).

Structure of the ITS2 gene
A completeITS-2, a part of 5.8S and a part of 28S gene fragment were amplified from each sample and expected to be approximately 500-538 bp in length.The sequenced ITS2 fragment was ca.525 bp for Fasciola sp.isolated from sheep, (accession number in Gen Bank: DQ385828), 509 bp for Fasciola sp.isolated from buffaloes (accession number DQ383512) and 538 bp for Fasciola spp.Isolated from cows (accession number DQ385829).
The ITS2 coding region was subjected to direct sequencing giving products approximately 343 bp.The average GC contents were nearly 48-50% in the three isolates.Few gaps were inserted in the most variable areas of the alignment.As demonstrated in Table (1), among the three samples under study, two individuals possessed ITS-2 sequences identical to that of pure F. gigantica.(isolate of cow & buffalo) On the other hand, Fasciola isolate of sheep had a different sequence variation in many sites from both F. hepatica and F. gigantica (Table 1).Also, many sites substitutions can be noticed in Egyptian Fasciola (cow and buffaloes isolates) as compared with F. gigantica and F. hepatica of different countries.Whereas, in sheep isolates there are many substitution sites that are different from all compared sequences.

Phylogenetic position of the studied species
The topologies of phylogenetic trees inferred from maximum likelihood and Bayesian inference were identical (Fig. 1).The tree revealed the existence of three main clades: A, B and C. Clade Acomprises sequences of F. gigantica and it receives the maximum support value (BS =100% /1.00=PP), clade B comprises representatives of F. Hepatica and it receivesa high support value (BS=75%/1.00=PP),while clad C comprises sequences of Fascioledes magna and F. jaksoni and it receives BS=85%/1.00=PP.
Our obtained partial ITS2 gene sequences fall within clade A, the obtained sequences of Fasciola isolates of buffalo and cow receive high support value (BS=100%/1.00=PP),while the obtained sequence of Fasciola obtained from sheep received weak support value.The phylogenetic tree revealed that F. gigantica has a high genetic diversity in comparison to F. hepatica.The intra-clade genetic variability within F. gignatica group (A) ranged between 0.3%-2%, while in F. hepatica group (B) is less than 0.7%.The phylogenetic tree also suggests a possible pattern of geographical origin with F. gigantica group.Concerning the results of the highly repeated fragments of DNA in the three isolates, the size of the smallest one is ~ 114 bp.Also the purified fragments were visualized on the agarose gel electrophoresis (Fig. 2).No band variations were noticed among the three isolates of F. gigantica.The smallest fragment was subjected to direct sequencing, as described in Fig. (3).The obtained results revealed sequence variations among the three isolates and other Fasciola of various countries with a percentage of 90%.Several previous studies have investigated that these sequences can give reliable genetic markers for the accurate identities of Fasciola spp.(Itagaki and Tsutsumi, 1998;Agatsuma et al., 2000;Huang et al., 2004;Itagaki et al., 2005Itagaki et al., a,b 2009;;Mas-Coma et al., 2009).The results of the present work confirmed that F. gigantica is the main species exists in Egypt, in addition to an intermediate form of Fasciola found in sheep.This disagree with the results of Amer et al. (2011) who reported that F. hepatica was more dominant in sheep than in other hosts.
Earlier studies recorded the presence of other species of Fasciola in African countries based on the phylogenetic position and interspecific variation, using ITS2 sequences (Adlard et al., 1993;Hashimoto et al., 1997;Itagaki and Tsutsumi, 1998;Itagaki et al., 2005 andSemyenova et al., 2005).
Differentiation of the ITS2 gene among populations and species has been reported to be dependent upon many factors, including genetic drift, the relative number and size of repeats, rates of unequal crossover, gene conserving, immigration, and the number of loci and now biogeographical distribution (Levinson and Gutman, 1987).In the present study, the nucleotide sequences of ITS2 were determined in three worms of Fasciola recovered from Egyptian livestock (buffaloes, cows and sheep).
Nowadays the genetic diversity of Egyptian Fasciola species is underestimated.Previously, F. gigantica was the only species reported in animals.When human fascioliasis emerged, it was reported to be due to F. hepatica and importation of infected animals was considered the source of propagation of infection (Lotfy et al., 2002).However, the absence of Lymnaea truncatula in an area endemic for human fascioliasis, posed a question about the possibility of local transmission.
