Genetic Characterization of Fasciola Isolates from West Azerbaijan Province Iran Based on ITS1 and ITS2 Sequence of Ribosomal DNA
Background: Fascioliasis, caused by Fasciola hepatica and F. gigantica, has medical and economic importance in the world. Molecular approaches comparing traditional methods using for identification and characterization of Fasciola spp. are precise and reliable. The aims of current study were molecular characterization of Fasciola spp. in West Azerbaijan Province, Iran and then comparative analysis of them using GenBank sequences.
Methods: A total number of 580 isolates were collected from different hosts in five cities of West Azerbaijan Province, in 2014 from 90 slaughtered cattle (n=50) and sheep (n=40). After morphological identification and DNA extraction, designing specific primer were used to amplification of ITS1, 5.8s and ITS2 regions, 50 samples were conducted to sequence, randomly.
Result: Using morphometric characters 99.14% and 0.86% of isolates identified as F. hepatica and F. gigantica, respectively. PCR amplification of 1081 bp fragment and sequencing result showed 100% similarity with F. hepatica in ITS1 (428 bp), 5.8s (158 bp), and ITS2 (366 bp) regions. Sequence comparison among current study sequences and GenBank data showed 98% identity with 11 nucleotide mismatches. However, in phylogenetic tree F. hepatica sequences of West Azerbaijan Province, Iran, were in a close relationship with Iranian, Asian, and African isolates.Conclusions: Only F. hepatica species is distributed among sheep and cattle in West Azerbaijan Province Iran. However, 5 and 6 bp variation in ITS1 and ITS2 regions, respectively, is not enough to separate of Fasciola spp. Therefore, more studies are essential for designing new molecular markers to correct species identification.
Sunita K, Singh DK. Fascioliasis control: In vivo and in vitro phytotherapy of vector snail to kill Fasciola larva. J Parasitol Res. 2011;2011:240807.
Nguyen S, Amer S, Ichikawa M, Itagaki T, Fukuda Y, Nakai Y. Molecular identification of Fasciola spp. (digenea: Platyhelminthes) in cattle from vietnam. Parasite. 2012;19:85-89.
Mas-Coma S, Valero MA, Bargues MD. Chapter 2. Fasciola, lymnaeids and human fascioliasis, with a global overview on disease transmission, epidemiology, evolutionary genetics, molecular epidemiology and control. Adv Parasitol. 2009;69:41-146.
Mas-Coma S, Bargues MD, Valero MA. Fascioliasis and other plant-borne trematode zoonoses. Int J Parasitol. 2005;35:1255-1278.
El-Rahimy HH, Mahgoub AM, El-Gebaly NS, Mousa WM, Antably AS. Molecular, biochemical, and morphometric characterization of Fasciola species potentially causing zoonotic disease in Egypt. Parasitol Res. 2012;111:1103-1111.
Khanjari A, Bahonar A, Fallah S, Bagheri M, Alizadeh A, Fallah M, Khanjari Z. Prevalence of fasciolosis and dicrocoeliosis in slaughtered sheep and goats in Amol Abattoir, Mazanda¬ran, northern Iran. Asian Pac J Trop Dis 2014; 4(2): 120-124.
Dar Y, Amer S, Mercier A, Courtioux B, Dreyfuss G. Molecular identification of Fasciola spp. (digenea: Fasciolidae) in Egypt. Parasite. 2012;19:177-182.
Amor N, Halajian A, Farjallah S, Merella P, Said K, Ben Slimane B. Molecular characterization of Fasciola spp. From the endemic area of northern iran based on nuclear ribosomal DNA sequences. Exp Parasitol. 2011;128:196-204.
Ashrafi K, Valero MA, Panova M, Periago MV, Massoud J, Mas-Coma S. Phenotypic analysis of adults of Fasciola hepatica, Fasciola gigantica and intermediate forms from the endemic region of Gilan, Iran. Parasitol Int. 2006;55:249-260.
Qureshi AW, Tanveer A, Qureshi SW, Maqbool A, Gill TJ, Ali SA. Epidemiology of human fasciolosis in rural areas of Lahore, Pakistan. Punjab Univ J Zool. 2005;20:159-168.
Over hJ, Jansen J, Van olm PW. Distribution and impact of helminth diseases of livestock in developing countries. FAO Animal Production and Health Paper. 1992: 96, Rome.
Mahdi NK, AL-Baldawi FA. Hepatic fasciolosis in the abattoirs of basrah. Ann Trop Med Parasit. 1987;81:377-379.
