Molecular Characterization of Fasciola Samples Using Sequences of Second Internal Transcribed Spacer-rDNA in Different Geographical Localities of Sistan and Balouchestan Province, Iran

Background: The Fasciola trematodes are the most common liver flukes, living in a range of animals with global distribution and resulting in profound economic loss and public health challenges. Previous studies have indicated that the sequences of the second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA) provide reliable genetic markers for molecular systemic studies of Fasciola. Objectives: The objective of the present study was to characterize Fasciola samples from different geographical regions of Sistan and Balouchestan province using sequences of second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA). Materials and Methods: Twenty adult trematodes were collected from the livers of slaughtered infected cattle. Total genomic DNA was extracted and ITS-2 rDNA targets were amplified by polymerase chain reaction (PCR). All samples were sequenced and investigated using the ClustalW2 sequence alignment tool and MEGA software. The sequences of some Iranian and non-Iranian isolates were used for comparison, in order to evaluate the variation in sequence homology between geographically different trematode populations. Results: The results of comparing the ITS-2 sequences with the BLAST GenBank database showed one type of sequence for F. hepatica and three different types of sequences for F. gigantica in the specimens. Conclusions: The present study demonstrated that Fasciola samples from cattle in two geographical locations in Sistan and Balouchestan province represented no genetic diversity in F. hepatica and high genetic variation in F. gigantica.


Background
The Fasciola trematodes are common liver flukes, living in a range of animals with global distribution and resulting in profound economic loss (1) and public health challenges (2)(3)(4).Human infection of F. hepatica and F. gigantica happens accidentally and occurs most frequently in sheep and cattle raising regions (5).In Iran, as a subtropical country, the distribution of these two species overlaps in most areas (6).In order to find the best understanding of fluke pathogenesis, life cycle and host-parasite relationship, proper identification of Fasciola species and strains is necessary.F. hepatica and F. gigantica can generally be identified based on morphology (6), but use of molecular methods could be helpful to distinguish intermediate types and subspecies.Previous studies have shown that the sequences of the second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA) provide reliable genetic markers for systemic molecular studies of Fasciola (7)(8)(9)(10)(11)(12).

Objectives
Therefore, the objective of the present study was to characterize Fasciola samples from different geographical regions of Sistan and Balouchestan province using sequences of second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA).The present findings provide a foundation for further molecular studies on F. hepatica and F. gigantica in the province and have implications for a better understanding of the disease they cause.

Parasites
The study was carried out in the Zabol and Iranshahr districts of Sistan and Balouchestan province in the southeast of Iran (May 2014 to April 2015).Twenty adult trematodes were collected from the livers of slaughtered native cattle.All fluke specimens were washed several times in physiological saline (37°C) and identified morphologically as Fasciola hepatica and Fasciola gigantica, according to morphological keys and descriptions (13), and stored in 70% ethanol until the extraction of genomic DNA.The sample codes, host species and geographical origins are listed in Table 1.

DNA Extraction, PCR and Sequencing
Total genomic DNA was extracted from individual flukes using a MBST (molecular biological system transfer) DNA purification kit, according to the manufacturer's recommendations.All the DNA samples were stored at -20°C until further use.ITS-2 rDNA plus primer flanking 5.8S and 28S sequences was amplified by polymerase chain reaction (PCR) from genomic DNA using primers (forward: 5'-TCTTGAACGCATATTGCGGC-3') and (reverse: 5'-AGTTCAGCGGGTAATCACGT-3') (14).A thermocycler (eppendorf, mastercycler EP gradient S) was used to amplify 50 μL in 10 mM Tris-HCl, pH 8.4, 50 mM KCl, 2 mM MgCl 2 , 200 μM each of dNTP, 50 pmol of each primer, and 0.25 U Taq polymerase under the following conditions: 95°C for 5 minutes (initial denaturation), followed by 35 cycles at 95°C for 30 seconds (denaturation), 55°C for 30 seconds (annealing), 72°C for 30 seconds (extension), and a final extension at 72°C for 7 minutes (15).One microliter of genomic DNA was used for each PCR reaction.Samples without genomic DNA were included in the PCR reaction as negative controls.An aliquot (5 μL) of each amplicon was examined on 1% agarose gel stained with ethidium bromide and photographed using a gel documentation system (Figure 1).

Sequencing and Data Analysis
All samples were sequenced in both directions by Pishgam biotechnology company.Multiple alignments of the ITS-2 sequences of the present sequences were then used to compare and calculate similarity scores.In this step, ITS-2 sequences of some Fasciola isolates from other parts of Iran and other countries around the world were also included.The ClustalW2 sequence alignment tool was used for all alignments and calculation of similarity scores (16).The phylogenetic trees were constructed based upon the ITS-2 sequences from Sistan and Balouchestan province using the maximum parsimony (MP) and distance methods, namely, neighbor-joining in MEGA 6.0 (17)(18)(19).Branch support was given using 1,000 bootstrap replicates in MEGA (18).

