Molecular characterization of Fasciola spp. from ruminants in Duhok province using the ITS1 ribosomal DNA marker

Rekani, Adnan M.; Mero, Wijdan M.

doi:10.33899/ijvs.2022.134290.2358

Journals List

Molecular characterization of Fasciola spp. from ruminants in Duhok province using the ITS1 ribosomal DNA marker

Iraqi Journal of Veterinary Sciences

Article 2, Volume 37, Issue 2, April 2023, Pages 315-323 PDF (531.49 K)

Document Type: Research Paper

DOI: 10.33899/ijvs.2022.134290.2358

Authors

Adnan M. Rekani^* ¹; Wijdan M. Mero²

¹Department of Animal Production, College of Agricultural Engineering Sciences, Duhok University, Duhok, Iraq

²College of Sciences, Nawroz University, Duhok, Iraq

Abstract

This study aimed to characterize and identify the genotypes of Fasciola spp. isolated from sheep, goats, and cattle in Duhok province based on the ITS1 region of rDNA. About 54 adult Fasciola flukes were individually isolated from the livers of naturally infected ruminants. After morphological identification, the genomic DNA of 54 isolated Fasciola spp. was successfully extracted, and the ITS1segment (518 bp) of rDNA was amplified. The amplicons were confirmed by gel electrophoresis and yielded mono cleared bands. Five amplicons from these samples (2 sheep, 2 cattle, and 1 goat) were selected for sequencing and then compared with NCBI-GenBank sequences for genotyping and phylogenetic analysis. Sequencing analysis and the BLAST results revealed that 3/5 of the resultant sequences were F. hepatica and 2/5 were F. gigantica. The ITS1 sequences were submitted to NCBI-GenBank with accession numbers: OM920533, OM920534, OM948733, OM948683, and OM918714. Alignment analysis of the current study and GenBank ITS1 sequences showed the presence of nucleotide variations between F. hepatica and F. gigantica species (interspecific), which were enough to separate them. At the same time, they were not observed within the same species of Fasciola (intraspecific). The pairwise identity percentage of intraspecific and interspecific Fasciola isolates was 100% and 99.2-99.6%, respectively. Phylogenetic analysis of the ITS1 sequences demonstrated that the Fasciola isolates of this study were clustered into two clades (hepatica and gigantica clades). The present study concluded that both Fasciola spp. (F. hepatica and F. gigantica) existed among the infected ruminants in Duhok province and are closely related to intraspecific Fasciola isolates from different countries in the Middle East, Asia, Europe, and Africa.

Highlights

Both F. hepatica and F. gigantica were isolated from infected ruminants (sheep, goats, and cattle) slaughtered at Duhok province abattoirs and they were confirmed by DNA sequencing.
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 genotype of Fasciola species of this study is closely related to the genotypes of the same species identified in other countries in the Middle East, Asia, Europe, and Africa.

Keywords

Fasciola spp; ITS1 region; PCR; Duhok; Iraq

Full Text

Introduction

Fascioliasis is a severe parasitic disease that affects ruminants (sheep, goats, and cattle), humans, and other mammals, caused by infection with F. hepatica and F. gigantica (1,2). These flukes cause severe public health issues and critical economic losses in livestock production (3,4). Fascioliasis in domestic ruminants leads to economic loss because of reduced production of milk, meat, and wool; reduced herd productivity; condemned livers; and raised mortality rates of animals (5,6). Fasciola gigantica is prevalent in tropical zones, while F. hepatica is in temperate areas, and both species can exist in subtropical regions (7,8). Previous studies have demonstrated that both species are common in the Middle East, particularly in Iran, Turkey, Saudi Arabia, and Iraq (9,10). The distribution of Fasciola species depends on the specific intermediate snail host, local plants, and environmental conditions on which their cercariae encyst (11,12). Duhok province consists of different geographical and climatic regions. There are some areas, especially in the highlands, which are suitable habitats for amphibian snails, such as Lymnaea truncatula, the intermediate host for F. hepatica (13), and others are aquatic environments for the distribution of aquatic snails like Lymnaea Auricularia (Radix Auricularia), the intermediate host of F. gigantica (14). Historically, morphological differences have been used to distinguish these two species (15). Compared to F. gigantica, adults of F. hepatica are smaller, with a broader shoulder, a smaller ventral sucker, and a more extended cephalic cone (16). Traditional morphological approaches were the only way to identify Fasciola species for a long time. Due to the limitations of morphological identification of Fasciola species, various molecular tools can be used to identify and differentiate Fasciola species (17-19). Furthermore, molecular methods and markers are required for species confirmation and distinguishing intermediate forms (20,21). Sequences of the first and second internal transcribed spacers of the rDNA (ITS1 and ITS2) can distinguish the two species and their intermediate forms (22,23). Siribat et al. (24) confirmed that the ITS1 region of rDNA is the most effective genetic marker for identifying Fasciola species.

