Morphological and molecular characterization of Fasciola isolates from livestock in Golestan province, northern Iran

Abstract Background Fascioliasis, caused by the liver flukes Fasciola hepatica and Fasciola gigantica, is a global zoonotic helminthic disease. The livestock and human are the final hosts of the parasites. Northern Iran is an important endemic region for fascioliasis. Few studies have been conducted on the characterization of Fasciola isolates from eastern regions of the Caspian littoral of the country. Objective The aim of the present study was to identify F. hepatica, F. gigantica and intermediate/hybrid forms of Fasciola isolates from livestock in Golestan province, northern Iran, using morphometric and molecular tools. Methods Livestock livers naturally infected with Fasciola spp. were collected from Golestan slaughterhouse during 2019–2020. The worms were morphometrically studied using a calibrated stereomicroscope. Genomic DNA was extracted from all samples, and polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) was performed on internal transcribed spacer (ITS1) region using Rsa1 restriction enzyme. All the isolates were then analysed by multiplex PCR on Pepck region. Results A total of 110 Fasciola isolates were collected from the infected livers, including 94 sheep, 12 cattle and 4 goats. Morphometric analysis of 61 adult Fasciola isolates indicated that, 44 and 17 isolates belonged to F. hepatica and F. gigantica, respectively. Eighty‐one and 29 isolates belonged to F. hepatica and F. gigantica using ITS1‐RFLP, respectively. However, Pepck Multiplex PCR indicated 72 F. hepatica, 26 F. gigantica and 12 intermediate/hybrid forms. All 12 hybrid isolates were found in sheep host. Two isolates were identified as F. gigantica using morphometry and F. hepatica using both molecular methods. Conclusion The present study confirmed the existence of both F. hepatica and F. gigantica species and reported the first molecular evidence of hybrid Fasciola isolates in ruminants of Golestan province.


Precise identification of
Traditionally, the distinction between Fasciola species has been made based on morphological features. Adults of F. hepatica are shorter but wider, with longer cephalic cone, and a bigger shoulder, smaller ventral sucker and more exterior testes, compared to F. gigantica (Muller & Wakelin, 2002). Nevertheless, precise differentiation of the two flukes is often difficult because of some morphological variations among different isolates. DNA-based molecular methods have been used to understand the nature and extent of inter-and intraspecific variations in Fasciola. Different tools and genetic markers have been used including polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) on the nuclear internal transcribed spacers (ITS1 and/or ITS2) (Anh et al., 2018;Mir et al., 2019;Rokni et al., 2010;Saadatnia et al., 2022;Shafiei et al., 2013) and mitochondrial cytochrome c oxidase subunit 1 and/or NADH dehydrogenase 1 (Farjallah et al., 2013;Itagaki et al., 2005) (Hayashi et al., 2018;Hayashi, Ichikawa-Seki, et al., 2016;Tang et al., 2016). Unfortunately, sufficient data and information on the molecular identity of

Morphometric study
For morphometric analysis, the worms were washed in PBS and fixed and tied between two slides. Morphology and morphometric characters of the flukes were studied using a microscope equipped with a drawing tube. The morphological parameters were recorded in an Excel spreadsheet. Morphometric analysis was performed using following criteria: body length (BL), body width (BW), body length to width ratio (BL/BW) and cephalic cone length.
Student's -test was employed for comparing the mean of different variables between F. hepatica and F. gigantica, and one-way analysis of variance was used to find any significant differences between the means of morphometric values in flukes separated from different hosts (Sahba et al., 1972;Yamaguti, 1958). Thereafter, the flukes were fixed in 70% ethanol and stored at refrigerator until further molecular analysis.