Results
The fragments of amplified DNA were 456 -457 bp long.Next, the ITS-2, PCR products were subjected to direct sequencing and one type of sequence for F. hepatica and three different types of sequences for F. gigantica were obtained.These sequences were deposited in GenBank under accession number (f.h.Zabol1 KT033696, f.g.Zabol KT223394, f.g.Iranshahr 1 KT223395, f.g.Iranshahr 2 KT223396 and f.g.Iranshahr 3 KT223397).The sequences were composed of partial 5.8S sequences of 63 bp, the complete ITS-2 sequences of 362 bp and partial 28S sequence of 75 bp for Fasciola samples.Table 2 lists the nucleotide variation at five variable sequence positions in the ITS-2 of the Fasciolidae species from different geographical regions that have been analyzed in the present study.Also, Table 2 shows the second and third sequences of F. gigantica from Iranshahr (f.g.Iranshahr 2, f.g.Iranshahr 3) that have a variation with F. gigantica from India, China and F. gigantica Iranshahr 1 and other geographical locations of Iran.The sequences that were obtained (ITS-2 rDNA of F. hepatica and F. gigantica) were compared with sequences of other F. hepatica and F. gigantica from Gen-Bank using the ClustalW2 tree building method.In this study, primary sequence analysis revealed a close relationship between the query sequence (F.hepatica from Zabol) and isolates of F. hepatica from China, Ireland, Ecuador, Egypt, Turkey, Austria and other geographical locations in Iran (Zanjan, Shiraz, Gachsaran) and a close relationship between F. gigantica samples and F. gigantica from China and India (Figure 2).The BLAST hit results indicated that our query ITS-2 sequences were identical to the sequences of various geographical isolates of F. hepatica and F. gigantica.

Discussion
Previous studies have characterized Fasciola from different countries and different regions of Iran using genetic approaches (15,(20)(21)(22)(23)(24)(25).However, before the present study, there have been no molecular investigations regarding the Fasciola species from Sistan and Balouchestan province.
Therefore, the objective of the present study was to characterize Fasciola samples from different geographical locations of Sistan and Balouchestan province, using sequences Int J Enteric Pathog.2016;4(1):e33362 of second internal transcribed spacer of ribosomal DNA, because this sequence has been shown to provide specific molecular markers for the identification of Fasciola hepatica and Fasciola gigantica and the intermediate forms (15,26).
The present results confirmed many previous findings around the world that have indicated the ITS-2 target is convenient for detection of the Fasciola species and subspecies (8,12).
F.hepatica and F. gigantica have been detected accurately in all specimens and morphologically intermediate forms.Two Fasciola flukes (11 and 12) were collected from Iranshahr and identified morphologically as F. hepatica, however, comparative sequence analysis specified them as F. gigantica.In the same study in Zanjan, Rahimi et al. (2009) showed that 7% of isolates were F. gigantica, based on the morphometric markers.However, ITS2-RFLP patterns and sequence analysis indicated that they were all identical to the F. hepatica species (14).Based on such overlaps between the two species, which could be observed anywhere in the world, it is suggested that molecular approaches be used for accurate identification (26).
In the present study one type of sequence for F. hepatica and three different types of sequences for F. gigantica have been detected.In Turkey, Erensoy et al. (2009) detected just one sequence for F. hepatica in all studied flukes (5).In addition, Ali et al. (2008) detected one type for F. hepatica and one type for F. gigantica in Niger (12).The variation of genetic types in F. gigantica in the present study is more than in previous studies in other geographical locations and could be due to the entrance of exotic cattle from India and Pakistan to the province, as well as the occurrence of Fasciola contamination in native flocks with exotic strains.
In this study, primary sequence analysis revealed a close relationship between the query sequence (F.hepatica from Zabol) and isolates of F. hepatica from China, Ecuador, Egypt, Turkey, Austria and other geographical location of Iran (Zanjan, Shiraz, Gachsaran).Also, a close relationship between F. gigantica samples and isolates of F. gigantica from China and India has been detected (Figure 2).
The BLAST hit results showed that our query ITS-2 sequences were much more similar to the sequences of various geographical isolates of F. hepatica and F. gigantica, in addition to some Fasciola spp.In conclusion, the present study demonstrated that Fasciola samples from cattle in two geographical locations in Sistan and Balouchestan province represented no genetic diversity in F. hepatica and high genetic variation in F. gigantica.The results of the present study provide the foundation for further studies on F. hepatica and F. gigantica in Sistan and Balouchestan province and have implications for the diagnosis and control of the disease they cause.

Figure 1 .
Figure 1.Agarose Gel Electrophoresis of ITS-2 PCR Products of Representative Fasciola Samples

Table 1 .
Geographical Locations and Host Origins of Fasciola Samples Used in This Study a a Host: cattle.
). Lane 12 represents the negative control.M is a DNA size marker (ordinate values in bp).

Table 2 .
Comparison of Nucleotide Variations at Seven Positions in the ITS-2 Sequences of F.hepatica and F.gigantica from Different Geographical Locations