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Genotyping and Phylogenetic Analysis of Fasciola Spp. Isolated from Sheep and Cattle Using PCR-RFLP in Ardabil Province, Northwestern Iran.

Abstract Background The aim of this study was to detect the genotype of Fasciola spp. in Meshkin-Shahr, Ardabil Province, northwestern Iran in different hosts using PCR-RFLP. Methods The parasite hosts included cattle, and sheep. Overall, 70 adult flukes from livers of slaughtered animals were collected from the abattoirs of aforementioned area. The included 35 samples from infected sheep and 35 samples from 35 infected cattle. PCR-RFLP and sequence analysis of the first nuclear ribosomal internal transcribed spacer (ITS 1) region from Fasciola species were used to conduct the study. Results The fragment of approximately 700bp in all of the Fasciola samples was amplified. PCR products of ITS 1 were subjected for digestion by restriction enzyme. RsaI restriction enzyme was selected for RFLP method that caused the separation specifically of Fasciola species. Amplicons with the sequences of F. hepatica had a pattern of about 360, 100, and 60 bp band size, whereas F. gigantica worms had a profile of 360, 170, and 60 bp in size, respectively. Results based on PCR-RFLP analysis were confirmed by sequence analysis of representative ITS 1 amplicons. No hybrid forms were detected in the present study. All sheep were infected with F. hepatica but cattle were infected with both species. Conclusion Both species of Fasciola are present in Ardabil. The method described here can be valuable for identification of Fasciola species in endemic parts for fasciolosis, regions with intermediate species and in that overlapping distribution area.


Introduction
Fascioliasis, caused by Fasciola spp., is one of the most zoonotic diseases with a global extension and of a significant concern in areas with suitable situations (1)." About 2.4-17 million cases of human fascioliasis are estimated in the world and 180 million exposed at risk of fascioliasis that demonstrates the importance of the disease (2)".
Two great epidemics of human fascioliasis with about 10,000 people infected in each case occurred in the north of Iran (3)(4)(5). According to WHO, fascioliasis is one of the most important diseases and parasitic infections transmitted by food (6). Considering the importance of fascioliasis in Iran, various studies especially in order to identify and genotyping of Fasciola seems indispensable. Morphological features of adult worms and eggs are influenced by the different factors such as host type, age of parasite, fixation of the samples and intensity of the infection (7(.Distribution overlap of F. hepatica and F. gigantica in Asia and simultaneous infection with these two parasites in cattle in northern Iran and bug reporting interface is also introduced in other provinces of Iran (8). It is usually difficult to accurate differentiation between Fasciola species based on phenotype criteria (9)(10)(11). Today, molecular methods based on DNA analysis is a great impact in many fields, including identifying systematic parasitology, parasite infection, parasite epidemic, analysis of the genetic structure of parasite populations, such as genetic differences between the sexes, gene expression and drug resistance of and the vaccine.Therefore, it seems that the use of the accurate and reliable method for identification and differentiation of Fasciola species is necessary. For this purpose, PCR-RFLP based on 28s rRNA, 18s rRNA, ITS 1 or ITS 2 has been used (12)(13). In the present study, region between 18S and 28S (ITS 1, 5.8s,) of ribosomal DNA was used by PCR-RFLP method. Since, ITS 1 sequence was suitable genetic markers for genotyping, interaspecific variations, and phylogenetic studies of parasites (14); we used only ITS 1 for molecular identification of Fasciola species.The adjacent of Ardabil Province to endemic areas of fasciolosis persuaded us to conduct the present study (15).

Parasite
Overall, 70 Fasciola isolates were studied from two hosts in Ardabil Province, northwestern Iran (Fig.  1). These parasites were isolated from 35 infected sheep and 35 infected cattle. Individual worms were washed extensively in PBS buffer (37°C) and subsequently fixed in 20% ethanol and maintained at room temperature for several weeks until extraction of genomic DNA (Table 1).

DNA extraction
Approximately 10 mg tissue was removed from a portion of the lateral zone of adult flukes and crushed. The ethanol in each of these samples was allowed to evaporate for a few minutes and washed in distilled water for 3 times and squashing before the genomic DNA was extracted with (DNG TM -PLUS) kit was used for genomic DNA extraction of Fasciola Parasites. The extracted DNA, split into aliquots, and stored at -20°C until used in the PCR.

PCR
To amplify a 700 bp region of the ITS 1 sequence, PCR was performed using a pair primer. Total volume of reaction was 15µl containing 1.5µl DNA template, 5µl distilled water, 10pmol of each primers (Forward and Reverse), and 7.5µl master mix (amplicon). PCR amplification was performed in Eppendorf Mastercycler Gradient thermocycler . The temperature profile was as follows: an initiation of 95ºC for 5 min, followed by 30 cycles of 94ºC for 30s (denaturation), 60ºC for 30s (annealing), 72ºC for 30s (extension) and a final extension of 72ºC for 5min followed by cooling at 4ºC.
PCR products were electrophoresed on 1.5% (for ITS 1 PCR) agarose gel in TAE buffer at 60 V for 40 min and visualized by UV illumination (UVITEC) after ethidium bromide staining. To estimate the size of the amplicons, a 100bp DNA ladder (Fermentas) was used in gels.

