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Molecular and Phylogenetic Status of Fasciola sp., of Cattle in Qena, Upper Egypt



Mosaab A. Omar, Asmaa M. Metwally and Khaled Sultan
 
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ABSTRACT

The species of liver fluke of the genus Fasciola (phylum platyhelminthes, order Digenea, Family Fasciolidae) are obligatory parasites that inhabit the large biliary ducts of herbivore animals as well as man. Reports on the species of Fasciola present in the Nile Delta, Egypt, appear controversial. In the current study a precise identification of Fasciola isolates from cattle in Qena province, Upper Egypt was done based on examination of the second Internal Transcribed Spacer (ITS2) and the mitochondrial cytochrome oxidase subunit I (COI). Amplification, sequencing and phylogenetic examination revealed that the collected Fasciola isolates represent only one species which is Fasciola hepatica.

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Mosaab A. Omar, Asmaa M. Metwally and Khaled Sultan, 2013. Molecular and Phylogenetic Status of Fasciola sp., of Cattle in Qena, Upper Egypt. Pakistan Journal of Biological Sciences, 16: 726-730.

DOI: 10.3923/pjbs.2013.726.730

URL: https://scialert.net/abstract/?doi=pjbs.2013.726.730
 
Received: December 08, 2012; Accepted: February 25, 2013; Published: April 04, 2013



INTRODUCTION

Fascioliasis is an important disease caused by members of genus Fasciola, transmitted by freshwater lymnaeid snails and infects many livestock species as well as man. Identification of Fasciola species depends mainly on morphological examination (Itagaki and Tsutsumi, 1998; Amer et al., 2011). Fasciola hepatica and F. gigantica thought to be the main causes of fascioliasis allover the world, the overlapping distribution both species has led to a long ranging controversy on the taxonomic identity of Fasciola species (Mas-Coma, 2004). The existence of a hybrid form of Fasciola complicates the problem of identification and subsequently misaddressing of exact epidemiological information on fascioliasis.

DNA studies and using of molecular tools aid in the exact identification of parasites. In ribosomal DNA (rDNA) the Internal Transcribed Spacer 2 (ITS2) gene have proven useful for diagnostic purposes at the level of species (Morgan and Blair 1995; Leon-Regagnon et al., 1999). ITS2 sequences have also been used to characterize and identify different Fasciola spp., (Huang et al. 2004; Amer et al., 2011). While, mitochondrial DNA (mt DNA) genes proves to be excellent markers to differentiate in-between closely-related species and testing the polymorphism of Fasciola species (Amer et al., 2011).

In Egypt, F. hepatica and F. gigantica are prevalent among livestock’s in the Nile delta (Lotfy and Hillyer, 2003) results showed that identification of Egyptian Fasciola based only on morpho-metric criteria is not a countable. Dar et al. (2012) confirmed the presence of F. hepatica, F. gigantica besides the hybrid form in Egypt. The pervious Egyptian studies focused on Fasciola isolates collected from Cairo and Delta region, with no information about situation in Upper Egypt.

So that in the present study, nucleotide sequences of the mitochondrial DNA, cytochrome oxidase subunit 1 (CO1) and Internal Transcribed Spacer 2 (ITS2) of the ribosomal RNA gene were used to identify Fasciola species that infects cattle in Qena, Upper Egypt.

MATERIALS AND METHODS

Parasitic samples: During visits to Qena city slaughter house, Upper Egypt (26°1012, N 32°4337 E), adult Fasciola specimens were obtained alive from biliary ducts of freshly slaughtered cattle. The collected specimens were washed thoroughly in physiological saline, preserved in ethyl alcohol 70% prior to genetic study.

Nucleic Acid extraction and amplification: Genomic DNA was extracted from two flukes by using a QIAGEN genomic DNA kit according to manufacturer protocol. The rDNA region spanning the ITS2 and a partial fragment of the mitochondrial CO1 gene were amplified by Polymerase Chain Reaction (PCR). The following universal trematode primer sets were used following Bowles et al. (1995) as follows for CO1 region: FH5 (forward): 5'-TTTTTTGGGCATCCTGAGGTTTA-3' and FH3 (reverse):5'-TAAAGAAAGAACATAATGAAAAT AATC -3' and for ITS2 region: 3S (forward):5'-GGTACCG GTGGATCACTCGGCTCGTG-3' and A28 (reverse): 5'-GGGATCCTGGTTAGTTTCTTTTCCTCCGC-3'.

