Molecular Identication and Phylogenetic Analysis of Fasciola Hepatica Isolates From Cattle and Sheep in Golestan Province, Northeast of Iran.

Background: Fascioliasis in livestock and humans, caused by Fasciola hepatica and Fasciola gigantica, has socioeconomic importance worldwide. Iran is one of the important endemic foci for fascioliasis. Molecular approaches can be precise and reliable for the identication and characterization of Fasciola spp. The aims of this study were molecular identication of Fasciola hepatica isolates in Golestan province, north of Iran, and then comparative analysis of results using GenBank sequences. Material and Method: Fasciola ukes were isolated from the livers of infected livestock. DNA was extracted from samples using the phenol-chloroform technique. RFLP-PCR using TasI restriction enzyme was performed along with morphometric evaluations for the detection of F. hepatica species. PCR was performed to amplify partial fragments of the internal transcribed spacer 1 (ITS1), cytochrome c oxidase sub.1 (CO1), and NADH dehydrogenase sub.1 (ND1) genes for samples that were conrmed as F. hepatica. Twenty-eight PCR products were sequenced. The phylogenetic tree with MEGA 7 software was drawn for all three genes. Results: Out of 271 ukes collected from sheep and cattle, 126 conrmed as F. hepatica by the PCR-RFLP method. Results based on PCR-RFLP analysis were conrmed by sequence analysis. Only one haplotype for the CO1 gene and four different haplotypes for the ND1 gene were identied. Seven sequences of each gene registered on GenBank and accession numbers were received. Conclusions: This study showed that F. hepatica is widely distributed among livestock in Golestan province. It was also found that genetic diversity among the ND1 region in isolates of our study was considerably higher than the CO1 sequence region.


Introduction
Fascioliasis is one of the most important diseases in domestic livestock and humans caused by the genus Fasciola. The disease causes signi cant economic loss and health problems in several regions of the world. Iran, it is important to detect the species of the genus of these helminths to do follow-up work, control, and treatment of the disease.
Due to the several variations in morphological speci cations, abnormal ploidy (diploidy, triploidy, and mixoploidy), morphological methods cannot guarantee the accurate differentiation between Fasciola species (Itagaki et al. 1998;Mas-Coma et al. 1999). Therefore, several studies, especially to genotyping Fasciola spp. have been done in different parts of Iran (Aryaeipour et al. 2014;Bozorgomid et al. 2016).
Over the last two decades, to overcome the limitation of the morphologic methods, several PCR-based methods, including speci c PCR assays, PCR-RFLP, and PCR-SSCP, have been developed for the identi cation and differentiation of Fasciola spp. PCR-RFLP technique is the one in which many researchers expressed its importance (Hashimoto et al. 1997 Another marker to differentiate Fasciola spp. is mitochondrial DNA (mtDNA). The mitochondrial genome, due to its faster evolving and elevated mutation rate in comparison with the nuclear genome, is a suitable target for discrimination among individuals from the same population and closed related species (Boore 1999

Study area and sample collection
In this eld study, from December 2014 to July 2016, 271 samples were collected in Golestan Province (Halakou et al. 2017). Liver ukes were collected from infected cattle and sheep in several abattoirs of Gorgan, Gonbad Kavus, Ramian, Aliabad, Aq Qala, Bander Turkman, and Kalaleh in Golestan province in the northeast of Iran (Fig. 1). The liver worms were identi ed to the species based on morphometric criteria according to standard taxonomic keys (Yamaguti 1958;Sahba et al. 1972) and the posterior parts (excluding the uterus) of worms' bodies were cut and put in ethanol 70% and stored at -80 °C until further use.

DNA extraction and PCR
Samples were washed three times with PBS buffer. Small posterior parts of the worms were subjected to DNA extraction using a conventional phenol-chloroform method with slight modi cations (Sambrook et al. 1989) and extracted DNA samples were stored at -20°C. PCR for partial sequences of ITS1, CO1, and ND1 genes was performed (Itagaki et al. 2005a) in 15 µl reactions containing 1μL (1ng) DNA template, 1μL primers (10 pmol of each primer), 5.5 μL distilled water, and 7.5μL master mix (Ampliqon, Denmark). Table 1 shows the sequences of primers, PCR pro les, numbers of cycles, and sizes of amplicons that were determined for F. hepatica ITS1, CO1, and ND1 genes. 5μL of each PCR product was examined in a 1.5% agarose gel and stained with Ethidium Bromide (EtBr) and visualized under a UV transilluminator. A total volume of RFLP reaction contained 0.5 μL of the TasI enzyme, 1.5 μL of enzyme buffer, 9mL distilled water, and 5μL ITS1-PCR product was incubated at 65° C for 2.5 h and 80° C for 20 minutes. 5μL of each reaction product mixed with 2μL of loading buffer was subjected to the gel electrophoresis on 2.5% agarose gel in TAE buffer and were stained with Ethidium Bromide.

Sequencing and Phylogenetic Tree Analysis
Due to budget constraints, 14 samples of RFLP con rmed F. hepatica samples were sequenced for ITS1, CO1, and ND1 genes. About 25μL of PCR products were sent to Bioneer Company (South Korea A maximum-likelihood (ML) phylogram based on ITS1, CO1, and ND1 genes was constructed using MEGA 7.
In addition to the ITS1, ND1, and CO1 haplotypes of the Fasciola ukes from Golestan province, those of F. hepatica from other parts of Iran and some countries were included as references (Figure 4-6). Nucleotide sequences of Fasciolopsis buski (GenBank:EF612477), and Fasciola gigantica (GenBank:AB385622, AB385617) were used as outgroups.

