A Consistent PCR-RFLP Assay Based on ITS-2 Ribosomal DNA for Differentiation of Fasciola Species.

OBJECTIVE(S): Fascioliasis is a zoonotic parasitic disease caused by liver fluke species of Fasciola hepatica and Fasciola gigantica. Differentiation of these two species, based on their morphological characteristics, is difficult. The current study aimed to use PCR-RFLP assay to distinguish between F. hepatica and F. gigantica, based on profiles of RFLP, produced by effect of endonucleases on ITS2 of the ribosomal DNA genes from these two species.
MATERIALS AND METHODS: Adult Fasciola spp. were isolated from bile duct of naturally infected animals. The species of Fasciola were confirmed by sequencing the 505 bp region of the ITS2 gene in the isolates. By running the sequences of the samples in NEBcutter, suitable restriction enzymes (MspI and KpnI) were selected. Eight F. gigantica and eighteen F. hepatica samples were evaluated.
RESULTS: While RFLP pattern with MspI produced a profile by which it was difficult to differentiate these two species, KpnI along with MspI, produced a consistent pattern of a 231, 212 and 93 bp fragments in F. hepatica. This pattern was not seen in F. gigantica.
CONCLUSION: Findings of this study demonstrated that RFLP with KpnI and MspI produce a suitable pattern which simply differentiates F. hepatica from F. gigantica.

Introduction

Fascioliasis is a zoonotic parasitic disease caused by the liver fluke species of the genus Fasciola. F. hepatica and F. gigantica are two species which infect human and animals. F. hepatica has a worldwide distribution and both species are present in the tropical and subtropical regions of Africa and Asia (1).

Differentiation of these two species, based on their morphological characteristics, is difficult. The differences in the intermediate hosts, control strategies, transmission patterns and also epidemiological characteristics which overlap in some area indicate that the proper differentiation of F. hepatica and F. gigantica infections in human or animals is crucial (2).

Due to the limitations of morphological methods, several molecular approaches, using different molecular targets, have been developed for the differentiation of F. hepatica and F. gigantica (3).

Several DNA-based approaches have been used for differentiation of Fasciola species (4-7). Among them, sequencing of the first and the second internal transcribed spacers (ITS-1 and ITS-2) of ribosomal DNA and mitochondrial DNA (mtDNA) provided reliable genetic markers for species-level identification of Fasciola species (3). ITS-2 sequence is located between the 5.8S and 28S coding regions of rDNA that are highly conserved and have few inter-specific nucleotides useful for genetic characterization and identification in both F. hepatica and F. gigantica (8-9).

Although sequencing of ribosomal or mitochondrial DNA which shows the differences between the nucleotides of the desired gene, offers reliable methods for differentiation of Fasciola species, RFLP-based approaches provide a relatively simple, cost-effective and appropriate method for differential diagnosis of F. hepatica and F. gigantica. In line with this, different restriction enzymes and target genes have been used (2). The current study was performed to examine the utility of a PCR-RFLP assay for differentiation of F. hepatica and F. gigantica, based on RFLP profiles, produced by the effect of endonucleases on ITS2 of the ribosomal DNA genes from these two species.

Materials and Methods

Adult Fasciola spp. were isolated from bile duct of naturally infected sheep, goat and cattle at the slaughterhouses from various regions of Kohgiluyeh and Boyer-Ahmad province in Iran, where human cases of fasciolosis has been recently reported (10). Flukes were washed extensively in PBS (37°C) and subsequently fixed in 70% ethanol and maintained at 4°C for several weeks until used.

For genomic DNA extraction, a portion of the apical and lateral zone of adult flukes was removed and crushed. DNA from the crushed materials was extracted with phenol–chloroform method. Briefly, 500 μl of lysis buffer and 8 μl of proteinase K was added to the sample and incubated overnight at 37°C. Afterward, 100 μl of phenol–chloroform was added and centrifuged at 1000 g for 10 min at 25°C. Top aqueous phase was removed and absolute alcohol was used to precipitate the DNA. Extracted DNA was diluted in double distilled water and preserved at 4°C until used.

Amplification of the DNA was performed as described by Itagaki et al (2005) using a pair primer to amplify a 505 bp region of the ITS2 sequence (11). PCR reaction contained 3 μl of DNA solution, 0.25 μl of Taq DNA polymerase (Cinnagen, Iran), 2.5 μl of 10x PCR buffer, 1 μl of MgCl2, 1 μl of each primers (Forward: 5′-TGTGTCGATGAAGAGCGCAG-3′ and Reverse: 5′-TGGTTAGTTTCTTTTCCTCCGC-3′), 1 μl of dNTPs and 15.5 μl of DDW. The DNA product was sequenced and Fasciola species were identified.

