Molecular identification of Fasciola species based on ribosomal and mitochondrial DNA sequences in Riyadh, Saudi Arabia

Fasciola species are the causative agents of liver fluke disease (fasciolosis) which is primarily found in domestic and wild ruminants but also are causal agents of fascioliasis in humans. Few reports for the genotyping of Fasciola species in Saudi Arabia were available. This study aimed to identify the Fasciola species infecting sheep using morphology and molecular tools. For that, livers of 500 imported sheep were slaughtered at abattoirs located in Riyadh (Saudi Arabia), and parasite species were isolated from 28 liver samples and then preserved for subsequent stages in this study. These parasite species were identified as Fasciola hepatica and Fasciolagigantica via morphology and confirmed via the 28S rRNA, ITS-1, COI, and NDI sequences with a close identity for other Fasciola species within the family Fasciolidae. Phylogenetic analysis of the obtained sequences showed that Fasciola isolates from the current study were clustered in one subclade closely related to isolates from Iran, Vietnam, Australia, South Africa, Eastern Europe, India, Egypt, Sudan, Japan, Poland, Spain, Armenia, and Turkey. A combination of data from morphology and molecular analysis could be considered a useful tool for identifying Fasciola species infecting sheep in Saudi Arabia.



Fasciolosis is considered the most important helminth infection disease in the world.Trematode parasites (liver flukes) are a food-Arq.Bras.Med.Vet.Zootec., v.76, n.1, p.55-66, 2024 with a further 180 million at risk in more than seventy countries around the world, especially where sheep or cattle are reared, that can be transmitted to humans (Erensoy et al., 2009;Keiser and Utzinger, 2009;Dar et al., 2012;Rokni, 2014).There are certain differences between the geographical distributions of F. hepatica and F. gigantica.Only F. hepatica is distributed in Europe, the Americas, and Oceania, whereas both species are commonly found in Asia and Africa (Mas-Coma et al., 2005;Caravedo and Cabada, 2020;Tang et al., 2021).Moreover, F. hepatica occurs in temperate areas, F. gigantica in tropical zones, and both species overlap in subtropical areas and have a global geographical distribution (Laloret al., 2021).Human and animal fascioliasis is a serious health and veterinary problem, of the two species, are a major cause of economic loss to the agricultural community in developing countries, such as weight loss and a decrease in meat production in livestock animals because of morbidity and mortality (Tınar and Korkmaz, 2003).
In the last decades, the prevalence of animal fascioliasis has been investigated in several parts of Saudi Arabia (Khanjariet al., 2014).Fasciola infection in sheep in some parts of Saudi Arabia is recorded as a very serious problem, where the consumer shifts from local to imported sheep in Riyadh markets (Degheidy and Al-Malki, 2012).
Morphological criteria such as body length to width and shape are among the traditional and important methods to distinguish between the two Fasciola species, but these methods are difficult to trust because of the presence of overlapping features and the presence of intermediate forms (Valero et al., 2001;Marcilla et al., 2002;Periago et al., 2006).In Saudi Arabia, animal fascioliasis has been reported with a prevalence of 19.97% and 52.9% in sheep and cattle, respectively.The liver condemnation of meat and offal results in a loss of 0.2 million dollars annually.Human cases of fascioliasis have also been reported among immigrant workers in Saudi Arabia (Sanad and Al-Megrin, 2005;Degheidy and Al-Malki, 2012).In 2020, reported in Europe fascioliasis caused losses of USD 750 million a year, with the largest impact on the dairy and meat cattle industries (Charlier et al., 2020).
Recent studies showed that the molecular methods and markers are often necessary for parasite species confirmation and to recognize the intermediate forms (Amoret al., 2011;Yuan et al., 2016).The nuclear ribosomal DNA (rDNA) is beneficial for molecular studies because it is highly repetitive and contains variable regions flanked by more conserved regions.The first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) which occur between the 18S, 5.8S, and 28S coding regions, have been used for diagnostic purposes at the level of species (Huang et al., 2004;Tamura et al., 2011).Analysis of mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1) and cytochrome c oxidase subunit I (COI) DNA sequence variation has been used as a tool to understand the phylogeny of species as well as the geographical distribution of genetic variation and demographic history of populations (Penget al., 2009).
Although significant DNA sequencing for F. hepatica and F. gigantica is available from several regions, there are few reports for the genotyping of Fasciola species in Saudi Arabia.Therefore, our study was conducted to investigate and identify the fasciolid species by morphometric and molecular methods.