Sequencing and analysis
To select a suitable restriction enzyme we sequenced PCR products of one Fasciola samples from sheep and one Fasciola samples from cattle. The sequences were analyzed and aligned using Blast (http://blast.ncbi.nlm.nih.gov/Blast) and multalin (http://multalin.toulose.inra.fr/ multalin ) software in comparison with sequences of Fasciola ITS 1, previously published from other countries in GenBank. Nucleotides of 700bp from fasciolid species were subjected to comparison of restriction sites and selection the appropriate enzyme by Webcutter 2 using the following website: (http://bio.lundberg.gu.se/cutter2) software.

Restriction Fragment Length Polymorphism (RFLP)
Rsa I enzyme (Fermentas) was selected for RFLP method that caused the separation specifically of Fasciola species. To performance RFLP, 5 µl of Fasciola ITS 1 PCR product, 2.5 µl of supplied restriction enzyme buffer, 5 µl of restriction enzyme diluted, and D.W up to 25 µl were provided. According to the manufacturer instruction, the tubes were incubated at 37 °C for 7 h, to ensure full cutting of fragments. For analyzing the digestion products, 15 μl of each product in addition to 2 μl of loading buffer were electrophoresed on either 3% agarose gel in TAE buffer at 60 V for 40 min and visualized by UV illumination (UVITEC) af-ter ethidium bromide staining. To estimate the size of the separation specifically of Fasciola species, a 100bp DNA ladder (Fermentas) was used in gels.

DNA Sequencing and Phylogenic Analysis
PCR products of ITS 1, of 20 isolates from two hosts (cattle and sheep) were sequenced .DNA Analyzers Sequencing, using the same primers which were used in the PCR.The sequences were aligned and compared with those of existing sequences from the region, related to Fasciola spp. available in the GenBank, using the BLASTprogram of NCBI GenBank. Multiple alignments were performed with data related to Fasciola spp. from Iran and other countries deposited in Gen-Bank. A maximum likelihood tree was constructed using Blast (http://blast.ncbi.nlm.nih.gov/Blast).

Results
Genomic DNA was extracted from 70 isolates, which could amplify a fragment approximately 700bp in all of the samples (Fig. 2). Negative control did not produce any band on the gels.All amplified products of Fasciola were digested with the Rsa I restriction enzyme. The band patterns of the fragments digested with Rsa I were accurately distinguished among the two forms of Fasciola.

PCR-RFLP Analysis
Based on RFLP pattern, from 70 Fasciola isolates, 4 isolates (11.4%) from cattle had a RFLP pattern corresponding to F. hepatica, 31 isolates (88.6%) from cattle had a RFLP pattern corresponding to F. gigantica.35 isolates (100%) from sheep had a RFLP pattern corresponding to F. hepatica. RFLP pattern from F. hepatica, which had 5 cutting sites, were predicted to be separated into fragments of 367, 104, 68,59, 54, and 28 bp in the amplicons . while 5 fragments were produced by 4 cutting sites from F. gigantica including 367, 172, 59, 54, and 28 bp in the amplicons (Fig. 3, 4). 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. These findings showed that the band patterns between the amplicons with the sequences of F. hepatica and F. gigantica were evidently different.  The sequences of 700 bp ITS 1 of the flukes were analyzed and aligned with those of available sequences in GenBank using multalin (http://multalin.toulose.inra.fr/ multalin) software in comparison (Fig. 5, 6). Alignment of the sequences of ITS 1 showed DNA variable sites in which nucleotides at the position of 59, 68, 186, 276, 370,448, and 468 were single-base substituted resulting in segregation of the specimens (Table 1). Some sequences of F. hepatica and F. gigantica were analyzed and deposited in GenBank (Table 2). Ninety-hundred percent similarities were obtained in comparison of these sequences with all available data of Fasciola spp. in GenBank. Restriction sites of the nucleotides in the Fasciola species were studied by computer software for selection a suitable enzyme. Accordingly, Rsa I was selected as one of the best restriction enzyme for differentiation between F. hepatica and F. gigantica which showed effect on GTAC nucleotides.

Phylogenic tree
Phylogenic Tree of ITS 1 showed that flukes were scattered as pure F. hepatica and F. gigantica clades, suggesting that two genotypes of Fasciola are able to infect animals and probably human in Northwestern of Iran (Fig. 7).    Using phylogenetic tree, we could detect a close relationship of our isolates with those of other countries. As mentioned in tables and figures already, we detected two haplotypes of F. gigantica as G1 and G2. It is worth mentioning that recently a hypothesis has been proposed that this F. gigantica might be considered as separate species (24). Our findings showed that all isolates belonging to F. hepatica were of common H1 haplotype. Figure 6 shows that this haplotype has been reported already from Yasuj and Guilan from Iran, as well as from Egypt, Ireland, China and Australia as well. Besides, haplotype G1 of F. gigantica has been reported from Egypt, Vietnam, Indonesia, China, and Yasuj plus Guilan from Iran. Haplotype G2 is reported from Iran and Egypt as well as Saudi Arabia (All references in Fig.  7). As mentioned already no intermediate isolates were detected in our study. The limitation of our study is that more samples should have been collected to be sure of the deduction as much as possible. The strength might be told that this is for the first time that this study has been conducted in this area in its kind.

Conclusion
Comparing F. hepatica and F. gigantica sequences showed 90-100% homology between our samples with those originated from other regions available in GenBank, but comparing F. hepatica and F. gigantica with each other showed a few nucleotide differences.