Tran VH, Tran TK, Nguyen HC, Pham HD, Pham TH. Fascioliasis in Vietnam. Southeast Asian J Trop Med Publ Hlth. 2001;32: 48-50.
Ai L, Chen MX, Alasaad S, Elsheikha HM, Li J, Li HL, Lin RQ, Zou FC, Zhu XQ, Chen JX. Genetic characterization, species differentiation and detection of Fasciola spp. By molecular approaches. Parasit Vectors. 2011;4:101.
Choe SE, Nguyen TT, Kang TG, Kweon CH, Kang SW. Genetic analysis of Fasciola isolates from cattle in Korea based on second internal transcribed spacer (its-2) sequence of nuclear ribosomal DNA. Parasitol Res. 2011;109:833-839.
Itagaki T, Kikawa M, Sakaguchi K, Shimo J, Terasaki K, Shibahara T, Fukuda K. Genetic characterization of parthenogenic Fasciola sp. In japan on the basis of the sequences of ribosomal and mitochondrial DNA. Parasitology. 2005;131:679-685.
Turhan O, Korkmaz M, Saba R, Kabaaaliogu A, Inan D, Mamikoglu L. Seroepidemiology of fascioliasis in the antalya region and uselessness of eosinophil count as a surrogate marker and portable ultrasonography for epidemiological surveillance. Infez Med. 2006;14:208-212.
WHO. Control of foodborne trematode infections. Tecnical Report Series. 1995;849. Geneva: WHO.
Massoud J. Fascioliasis outbreak of man and drug test (triclabendazole) in caspian littoral, northern part of Iran. Bull Soc Fran Parasitol. 1989;8:438.
Lotfy WM, El-Morshedy HN, Abou El-Hoda M, El-Tawila MM, Omar EA, Farag HF. Identification of the egyptian species of fasciola. Vet Parasitol. 2002;103:323-332.
Marcilla A, Bargues MD, Mas-Coma S. A pcr-rflp assay for the distinction between Fasciola hepatica and Fasciola gigantica. Mol Cell Probes. 2002;16:327-333.
Agatsuma T, Arakawa Y, Iwagami M, Honzako Y, Cahyaningsih U, Kang SY, Hong SJ. Molecular evidence of natural hybridization between Fasciola hepatica and F. gigantica. Parasitol Int. 2000;49:231-238.
Itagaki T, Kikawa M, Terasaki K, Shibahara T, Fukuda K. Molecular characterization of parthenogenic Fasciola sp. In korea on the basis of DNA sequences of ribosomal its1 and mitochondrial ndi gene. J Vet Med Sci. 2005;67:1115-1118.
Peng M, Ichinomiya M, Ohtori M, Ichikawa M, Shibahara T, Itagaki T. Molecular characterization of Fasciola hepatica, Fasciola gigantica, and aspermic Fasciola sp. In china based on nuclear and mitochondrial DNA. Parasitol Res. 2009;105:809-815.
Le TH, De NV, Agatsuma T, Thi Nguyen TG, Nguyen QD, McManus DP, Blair D. Human fascioliasis and the presence of hybrid/introgressed forms of Fasciola hepatica and Fasciola gigantica in Vietnam. Int J Parasitol. 2008;38:725-730.
Periago MV, Valero MA, El Sayed M, Ashrafi K, El Wakeel A, Mohamed MY, Desquesnes M, Curtale F, Mas-Coma S. First phenotypic description of Fasciola hepatica/Fasciola gigantica intermediate forms from the human endemic area of the nile delta, Egypt. Infect Genet Evol. 2008;8:51-58.
Farjallah S, Sanna D, Amor N, Ben Mehel B, Piras MC, Merella P, Casu M, Curini-Galletti M, Said K, Garippa G. Genetic characterization of Fasciola hepatica from tunisia and algeria based on mitochondrial and nuclear DNA sequences. Parasitol Res. 2009;105:1617-1621.
Huang WY, He B, Wang CR, Zhu XQ. Characterisation of Fasciola species from mainland china by its-2 ribosomal DNA sequence. Vet Parasitol. 2004;120:75-83.
Amer S, Dar Y, Ichikawa M, Fukuda Y, Tada C, Itagaki T, Nakai Y. 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. 2011;60:5-12.
Sahba GH, Arfaa F, Farahmandian I, Jalali H. Animal fascioliasis in Khuzestan, southwestern Iran. J Parasitol. 1972;4:712-716.
Collins FH, Petrarca V, Mpofu S, Brandling-Bennett AD, Were JB, Rasmussen MO, Finnerty V. Comparison of DNA probe and cytogenetic methods for identifying field collected Anopheles gambiae complex mosquitoes. Am J Trop Med Hyg. 1988;39:545-550.
