Figure 1: Fasciola gigantica in the bile duct of an infected cattle liver.

Figure 2: Fasciola spp. isolated from the liver of infected ruminants in Duhok province (A: F. gigantica, B: F. hepatica).

Figure 3 shows the adult flukes of Fasciola ssp. that were selected for rDNA ITS1 sequencing and used for phylogenetic analysis. These flukes were identified according to their morphology and shape before the DNA extraction. The flukes of the figures A and C were similar to F. heatica, and those in figures D and E were similar to F. gigantica, while the fluke of figure B looks like a hybrid or intermediate form. Their details are demonstrated in table 2.

Figure 3:Adult flukes of Fasciola spp. isolated from ruminants in Duhok province (A, B, C, D represent stored parasites after removing them from ethanol 70%, and E is a unpreserved parasite). Amplicons of these isolates were selected for sequencing. (*= Isolate code)

The DNA of all isolates (54 samples) of the Fasciola was successfully extracted. The DNA concentration ranged between 35 and 310 ng/µl with a purity of 1.85 to 2.1 measured at A260/A280 nm. In almost all DNA isolated samples, a clear band of DNA with no smearing was visible in agarose gel electrophoresis, indicating that it had not been degraded.

Designed primers FITS1-(F&R) successfully amplified the ITS1 segment of ribosome DNA of all Fasciola specimens isolated from local domestic ruminants (sheep, goats, and cattle) in different locations of Duhok province. A single band (a monomorphic DNA fragment) from approximately 518 bp was apparent in all amplified ITS1 sequence lanes, as shown in figure 4.

Five PCR product samples with both forward and reverse primers were sent to the Macrogen Company for sequencing. The PCR products of the ITS1 segment were successfully sequenced in the resulting forward and reverse sequences for all the selected Fasciola isolates. Five sequences with a length ranging from 477 minimum to 495 maximum base pairs (bp) were obtained. GenBank's sequence alignment and the blast hit results for each of the obtained ITS1 sequences revealed that three isolates belonged to F. hepatica and two to F. gigantica. These five ITS1 sequences were deposited in NCBI-GenBank under Accession Numbers: OM920533; OM920534; OM948733 for F. hepatica and OM948683; OM918714 for F. gigantica, as demonstrated with their information in table 2. The variable sites of nucleotides (nitrogen bases) between F. hepatica and F. gigantica ITS1 sequences of the current study were 4 bases located at positions 13, 107, 185, and 205. In contrast, no nucleotide variation was detected in the ITS1 within intraspecific (within the same species) of each Fasciola species. The substitutions A to T at the 13th position, C to T at the 107th and 205th positions, and T to A at the 185th position clearly distinguished F. gigantica from F. hepatica and were sufficient to differentiate both species, as shown in table 2.

Table 3 shows the pairwise identity percentage and the genetic distance (p-distance) based on ITS1 sequences (targeting 495 bp) of the Fasciola isolates and sequences of other isolates' that are available in NCBI-GenBank. The pairwise identity percent of ITS1 sequences within the same species of Fasciola (intraspecific) from this study isolates and others from Iran, India, China, Pakistan, Spain, and Kenya was 100% with a 0.00 p-distance value and no genetic diversity. While the pairwise identity percent of ITS1 sequences between the F. hepatica and F. gigantica was 99.2 %, and the genetic distance (p-distance) was 0.082. The results indicate that there was close (identity) similarity between the ITS1 sequences of the same species (intraspecific) of Fasciola, which shows the purity of the isolates in this study with the Fasciola species.

Figure 4: Gel electrophoresis of Fasciola spp. rDNA ITS1 segments isolated from Duhok province. Lane L: DNA ladder (100+ bp); Lanes 1-5: amplified samples of rDNA ITS1 region (518 bp); Lane 6: blank.

 The phylogenetic relationship tree was constructed using the Neighbor-Joining method based on a comparative study of ITS1 sequences of Fasciola spp. (n = 5) obtained in the present study with closely related sequences from different countries submitted to GenBank (n = 19). The evolutionary distances were computed using the Kimura 2-parameter method and are in the units of the number of base substitutions per site. The result showed that three main groups of Fasciola species were clustered. The first was the F. hepatica group, including twelve nucleotide sequences with 0.00 evolutionary distance (p-distance) and a similarity of 100%; the second was the F. gigantica group, including seven nucleotide sequences (NS) with (0.00) evolutionary distance and similarity of 100%; the third was an intermediate group that was generated into two subgroups; the first was Fasciola spp. sequence from China and the second was composed of three sequences from Zimbabwe. The ITS1 sequences clustered into F. hepatica and F. gigantica groups were genetically highly homogeneous, and no polymorphism was observed within the same species (intraspecific) while it was present between the different species (interspecific). The sequences of the F. hepatica group were separated from the intermediate and F. gigantica groups by a value of 0.8 and 0.4% in genetic distance, respectively. The genetic distance between F. hepatica and the intermediate form is lower when compared with the distance value between F. hepatica and F. gigantica. Therefore, the intermediate group is located between these two groups, as illustrated in figure 2.

Table 2: Details and comparison of Fasciola species rDNA ITS1 sequences from Duhok province

Table 3: Pairwise identity percentage (%) of Fasciola spp. ITS1 sequences of this study and compared with NCBI-GenBank references (in left lower matrix), and genetic distance only for this study's ITS1 sequences (in suitable upper matrix).