However, so far, limited studies have been conducted in Iraq for molecular characterization and genotyping of Fasciola spp. Hence, this study aims to use molecular markers to characterize and identify Fasciola spp. isolated from different hosts in different localities of Duhok province based on the first internal transcribed spacer (ITS1) rDNA sequence and to find the phylogenetic relationships between the isolated specimens of the present study and other isolates from various parts of the world.

Material and methods

Samples and data collection permit

Approval was obtained from the Veterinary Directorate in Duhok Province (No. 1017, dated 9/13/2020) to examine and collect liver samples of slaughtered ruminants at Duhok Province Abattoirs infected with Fasciola spp.

Samples (parasites) collection

Adult Fasciola spp. flukes were collected from the livers of 54 naturally infected local ruminants (sheep, goats, and cattle) slaughtered at various abattoirs in Duhok province from November 2020 to October 2021. The flukes were isolated from infected livers using forceps and placed into Petri dishes containing 0.9% normal saline solution, washed with PBS (pH 7.2), preserved in 70% ethanol, and kept frozen at-20 ^oC to be used for genomic DNA extraction.

Morphological identification

Each fluke was placed between two slides for morphological identification of the species of liver flukes and identified according to the descriptions of (25,26).

DNA extraction

All individual worms were washed three times with PBS before DNA extraction to remove the ethanol, and then a small piece of the posterior region of an adult fluke was cut and weighted (about 20-25 mg). Total DNA was extracted from 54 adult flukes individually (one adult fluke was taken for each case of infection) using an AddPrep Genomic DNA Extraction Kit (Add-bioInc., Korea) and according to the manufacturer's reference protocols. The DNA concentration and purity of the extracted DNA were evaluated by the NanoDrop Spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific Inc., USA), and the samples were stored at -20 °C for further analysis.

Primer’s design and preparation:

To amplify the DNA fragment of ITS1 regions of Fasciola spp. isolates, a pair of primers were designed using Geneious (version: 2016.3.4), primer BLAST software [available at], from the consensus sequence obtained by the multiple alignments, and with an expected amplicon size of 518 bp of ITS1 sequence. These primers were named FITS1-F as the forward primer (5-CATTGAGGTCACAGCATATCCG-3) and FITS1-R as the reverse primer (5-GGCGTCGTGATAGTTTATAAGCC-3). The primers were synthesized in Germany.

DNA amplification using PCR

DNA fragments of ITS1 regions were amplified by the FITS1-(F and R) primers through the polymerase chain reaction (PCR). The PCR reaction was carried out in a 20 μl reaction volume that included 10 μl of Hot Start DNA Master Mix (2X), 5 μl of DNA template, 1 μl of each forward and reverse primer (= 2 μl), and 3 μl of nuclease free deionized water. A thermocycler (Gene AMP PCR System 9700 Thermocycler) was used to perform the amplification. The PCR cycling protocol for ITS1 was as follows; an initial denaturation step at 95 ˚C for 10 min, followed by 35 cycles of denaturation at 95˚C for 30 sec, annealing at 58˚C for 30 sec, and extension at 72˚C for 30 sec; followed by one cycle of final extension at 72˚C for 5 min, and hold at 4˚C. The amplification of the ITS1 fragments by PCR was confirmed by electrophoresis on a 1.5% agarose gel in 100 ml of 1X TAE buffer, subsequent staining with RedSafe Dye, and visualization with a UV Transilluminator.

DNA sequencing and phylogenetic analyses

Five amplicons (PCR products) of Fasciola isolates were selected depending on different hosts (2 sheep, 2 cattle, and 1 goat) and localities (Duhok, Zakho, Akre). The selected samples with the amplifying primers as sequencing primers were sent to Macrogen Company (Korea) for DNA sequencing. All sequences were cleaned up and aligned by BioEdite and Geneious software programs. The Basic Local Alignment Search Tool (BLAST) program in NCBI-GenBank was applied to identify and compare the obtained nucleotide sequences based on the ITS1 region [available at]. Pairwise identity percent of isolated Fasciola species sequences from this study and blast hits were aligned and analyzed using the ClustalW method (27) and Geneious (version: 10.2.3.) software. The sequences were entered into MEGA 11 (28) software for constructing a phylogenic tree using the Neighbor-Joining method (29), and for the calculation of evolutionary distance, the Kimura 2-parameter method was used (30). Also, the pairwise genetic distances (evolutionary divergence) between the ITS1 sequences were conducted using the proportion distance (p-distance) method in MEGA 11 software and used as percentage values. The details of Fasciola species rDNA ITS1 sequences from different countries available in NCBI-GenBank that were used for comparison of similar rates and constructing a phylogenic tree with sequences of current study are shown in table (1).