MATERIAL AND METHODS
Liver samples of 500 imported sheep (from Sudan and Somalia) were randomly collected from slaughterhouses in Riyadh (Saudi Arabia), during the period of August to November 2022.Macroscopic examination was done for parasitic infections.
Adult trematodes were collected, washed in physiological saline solution (0.9%), and examined to identify morphometric characteristics using described standard taxonomic keys to the species level according to previous studies (Sahbaet al., 1972;Toft and Eberhard, 1998).Fasciola specimens were fixed in 70% ethanol and stored at -80 o C until used for molecular identification.The samples were examined and photographed using a Nikon microscope (SMZ18, DS-Ri2, Japan) using NIS Elements software.

57
Total DNA was extracted from ethanolpreserved samples, and a small part from each ethanol-preserved Fasciola specimen was cut, washed in distilled H 2 O before DNA isolation, allowed to dry, and crushed in sterile 1.5 ml micro-centrifuge tubes.A portion of the apical and lateral zone of adult flukes, not including the reproductive organs, was removed, and crushed.
DNA from the crushed materials was extracted using a QIAGEN DNeasyBlood and Tissue Kit (Catalogue # 69504) according to the manufacturer's instructions.PCR was performed to amplify the target genes, COI, ND1, ITS1, and 28S, using four pairs of primers as shown in Table (1) The polymerase chain reaction (PCR) was performed in 20µL reaction volume, comprising of 4.0μL of Solis BioDyne 5× FIREPolMaster Mix, 12.5mM MgCl 2, and 2 mM dNTPs of each, 0.6 μL of each primer, 1.0μL of DNA template and finally 12.8µL of nuclease-free water.PCRs were carried out in a Bio-Rad thermocycler and the thermal cycling conditions involved were an initial denaturation of 3 min at 95°C, followed by 35 cycles of 30 s at 95 °C as the denaturation step, an annealing step for 45s at 56 o C (28S), 57 o C (ITS-1, COI), 60 o C (NDI), and an extension for 45s at 72 o C.This was followed by a final extension of 5 min at 72°C.Agarose gel electrophoresis was performed to confirm amplification and 1.5% agarose gel was used to separate the PCR products.
All nucleotide sequences were determined through direct sequencing of the PCR products with the ABI Prism BigDye Terminator Cycle Sequencing Kit, using ABI 31301 DNA Analyzer (Applied Biosystems, Thermo Fisher Scientific, USA).The sequences were blasted with the GenBank TM nucleotide databases.The aligned sequences were trimmed in BIOEDIT 4.8.9 (Hall, 1999).Phylogenetic analysis was performed using the Maximum likelihood, and Tamura-Nei model methods.The software for Molecular Evolutionary Genetics Analysis (MEGA v.11) was used to perform this phylogenetic analysis (Tamuraet al., 2021).Boostrap values were included based on 1.000 replicates.

RESULTS
A total of 500 slaughtered sheep were examined, 28 sheep were found to be infected with F. hepaticarepresenting 5.6% and F. gigantica represented the same percentage.
Microscopic examination showed that hepatic samples were infected with Fasciola species, according to the measurements and characteristics of the different Fasciola spp., the two Fasciola species with different lengths as F. hepatica (2.5cm) and F. gigantica (5 cm).Both species are characterized by the main diagnostic features such as a flattened body with brown color and leaf-like shape, as well as the presence of two suckers (oral and ventral) (Figure 1).The higher frequency of fascioliasis in imported sheep from Sudan and Somalia is likely because rainfall is higher and irrigation schemes are more developed in that area so that habitats for the intermediate snail hosts are more plentiful there.
PCR products revealed the presence of different bands specified the presence of Fasciolaspecies by using the recommended primers as 618 bp (28S rRNA), 680 bp (ITS-1), 500 bp (COI), and 700 bp (NDI) (Figure 2).For analysis of the phylogenetic diversity of the flukes, phylogenetic trees were built, using the partial 28S rRNA, ITS1, COI, and NDI sequences of F. gigantica and F. hepatica from the present study along with other available sequences from the region, Asia, Africa and from another region, Europa (Figures 3-6).
Molecular analysis of the partial 28S rRNA region for the recovered specimens revealed the obtaining of 17 sequences which were submitted to GenBank to get accession numbers (OP800250 to OP800266).All sequences are compared with other databases on GenBank and identified as F. hepatica and F. gigantica within the family Fasciolidae.Phylogenetic analysis revealed that the closest related specimens in the same clade Fasciola taxa were collected previously from Iran, Vietnam, Australia, South Africa, and Eastern Europe (Figure 3).
The partial ITS-1 region for the retrieved specimens underwent molecular analysis, and the results showed that 17 specimens were obtained and submitted to the GenBank database to receive accession numbers (OP802570 to OP802574) and (OP866740 to OP866750).All sequences were identified as belonging to the F. hepatica and F. gigantica species within Fasciolidae after being compared to other databases on GenBank.The most closely related Fasciola taxa in the same clade, according to the constructed dendrogram in Figure ( 4), were previously collected specimens from Iran, Vietnam, India, Egypt, and Japan.