In the present study, three isolates of the Egyptian Fasciola were characterized using well-defined ITS-2 sequence which emphasize the suggestions of earlier investigators that this rDNA sequence provides reliable genetic markers for the accurate differentiation and identification of Fasciola species.Previously published data (Adlard et al., 1993;Hashimoto et al., 1997;Itagaki and Tsutsumi, 1998;Agatsuma et al., 2000), demonstrated that Fasciola from buffalo from Guangxi represented F. gigantica.The ITS-2 sequences of Fasciola from sheep and cows were quite different, where ITS-2 sequences of Fasciola species from Koyoto has not been identified as F. hepatica nor F. gigantica, so the isolates of Fasciola from cows and sheep could represent an intermediate genotype or even a "hybrid" between F. hepatica and F. gigantica.
So far, for the best of our knowledge, this is the first time that molecular evidence have suggested the possible existence of an "intermediate genotype" of Fasciola in Egypt, in addition to F. hepatica and F. gigantica.This was confirmed by Agatsuma et al. (2000) who suggested the existence of natural hybridization between F. hepatica and F. gigantica, co-existing.This is the case of Fasciola spp., in Japan and Korea and perhaps, in Egypt, where F. hepatica and F. gigantica overlap.In Japan and Korea a triploid Fasciola spp.has been the subject of intense study to define its speciation.In a series of articles, Itagaki and collaborators (1998) showed the complexity of this issue.First, they compared the mitochondrial NADH dehydrogenase subunit 1 (ND1) and cytochrome oxidase subunit 1 (CO1) gene sequences of F. hepatica (Uruguay), F. gigantica (Zambia) and the triploid form of Japanese Fasciola spp.They found that intraspecific variation in the sequences was generally low, but the sequences of the Japanese triploid form were identical to those found in F. gigantica but were different from those of F. hepatica.This leads to the conclusion that Japanese Fasciola can be categorized as F. gigantica.
Second, they examined the ITS2 sequence of the nuclear RNA using the fluke population described above and concluded that the Japanese triploid worms were divided into two distinct types, one of which was almost identical to F. hepatica and the other to F. gigantica (Itagaki and Tsutsumi, 1998).Third, Itagaki et al. (2001) examined the mitochondrial DNA polymorphism detected by restriction fragment length polymorphism (RFLP) using 8 restriction enzymes and detected 3 different types in 76 Fasciola flukes studied.
The authors concluded that the Fasciola Japanese triploids are composed of two lineages having a genetic structure identical to F. hepatica and F. gigantica and hybrids between the two species.Whether a similar situation is occurring in the Nile delta of Egypt remains to be ascertained.Further investigations are warranted to clarify the phylogenetic position of Egyptian Fasciola spp.using other molecular markers.
Previous studies documented a high level of genetic diversity of Egyptian Fasciola at both NDI and COI (Walker et al., 2007;Peng et al., 2009).In the present study it was observed that Egyptian F. gigantic has a separate position on both trees which supported by high bootstrap values reflecting the prominent nucleotide differences from homologous sequences in data base especially those isolated from sheep.As stated by Constantine (2003) and Criscione et al. (2005), these differences may be attributed to the host range, long persistence of infection in the country.Moreover, regular import strategies of cattle from different countries may have an impact on the stability and consistency of the parasite lineages.As the molecular variation may reflect differences in virulence, host specificity and drug susceptibility/resistance population genetics of Fasciola parasites in Egypt needs close surveillance In conclusion, the present study revealed profound genetic heterogeneity in the Fasciola spp.populations in Egypt and provided the first molecular clue on existence of hybrid form.The results indicated that hybridization might be an active process in liver flukes due to the mixed infection.

Fig. 1 :
Fig.1: The phylogenetic relationships of the Fasciola spp.collected from buffalo, cow and sheep in Egypt and other representative isolates (F.gigantica, F. hepatica and Fasciola spp.) in different localities in the world based ITS2 region.

Table 1 :
Comparison of ITS-2 variable sites in different Fasciola isolates.