Table 1:Details of Fasciola species rDNA ITS1 sequences from different countries available in NCBI-GenBank that were used for phylogenic analysis.

Species	Country	Accession numbers	Host	Isolate code	Locality
F. heptica	Iran	MN784625	Cow	SanandajV2
F. hepatica	India	KX198628	Sheep	8772	Kashmir
F. hepatica	Chine	KJ789331	Cattle	YW2014.5.4
F. hepatica	Spain	MG569981		FhH2ASpain	La corunya
F. hepatica	Switzerland	MK321601	Cattle	ChE 3-4
F. hepatica	Tunisia	GQ231547		Ari 11
F. hepatica	Aljeria	MK212150	Bos taurus	F12-Algeeria
F. hepatica	North Africa	MN559387
F. gigantica	Iran	MN784632	Sheep	AhavzV2
F. gigantica	Pakistan	KF638561	Buffalo	bdnfgPKI
F. gigantica	India	KX198619	Cattle	76555	Kashmir
F. gigantica	China	KJ789336		YW2014.5.4.5
F. gigantica	Kenya	MZ396913		60_ITS1_CO8
F. sp.	China	MN310034	Ovis aries	HLJI	Heilongjiang
F. sp.	Zimbabwe	MK330624	P. Columella
F. nyanzae	Zimbabwe	MW046870	R. Natalensis
F. nyanzae	Zimbabwe	MT893595	R. Natalensis

F = Fasciola, P = Pseudosaccinea, R = Rax,

Results

Figure 1 shows Fasciola gigantica in the bile duct of the infected local cattle slaughtered at Zakho abattoir. Calcification, dilatation, and hyperplasia appear in the biliary duct. The morphology and shape of F. hepatica and F. gigantica are shown in (Figures 2 and 3). F. gigantica is longer than F. hepatica, while its shoulders are wider. The width of the F. hepatica is irregular along the length of the parasite’s body; the posterior part is pointed and has prominent shoulders, while in F. gigantica, the width is regular along the length of the parasite’s body with no protruding shoulders.

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

Species	Country	Accession numbers	Variable sites in the ITS1	Host	Isolate code	Locality
Species	Country	Accession numbers	13 90 107 185 205 341	Host	Isolate code	Locality
F. hepatica	Iraq/ Duhok	OM948733	- A C T C A	Cattle	AC58	Akre
F. hepatica	Iraq/ Duhok	OM920534	- A C T C A	Sheep	ZS27	Zakho
F. hepatica	Iraq/ Duhok	OM920533	A A C T C A	Cattle	ZC63	Zakho
F. gigantica	Iraq/ Duhok	OM948683	T A T A T A	Sheep	DS29	Duhok
F. gigantica	Iraq/ Duhok	OM918714	T A T A T A	Goats	ZG62	Zakho

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).

No.	Code and (Acc. No.)	1	2	3	4	5
No.	Code and (Acc. No.)	Fh ZC63	Fh ZS27	Fh AC58	Fg ZG62	Fg DS29
1	Fh ZC63 (OM920533)		0	0	0.082	0.082
2	Fh ZS27 (OM920534)	100		0	0.065	0.065
3	Fh AC58 (OM948733)	100	100		0.065	0.065
4	Fg ZG62 (OM918714)	99.2	99.4	99.4		0
5	Fg DS29 (OM948683)	99.2	99.4	99.4	100
6	Fh Iran (MN784625)	100	100	100	99.2	99.2
7	Fh India (KX198628)	100	100	100	99.2	99.2
8	Fh Spain (MG569981)	100	100	100	99.2	99.2
9	Fh.Chine (KJ789331)	100	100	100	99.2	99.2
10	Fg Iran (MN784632)	99.2	99.4	99.4	100	100
11	Fg India (KX198619)	99.2	99.4	99.4	100	100
12	Fg Chine (KJ789336)	99.2	99.4	99.4	100	100
13	Fg Kenya (MZ396913)	99.2	99.4	99.4	100	100

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.

3000 bp ----------------

1000 bp ----------------

500 bp --

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.

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