ITS1 PCR-RFLP and Pepck Multiplex PCR
A small piece of lateral parts of each worm was removed and squashed inside a clean porcelain mortar containing 500 µL lysis buffer (0.1 M NaOH, 0.5 M Tris-HCl, 0.05 M EDTA and 1% SDS) using a pestle. Then the lysate transferred to a micro tube and incubated at 60 • C for 3 h after adding 20 µL proteinase K. The lysate from each sample was subjected to DNA extraction using a conventional phenol-chloroform method with minor modifications (Sambrook et al., 1989). In the final step, the DNA was eluted in 50 µL deionized distilled water (DDW) and frozen at 20 • C until the next step.

Morphometric analysis
Of 110 Fasciola isolates, morphometric analysis was performed on 61 specimens as some of the helminths were juvenile worms without full-grown reproductive organs. The morphometric characteristics of isolated flukes are summarized in

Molecular analysis
In ITS1-PCR a 680 bp fragment was visualized after gel electrophoresis   F. gigantica using morphometry, were characterized as F. hepatica by the both molecular methods. Moreover, a sheep isolate identified as F. hepatica using ITS1-RFLP was characterized as F. gigantica using morphometry and Pepck multiplex-PCR. Another sheep isolate, indicated as F. gigantica using ITS1-RFLP, was found to be F. hepatica by using morphometry and Pepck multiplex-PCR.

DISCUSSION
Traditionally, identification of F. hepatica and F. gigantica, the main Fasciola species infecting human and animals, has been performed using morphologic and morphometric methods. However, the two species cannot be accurately discriminated by these methods. In addition, during past decade, the intermediate forms of F. hepatica is more frequent than F. gigantica in endemic regions of the world (Mucheka et al., 2015;Tolan, 2011). However, the results of a study support that F. gigantica might be the main causal agent of fascioliasis in some northern regions of the Iran (Ashrafi et al., 2004).
The life cycle of Fasciola spp. involves a very wide range of domestic and

F I G U R E 2 Host-based geographical distribution of Fasciola hepatica, Fasciola gigantica and the hybrid forms in different livestock species in
Iran (see Table 2 for the details and references).
wild mammals, including sheep, cattle, horse, camel, coypu, rabbit as well as humans as definitive hosts (Akhlaghi et al., 2018;Beesley et al., 2018;Mas-Coma et al., 2020;Sabourin et al., 2018). Previous study in Golestan province using morphological method indicated 11.84% of sheep and cattle isolates belonged to Fasciola sp.; however, this was not confirmed by molecular methods (Halakou et al., 2017). We found two sheep isolates identified as F. gigantica using morphometry and as F. hepatica using both molecular methods. Moreover, we observed discrepancy among results of other two sheep isolates using three methods. It is recommended to follow a multilocus approach for molecular characterization of helminth parasites of human and veterinary importance. human isolate, belonged to F. hepatica species (Javanmard et al., 2020).

Another study in the eastern provinces of Khorasan Razavi and North
Khorasan indicated all 90 cattle isolates belonged to F. hepatica species (Raeghi et al., 2016). A study from East Azerbaijan province, northwestern Iran, reported all 72 stool isolates from sheep and cattle as the F. hepatica (Baran et al., 2017). In West Azerbaijan, north-western Iran, all 40 sheep and 50 cattle isolates were F. hepatica (Galavani et al., 2016). There are similar results in other countries. For example, all the 225 Fasciola isolates from 7 sheep, 73 cattle and 1 pig from 18 distinct geographic areas in Ecuador, South America, identified as F. hepatica (Kasahara et al., 2021).
There are some other studies in Iran that reported F. hepatica as the more prevalent species in all hosts (
However, there are a few studies in Iran reported F. gigantica as the more prevalent species in livestock (Figure 2). In a study in Zabol, a city of Sistan and Baluchestan province, south-eastern Iran, 88.6% and 91.5% of sheep and cattle isolates, belonged to F. gigantica, respectively (Mir et al., 2019). Another study in Ardabil province, north-western Iran, reported F. gigantica in 88.6% of cattle isolates, whereas 100% of sheep isolates were F. hepatica (Aryaeipour et al., 2014).
In some countries, F. gigantica is the dominant species in livestock.