Results
The primers used in this study successfully ampli ed a fragment of 463 bp in all Fasciola spp. samples for the ITS1 gene ( Figure 2). PCR products of all ITS1 samples were digested with TasI enzyme. TasI has one cutting site for F. hepatica and two cutting sites for F. gigantica. Samples identi ed as F. hepatica were selected for following sequencing analysis, and the rest samples (F. gigantica samples) were stored at -20℃ for further eventual studies. Figure 3 shows the PCR-RFLP pattern after digestion with the TasI enzyme. Table  2 shows the number of Fasciola spp. infected sheep and cattle con rmed by RFLP-PCR.  (Figs. 4, 5, and 6). The phylogenetic analysis of ITS1 sequences revealed that all isolates from the present study were very similar to each other and closely related to the references from Uruguay, Japan, Australia, the United Kingdom, and also some other parts of Iran. The results showed that all samples from this Golestan province were composed of 100% homologous sequences, and only the Bandar Turkman sample was recorded with 82% homology compared to other samples. (Figure 4).
All isolates of F. hepatica positive for CO1 gene in the present study were closely related to the references from Ecuador, Poland, and some other parts of Iran ( Figure 5). Results of the phylogenetic tree analysis for F.hepatica ND1 indicated that isolates from Gonbad Kavus, Gorgan, and Ramian were similar to two isolates from Iran (GenBank GQ175362-63 ), and two isolates from Egypt (GenBank LC076257 and AB554177).
Isolates from Bandar Turkman and Aliabad were very similar to each other. Finally, an isolate from Gorgan (GenBank MN594514) was very similar to a sequence from China ( Figure 6).

Discussion
Fasciola species, including F. hepatica and F. gigantica, were considered as a critical veterinary problem until the end of the 1980s, mainly due to considerable economic losses which these parasites cause in livestock (Mas-Coma et al. 1999). Meanwhile, human infections were rare and sporadic (Cook 1996 identical. The only exception was a sample from Bandar Turkman, which showed 82% identity to other selected isolates. High similarity between the isolates in this region may cause a great variation even due to one or two nucleotide change. Results obtained from the sequencing of ITS1, CO1, and ND1 genes con rmed that F. hepatica is the main uke of sheep in the Golestan province in the north of Iran. Sarkari et al. (2017) showed that 78% of ukes isolated from sheep in Talesh county located at Guilan province (neighboring province in Northern Iran) were F. hepatica using PCR-RFLP assay. Comparing CO1 sequences of F. hepatica isolated from the present study with other sequences revealed that all isolates of our study showed 100% identity with some recorded sequences from Iran, Ecuador, and Poland. Based on our ndings, there were no differences between CO1 sequences in our studied isolates. According to comparing our isolates sequences with the GenBank database using the BLAST tool, the main haplotype of the CO1 gene was FhC2.
According to the results of the phylogenetic tree, F. hepatica ND1 sequences of isolates from Aqqala showed 85% identity with sequences from Gonbad Kavus, Gorgan, Ramian, Egypt, and some other parts of Iran which had 100% identity with each other their selves. Two isolates from Bandar Turkman and Aliabad showed 100% identity with each other and over 80% identity with isolates from Aqqala, Gonbad Kavus, Gorgan, Ramian, Egypt, Iran, Uruguay, Ireland, and Italy. One reminded isolate from Gorgan showed 100% identity with an isolate from China. The Findings of the present study represent substantial differences between sequences of ND1 F. hepatica isolates from various regions of Golestan province. These ndings were far different from results in which Sarkari et al. (2017) presented about substantial diversity of CO1 sequences compared to ND1 sequences. Based on the GenBank database and sequence similarity of ND1 gene, haplotypes Fh20 from Gonbad Kavus, haplotypes Fh04 and (Fh27/Fh26/Fh18/Fh08/Fh01) from Gorgan, and haplotypes (FhN21/FhN2) and (Fh27/Fh26/Fh18/ Fh08/Fh01) from Ramian were found (

Conclusion
Overall, the ndings of the present study suggested that F. hepatica is the main liver uke of the sheep population of Golestan province, North of Iran. This study indicated that the sequence differences between isolates among ND1 were much more than CO1 individuals. All samples were identi ed by morphometric and ITS1 RFLP-PCR evaluations, con rmed by ITS1, CO1, and ND1 genes sequencing as F. hepatica. Sequencing analysis of the ITS1, CO1, and ND1 regions showed no intermediate form of Fasciola in our research. Figure 1 Golestan province (North-East of Iran) and locations where the samples were collected Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.   Maximum-likelihood tree of F. hepatica ITS1 gene from Golestan Province of Iran. The nucleotide sequence of the F. buski (GenBank accession no EF612477) detected in Vietnam was considered outgroup.

Figure 5
Maximum-likelihood tree of F. hepatica CO1 gene from Golestan Province of Iran. The nucleotide sequence of the F. gigantica (GenBank AB385622) detected in Vietnam was considered as an outgroup Maximum-likelihood tree of Fasciola hepatica ND1 gene from Golestan Province of Iran. The nucleotide sequence of the F. gigantica (GenBank AB385617) detected in Vietnam was considered as an outgroup