DISCUSSION
Fascioliasis control is intricate due to the parasite's complicated life cycle and emerging drug resistance (Kaplan, 2001).To develop effective control measures, it would be necessary to understand the parasite life cycle and evaluate its genetic diversity and population structure (Kaplan, 2001;Itagaki et al., 2005).In this study, the natural occurrence of fascioliasis in sheep was reported, and its taxonomic identification via molecular analysis.A total of 28 (5.6%) out of 500 imported sheep were found to be infected with F. hepatica and F. gigantica.al., 2022).Many studies based on both nuclear and mitochondrial sequences have shown that F. hepatica is the species that is most prevalent and abundant in temperate areas, while F. gigantica is found in tropical countries of Africa (Alajmi, 2019).In this study, four genes of 28S, ITS-1, COI, and NDI were selected to amplify and identify the recovered parasite species.We employed the genes that evaluate the existing primers targeting eukaryotic.Briefly, the nuclear large subunit rRNA (28S rRNA) has been used for identification, genotyping, intra-and interspecific variations, and phylogenetic studies of these parasites (Conceição et al., 2004;Huang et al., 2004;Kleiman et al., 2007;Prasad et al., 2008;Chamuah et al., 2016).The first internal transcribed spacers (ITS-1) of nuclear ribosomal DNA (rDNA), have been used for diagnostic purposes at the level of species (Tkach et al., 2000;Kostadinova et al., 2003;Tandon et al., 2007;Ali et al., 2008).Mitochondrial DNA such as COI and ND1 has been used to find out the genetic variation of Fasciola species (Itagaki et al., 2005;Peng et al., 2009;Shoriki et al., 2014).Based on intra-and interspecific variations between comparable isolates, the recovered Fasciola worms were identified as F. hepatica and F. gigantica (Alajmi, 2019) reported both variations were studied, where interspecific variations range between 0 and 1%, while, variations between studied species were 1%.Similar intraspecific variations were obtained by Galavaniet al. (2016) in their genetic characterization of Fasciola species (F.hepatica and F. gigantic) isolates from Iran.While interspecific variation reaches 3% by Marcilla et al. (2002) which reported few interspecific nucleotide variations between F. hepatica and F. gigantica.The molecular origin of the Family of Fasciolidae is represented as paraphyletic (Lotfy et al., 2008).

CONCLUSION
It could be concluded that there is a low infection with Fasciola species in the studied area within Saudi Arabia, also, molecular methods are more effective in identifying these species.Further studies are recommended to control this infection.

Figure 4 .
Figure 4. Phylogenetic relationship of ITS1 sequences of isolates of F. hepatica and F. gigantica from Saudi Arabia using maximum likelihood method.P. westermani as an out-group strain.Numbers at the nodes indicate the percentage of bootstrap support.

Figure 5 .
Figure 5. Phylogenetic relationship of COI sequences of isolates of F. hepatica and F. gigantica from Saudi Arabia using maximum likelihood method.P. westermani as an out-group strain.Numbers at the nodes indicate the percentage of bootstrap support.

Figure 6 .
Figure 6.Phylogenetic relationship of NDI sequences of isolates of F. hepatica from Saudi Arabia using maximum likelihood method.P. westermani as an out-group strain.Numbers at the nodes indicate the percentage of bootstrap support. .