Acc. No. = Accession numbers. Fh= Fasciola hepatica, Fg= Fasciola gigantica. 1-5 represent the isolates from this study (bold writing)

Figure 5: Phylogenetic relationship tree based on the ITS1 sequences of Fasciola species isolates from Duhok province and references in the GenBank database using the Neighbor-Joining method. Fasciola jacksoni (AN: EF612473) was used as the out-group. This analysis involved 24 nucleotide sequences: 5 from this study highlighted by a red color marker and 19 from GenBank. The phylogenetic tree was conducted in MEGA11.

Discussion

The morphology of F. hepatica and F. gigantica can generally be used for distinguishing between them, but for detecting and distinguishing intermediate forms between Fasciola species, one need to use molecular methods, and genetic markers (20). Aryaeipour et al. (31) concluded that the PCR-RFLP method was a more reliable method than morphology for the differentiation of Fasciola species and the morphological methods were inefficient for determining genetic diversity. The first and the second internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal DNA (rDNA) were used as genetic markers for genetic characterization and differential between the species of Fasciola that have been confirmed by several previous studies (32-34). These markers are non-coding regions between the 18S, 5.8S, and 28S coding regions and have been used for diagnostic purposes at the species level (35-38). In the Middle East, many studies relied on the ITS1 marker to identify Fasciola species (39,40). In this research, Fasciola spp. were diagnosed and characterized using the ITS1 region, which is in line with the previous studies conducted by other researchers (41-46).

In the present study, a 518 bp segment (encompassing ITS1 and 5.8 RNA gene) was amplified using primers ITS1, and five amplicons were sequenced by Macrogen company (using the Sangar method). According to the findings of the current study, F. hepatica and F. gigantica are the only two Fasciola species isolated from infected ruminants (sheep, goats, and cattle) in the Duhok province as described by other research in the Erbil, like Mohammed et al. (47) in Duhok province based on ITS1 and ITS2 markers, Raoof et al. (48) in Sulaymaniyah province based on the mitochondrial 28S rRNA gene, and others from different parts of Iraq. Previous studies in neighboring countries stated that both Fasciola species (F. hepatica and F. gigantica) were present in domestic ruminants from different geographical and climatic areas (49,50).

Alignment analysis revealed very close similarity among the nucleotide sequences, and genetic variation was not observed within the same species (no intraspecific variation within species) of the current study and BLAST hits in GenBank. On the other hand, significant genetic variability was detected between F. hepatica and F. gigantica, and these results agree with those of Shaldoum et al. (51) and Mir et al. (2), which used the same marker (ITS1) to distinguish between the two Fasciola species.

 The sequence variation between F. hepatica and F. gigantica was not related to the geographical origins and host species of isolates, as reported by Alasaad et al. (32). The sequences of the ITS1 region from nuclear ribosome DNA are reliable genetic markers for systematic molecular studies of Fasciola spp. and interspecific variations as suggested by Huang et al. (43).

The presence of hybrid and intermediate form genotypes of Fasciola were not observed in this study using ITS1 sequences. This result is in line with Shaldoum et al. (51) from Egypt and Rokni et al. (39) from Iran, who also identified Fasciola spp. as F. gigantica and F. hepatica. 

Phylogenetic analyses support understanding species information, population differentiation, and ecological adaptation (46). According to the phylogenetic tree depended on the ITS1 sequence of rDNA, isolated Fasciola species from slaughtered ruminants in Duhok province were clustered into two clades (hepatica and gigantica clade) with a remarkable genetic divergence between them. Phylogenic analysis indicated that the ITS1 sequences of this study were identical to those from Iran, India, China, Spain, Switzerland, Tunisia, Algeria, and North Africa forthe F. hepatica as reported by Muhammad et al. (52) in Erbil province. Results were also identical to those from Iran, Pakistan, India, China, Nigeria, and Kenya for F. gigantica as reported by Hamoo et al. (53) in Duhok province (in Aqrah city), and also this close relationship between the same species of Fasciola has been shown by Nguyen et al. (54). The analysis of the rDNA ITS1 sequence of Fasciola species was commonly reported in Asia, Europe, and African countries (2,18,49). The present study results and those of other studies demonstrate the diagnostic power of the ITS1 region in differentiating the Fasciola species (39,55).

Conclusion

The designed primers that were used for amplifying ITS1 nuclear rDNA of Fasciola spp. in the present study can be used to detect and diagnose Fasciola species. The genotypic and phylogenic analysis indicated that F. hepatica and F. gigantica were the two Fasciola species isolated from infected ruminants (sheep, goats, and cattle) in Duhok province. The genotype of these species is closely related to the genotypes of the same species identified in other countries in the Middle East, Asia, Europe, and Africa. In this study, hybrid forms of Fasciola were not detected, and the nucleotide variation of ITS1 sequences between the two species was enough to separate them.

Acknowledgments

The authors express their sincere thanks to the Department of Animal Protection, College of Agricultural Engineering Science, and the University of Duhok for offering the laboratory facilities and financial support. Great thanks and appreciation to the veterinarian and meat inspection staff at the Duhok and Zakho slaughterhouses, especially Dr. Azad Mahdi Azo, for his assistance and to all those who helped complete this study.

Conflict of interest

There is no conflict of interest in the current study.