After sequencing, using NEBcutter V2.0 software (12), the cutting sites of commercially available restriction enzymes on ITS2 sequences of F. hepatica and F. gigantica were assessed (Figure 1). MspI and KpnI were selected as the enzymes which might produce the most informative profile. For RFLP, a total volume of 20 μl, including 10 μl of ITS2 PCR product was added with 1 μl of either MspI or KpnI, 4 μl of 10x Tango buffer (Fermentas, Lithuania) and 4 μl of DD-H2O. The tubes were incubated at 37°C for 12 hr, according to the manufacturer instruction to ensure full cutting of fragments. For analyzing the digestion products, 15 μl of each product in addition to 2 μl of loading buffer were run in 2% gel electrophoresis.

Figure 1

Cutting sites of MspI restriction enzyme on a) Fasciola gigantica, b) F. hepatica, C) cutting sites of KpnI on F. hepatica. F. gigantica has no cutting sites for KpnI

Results

The species of Fasciola were confirmed by sequencing the 505 bp region of the ITS2 gene in the isolates. All amplified products of both F. hepatica and F. gigantica were digested with the MspI restriction endonuclease. Eight F. gigantica and eighteen F. hepatica samples were evaluated. RFLP pattern with MspI produced a 212 and 324 bp fragments for F. hepatica and 218 and 322 bp for F. gigantica (Figure 2). Based on these profiles, it was quite difficult to differentiate these two species. Using KpnI along with MspI (double digestion), a consistent pattern was found where KpnI cut ITS2 fragment of F. hepatica and produced a 231, 212 and 93 bp fragments (Figure 3). This pattern was not seen in F. gigantica (Figure 3).

Figure 2

RFLP pattern of PCR products of F. hepatica and F. gigantica digested by MspI. L) 100-bp DNA ladder, C) undigested PCR product for control, lane 1, 2, 4, 10, 12, 14, 15 and 19 are F. gigantica; lane 3, 5-7, 11, 13, 16-18, 20 and 21 are F. hepatica

Figure 3

RFLP pattern of PCR products of F. hepatica and F. gigantica digested by KpnI and MspI. L) 100-bp DNA ladder, C) undigested PCR product for control; lane 1, 2, 6, 7, 8, 11, 15 are F. gigantica and the rest are F. hepatica

Discussion

Differentiation of Fasciola species is crucial for proper implementation of any control measurement about both human and animal fascioliasis (1). Molecular markers based on DNA analysis have been employed for genetic characterization and identification of parasites especially helminthes (13, 14). In the present study, a rapid and simple method of RFLP assay, based on the partial rDNA of ITS2 gene, was utilized for the accurate differentiation and identification of Fasciola species.

Restriction enzymes are powerful and simple approaches toward the characterization of parasite species based on differences in their genomes. These methods have been used for differentiation among Fasciola species based on the profiles generated by the effects of endonucleases on ITS genes of these parasites (4, 5). In a study in China, effect of Hsp92II or RcaI on ITS2 region, Fasciola spp. were differentiated from one another by their unique restriction patterns (8). Isolates of Chinese Fasciola produced a mixture of patterns in two Fasciola species. Huang et al (2004) used the restriction endonucleases, Hsp92II and RcaI, on ITS2 region for molecular differentiation of Fasciola spp. and showed that Hsp92II produces different profiles in different isolates from different hosts. They reported that Hsp92II is better than RcaI for differentiating between these two species (8).

Fasciola species have been traditionally classified based on their morphological features, such as width and body length. Because of the size variations of these two species, the discrepancy of morphological features, and the presence of intermediate forms, it is difficult to distinguish the two species, solely based on these characteristics. RFLP is a powerful approach for discriminating these two species. RFLP pattern have been used for characterization of Fasciola species and also other organisms in Iran (15-18). Karimi (2008) showed that in 18S DNA region, BfrI restriction enzyme produce similar profile for both F. hepatica and F. gigantica whereas DraI generates different patterns for two species of Fasciola (15). Rokni et al (2010) used TasI restriction enzyme for ITS1 region which properly differentiated F. hepatica from F. gigantica (2). In another study, Saki et al (2011) showed that AvaII and DraII restriction enzymes in 28S DNA appropriately differentiate these two species (16). However, Ghavami et al (2009) showed that the pattern of restriction digestion in ITS2 sequence of Fasciola samples which was seen with BamHI and PagI restriction enzymes at the nucleotide positions of 230, 340 and 341 bp are specific to F. hepatica species and has no effect on F. gigantica (17).

Conclusion

In the current study, considering the sequences of ITS2 of F. hepatica and F. gigantica, two endonucleases, MspI and KpnI, were used to differentiate Fasciola spp. For MspI, the RFLP profiles of two species were very similar while for KpnI the profile was quite different and this enzyme was able to cut the ITS2 of two species of Fasciola at different sites which may be utilized for the differentiation of two species. The evaluation of the Fasciola spp. in the areas where two species of the fluke coexist is important and this simple RFLP can be used for discerning these two species.