The PCR amplification was performed following the standard protocol (White, 1993) with minor modifications as described elsewhere (Prasad et al., 2007). For DNA sequencing the PCR products were purified using the Geneclean2 PCR purification kit according to manufacturer protocol and sequenced in both directions using PCR primers.

Phylogenetic analysis: For phylogenetic analysis, the sequences were aligned using ClustalW multiple alignments W, trees were constructed using MEGA4 (Higgins and Sharp, 1988; Tamura et al., 2007). Amino acid sequences of the mitochondrial COI gene were inferred using the codon tables of Garey and Wolstenholme (1989).

RESULTS AND DISCUSSION

To identify and study the phylogenetic relationship of Fasciola sp., collected from cattle in Qena, Upper Egypt. Amplification and sequencing of ITS2 rDNA and COI mt DNA genes was done. ITS2 of rDNA of our isolate was successfully obtained, Genbank accession number is (AB510492.). ITS2 fragments of were estimated to be 481 bp long. For comparative purposes, the ITS2 sequences of fasciolids from various hosts and geographical regions were obtained from GeneBank. No difference was observed between the examined Fasciola samples and F. hepatica from other hosts from Delta of Egypt and other parts of the world (Fig. 1). Also, Upper Egypt Fasciola isolate differs than ITS2 of F. gigantica by percentage agrees as previously reported by Adlard et al. (1993). The phylogenetic tree based on ITS2 region showed a close relationship with F. hepatica from Egypt, Iran and Turkey (Fig. 2). It was remarkable that F. giagnatica “hybrid form” (EU260069) from human case in Vietnam clustered with F. hepatica.

Mitochondrial CO1 gene of our Fasciola isolate was successfully amplified and sequenced. Its Genbank accession number is (AB510491).The CO1 alignment consisted of 421bp. The G+C content were close to 37%. ‘Variation’ between sequences of Upper Egypt Fasciola sp. and F. hepatica from Genbank sequences was found at only one nucleotide site. On the other hand, F. gigantica deposited sequences differed from the examined Fasciola samples at 28±50 sites.

Fig. 1: Neighbor-Joining phylogenetic tree (1,000 bootstraps) based on ITS2gene sequences of Fasciola spp., Fascioloides magna (Accession No. EF534992) used as outgroup. Tree was constructed using MEGA4. Bootstrap values showed on internal nodes. The scale bar indicates the proportion of sites changes along each branch. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated from the dataset. There were a total of 347 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4

Fig. 2: Multiple alignments done of ITS-2 sequences of Fasciola spp.

Fig. 3: Maximum parsimony phylogenetic tree (1, 00 bootstraps) based on COIgene sequences of Fasciola spp., Fasciolopsis buski (Accession No. EF027094) used as outgroup. Tree was constructed using MEGA4. Bootstrap values showed on internal nodes. The scale bar indicates the proportion of sites changes along each branch. Codon positions included were 1st+2nd+3rd+Noncoding. Complete deletion option was used. There were a total of 310 positions in the final dataset, out of which 162 were parsimony informative. Phylogenetic analyses were conducted in MEGA4

Thus, our specimens of Fasciola sp., represent F. hepatica. Comparison of the newly obtained CO1 sequence with other Fasciola sequences on Genbank through BLAST indicates that this new isolate is pure F. hepatica. The inferred maximum parsimony phylogenetic tree based on CO1 sequences showed that our samples are F. hepatica (Fig. 3). When compared with the COI sequence reported for F. hepatica by Garey and Wolstenholme (1989). Our Fasciola sp., isolate differed at seven nucleotide sites with F. gigantica from Indonesia (AB207179). The phylogenetic analysis based on CO1 gene agreed with the conclusions drawn from ITS2 data.

In the present study, sequence analysis revealed a close relationship between the query sequence from Upper Egypt and those of F. hepatica from Egypt and other parts of the world. In Egypt molecular approaches (e.g., Dar et al., 2012; El-Rahimy et al., 2012) have been utilized to specify Fasciola species. The results are also in line with these results that confirm the presence of F. hepatica in Egypt. At the same time we note that our isolates differ from F. gigantica from Egypt, Japan and Indonesia in more than six sites (Adlard et al., 1993) and also differs that the hybrid form. According to ITS2, the Fasciola sp., collected from cattle in Qena, Upper Egypt showed itself to be almost a perfect match with F. hepatica.

CONCLUSION

The results represented herein demonstrate that the species of Fasciola infects cattle in Qena, Upper Egypt is F. hepatica. Sequencing and amino acids analysis is a precise tool for identification of Fasciola. Despite the limitation of hosts included in this study, it will be a basic stone in the future works about fascioliasis in Egypt which is strongly engorged.

