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). Rising cases of human fascioliasis over the 1970-90 years in 42 countries convinced the World Health Organization to consider fascioliasis as an important human disease (Mas-Coma et al. 1999). Both Fasciola species including F. hepatica and F. gigantica can infect humans and animals. Many techniques including morphometry and molecular-based methods have been developed to differentiate these species (Mas-Coma and Bargues 1997; Itagaki et al. 1998; Marcilla et al. 2002; Ashrafi et al. 2006; Amer et al. 2011). DNA-based techniques like PCR-RFLP are the most appropriate methods for differentiation Fasciola flukes in comparison with other diagnostic methods (Marcilla et al. 2002). Moreover, sequence analysis of both ribosomal and mitochondrial DNA of Fasciolaspp. is a reliable method for genotyping, phylogenetic studies, and inter/intra-species investigation (Boore 1999; Le et al. 2001; Itagaki et al. 2005b; Semyenova et al. 2006; Prasad et al. 2008; Rokni et al. 2010; Ladoukakis and Zouros 2017)
In the present study, the TasI restriction enzyme was used for the RFLP technique based on the 463 bp sequence of the ITS1 gene. These F. hepatica sequences revealed two fragments of 151 and 312 bp, while F. gigantica producing three fragments of 151, 219, and 93 bp. Our samples based on the hosts showed that both species of Fasciola exist in Golestan province, and the majority of samples isolated from sheep were F. hepatica (69.65%). Many studies using PCR-RFLP have been done for detecting Fasciola spp. by targeting the ITS1 gene in Iran (Yamaguti 1958; Mas-Coma et al. 1999; Marcilla et al. 2002; Itagaki et al. 2005a; Kumar et al. 2016). Morphometric and PCR-RFLP results in the present study were consistent with some of the mentioned studies (Marcilla et al. 2002). In 2015, Yakhchali et al. used RFLP for identification and sequencing for the phylogenetic tree construction. They reported that all species of Fasciola worms found in West Azerbaijan were F.hepatica (Yakhchali et al. 2015). The RFLP-PCR and sequencing results of the present study using the ITS1 gene showed that most of the sheep were infected with F. hepatica, while the predominant species in cattle was F. gigantica.
ITS1 sequence analysis comparing to other registered sequences on GenBank databases showed that almost all of the isolates of the present study and selected isolates from different parts of the world were 100% 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 confirmed that F. hepatica is the main fluke of sheep in the Golestan province in the north of Iran. Sarkari et al. (2017) showed that 78% of flukes 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 findings, 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 findings 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 (Table 4). Haplotypes that are separated with a slash (/) had completely identical sequences, and it is not clear why authors selected different names for them.
Sharifiyazdi et al. (2010) used ribosomal ITS1 and mitochondrial DNA (CO1 and ND1 genes) for genetic characterization of two Fasciola samples directly isolated from humans in Guilan province, North of Iran. Both samples were confirmed as F. hepatica and showed 100% identity to an isolate from a Japanese patient (Sharifiyazdi et al. 2012).
Sarkari et al. (2015), using ITS1, ITS2, ND1, and CO1 genes, showed that over 87% of liver flukes that were isolated from sheep in Guilan province, were F. hepatica. They indicated that CO1 region sequences exhibit considerable variation among different isolates (Sarkari et al. 2017). The present study shows substantial diversity over the ND1 region instead. Raeghi et al. (2016) used ND1 and CO1 sequence regions for phylogenetic analysis of 90 F. hepatica flukes collected from 30 cattle in the North-East of Iran. Their study revealed significant genetic differences of the ND1 region among the analysed isolates (Raeghi et al. 2016). The overall result of the present study shows a correlation with what they concluded.
In a study by Shafiei et al. (2014) on different Fasciola hosts in Kohgilouyeh and Boyer-Ahmad province, in the southwest of Iran, ribosomal ITS1, ITS2, mitochondrial ND1, and CO1 sequencing were used for the analysis of genetic variations within and between isolates. ITS1 sequences showed six single base substitutions for F. hepatica isolates. Also, CO1 sequences showed seven DNA polymorphic sites for F. hepatica samples.
Aghayan et al. (2019) used 28s rRNA an ND1 for evaluating the genetic diversity of Fasciolaspp. in livestock from central Armenia. They found 29 haplotypes of the F. hepatica ND1 gene among 55 flukes and Fh01was the dominant haplotype (Aghayan et al. 2019).