REFERENCES
1:  Adlard, R.D., S.C. Barker, D. Blair and T.H. Cribb, 1993. Comparison of the second internal transcribed spacer (Ribosomal DNA) from populations and species of fasciolidae (Digenea). Int. J. Parasitol., 23: 423-425.
CrossRef  |  

2:  Amer, S., Y. Dar, M. Ichikawa, Y. Fukuda, C. Tada, T. Itagaki and Y. Nakai, 2011. Identification of Fasciola species isolated from Egypt based on sequence analysis of genomic (ITS1 and ITS2) and mitochondrial (NDI and COI) gene markers. Parasitol. Int., 60: 5-12.
CrossRef  |  PubMed  |  

3:  Bowles, J., D. Blair and D.P. McManus, 1995. A molecular phylogeny of the human schistosomes. Mol. Phylogenet. Evol., 4: 103-109.
CrossRef  |  PubMed  |  Direct Link  |  

4:  Dar, Y., S. Amer, A. Mercier, B. Courtioux and G. Dreyfuss, 2012. Molecular identification of Fasciola spp. (Digenea: Fasciolidae) in Egypt. Parasite, 19: 177-182.
PubMed  |  

5:  El-Rahimy, H.H., A.M. Mahgoub, N.S. El-Gebaly, W. M. Mousa and A.S. Antably, 2012. Molecular, biochemical and morphometric characterization of Fasciola species potentially causing zoonotic disease in Egypt. Parasitol. Res., 111: 1103-1111.
CrossRef  |  PubMed  |  

6:  Garey, J.R. and D.R. Wolstenholme, 1989. Platyhelminthes mitochondrial DNA evidence for early evolutionary origin of a tRNAser-AGN that contains a dihydrouridine arm replacement loop and of serine-specifying AGA and AGG codons. J. Mol. Evol., 28: 374-387.
PubMed  |  

7:  Higgins, D.G. and P.M. Sharp, 1988. CLUSTAL: A package for performing multiple sequence alignment on a microcomputer. Genetics, 73: 237-244.
CrossRef  |  Direct Link  |  

8:  Huang, W.Y., B. He, C.R. Wang and X.Q. Zhu, 2004. Characterization of Fasciola species from Mainland China by ITS-2 ribosomal DNA sequence. Vet. Parasitol., 120: 75-83.
CrossRef  |  PubMed  |  

9:  Itagaki, T. and K. Tsutsumi, 1998. Triploid form of Fasciola in Japan: Genetic relationships between Fasciola hepatica and Fasciola gigantica determined by ITS-2 sequence of nuclear rDNA. Int. J. Parasitol., 28: 777-781.
CrossRef  |  

10:  Leon-Regagnon, V., D.R. Brooks and G.P.P. de Leon, 1999. Differentiation of Mexican species of Haematoloechus Looss, 1899 (Digenea: Plagiorchiformes): Molecular and morphological evidence. J. Parasitol., 85: 935-946.
PubMed  |  

11:  Lotfy, W.M. and G.V. Hillyer, 2003. Fasciola species in Egypt. Exp. Pathol. Parasitol., 6: 9-22.
Direct Link  |  

12:  Mas-Coma, S., 2004. Human Fascioliasis. In: Waterborne Zoonoses: Identification, Causes and Control, Cotruvo, J.A. (Ed.). IWA Publishing, London, UK., ISBN-13: 9781843390589, pp: 305-322.

13:  Morgan, J.A. and D. Blair, 1995. Nuclear rDNA ITS sequence variation in the trematode genus Echinostoma: An aid to establishing relationships within the 37-collar spine group. Parasitology, 111: 609-615.
PubMed  |  

14:  Prasad, P.K., V. Tandon, A. Chatterjee and S. Bandyopadhyay, 2007. PCR-based determination of internal transcribed spacer (ITS) regions of ribosomal DNA of giant intestinal fluke, Fasciolopsis buski (Lankester, 1857) Looss, 1899. Parasitol. Res., 101: 1581-1587.
CrossRef  |  PubMed  |  Direct Link  |  

15:  Tamura, K., J. Dudley, M. Nei and S. Kumar, 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol., 24: 1596-1599.
CrossRef  |  PubMed  |  Direct Link  |  

16:  White, B.A., 1993. PCR Protocols: Current Methods and Applications. Vol. 15, Springer, New York, ISBN-13: 9781592595020, Pages: 392.

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