Djadid ND, Gholizadeh S, Aghajari M, Zehi AH, Raeisi A, Zakeri S. Genetic analysis of rdna-its2 and rapd loci in field populations of the malaria vector, Anopheles stephensi (diptera: Culicidae): Implications for the control program in iran. Acta Trop. 2006;97:65-74.
Koetschan C, Forster F, Keller A, Schleicher T, Ruderisch B, Schwarz R, Muller T, Wolf M, Schultz J. The its2 database iii--sequences and structures for phylogeny. Nucleic Acids Res. 2010;38:D275-279.
Thompson JD, Higgins DG, Gibson TJ. Clustal w: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22:4673-4680.
Tamura K, Dudley J, Nei M, Kumar S. Mega4: Molecular evolutionary genetics analysis (mega) software version 4.0. Mol Biol Evol. 2007;24:1596-1599.
Gholizadeh S, Djadid ND, Nouroozi B, Bekmohammadi M. Molecular phy-logenetic analysis of anopheles and cellia subgenus anophelines (diptera: Culicidae) in temperate and tropical regions of Iran. Acta Trop. 2013;126:63-74.
Saitou N, Nei M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4:406-425.
Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution. 1985;39:783-791.
Moghaddam AS, Massoud J, Mahmoodi M, Mahvi AH, Periago MV, Artigas P, Fuentes MV, Bargues MD, Mas-Coma S. Human and animal fascioliasis in Mazandaran province, northern Iran. Parasitol Res. 2004;94:61-69.
Ghavami MB, Rahimi P, Haniloo A, Mosavinasab SN. Genotypic and phenotypic analysis of Fasciola isolates. Iran J Parasitol. 2009;4:61-70.
Shahbazi A, Akbarimoghaddam M, Izadi S, Ghazanchaii A, Jalali N, Bazmani A. Identification and genetic variation of Fasciola species from tabriz, north- western Iran. Iran J Parasitol. 2011;6:52-59.
Mahami-Oskouei M, Dalimi A, Forouzandeh-Moghadam M, Rokni MB. Molecular identification and differentiat-ion of Fasciola isolates using pcr- rflp method based on internal transcribed spacer (its1, 5.8s rdna, its2). Iran J Parasitol. 2011;6:35-42.
Karimi A. Genetic diagnosis of Fasciola species based on 18s ribosomal DNA sequences. J Biol Sci. 2008;8:1166-1173.
Rokni MB, Mirhendi H, Mizani A, Mohebali M, Sharbatkhori M, Kia EB, Abdoli H, Izadi S. Identification and differentiation of Fasciola hepatica and Fasciola gigantica using a simple pcr-restriction enzyme method. Exp Parasitol. 2010;124:209-213.
Alasaad S, Huang CQ, Li QY, Granados JE, Garcia-Romero C, Perez JM, Zhu XQ. Characterization of Fasciola samples from different host species and geographical localities in spain by sequences of internal transcribed spacers of rDNA. Parasitol Res. 2007;101:1245-1250.
Lin RQ, Dong SJ, Nie K, Wang CR, Song HQ, Li AX, Huang WY, Zhu XQ. Sequence analysis of the first internal transcribed spacer of rdna supports the existence of the intermediate Fasciola between F. hepatica and F. gigantica in mainland china. Parasitol Res. 2007;101:813-817.
Lotfy WM, Brant SV, DeJong RJ, Le TH, Demiaszkiewicz A, Rajapakse RP, Perera VB, Laursen JR, Loker ES. Evolutionary origins, diversification, and biogeography of liver flukes (digenea, fasciolidae). Am J Trop Med Hyg. 2008;79:248-255.
Ali H, Ai L, Song HQ, Ali S, Lin RQ, Seyni B, Issa G, Zhu XQ. Genetic characterisation of Fasciola samples from different host species and geographical localities revealed the existence of F. hepatica and F. gigantica in niger. Parasitol Res. 2008;102:1021-1024.
Kralova-Hromadova I, Spakulova M, Horackova E, Turcekova L, Novobilsky A, Beck R, Koudela B, Marinculic A, Rajsky D, Pybus M. Sequence analysis of ribosomal and mitochondrial genes of the giant liver fluke Fascioloides magna (trematoda: Fasciolidae): Intraspecific variation and differentiation from Fasciola hepatica. J Parasitol. 2008;94:58-67.
Prasad PK, Tandon V, Biswal DK, Goswami LM, Chatterjee A. Molecular identification of the indian liver fluke, Fasciola (trematoda: Fasciolidae) based on the ribosomal internal transcribed spacer regions. Parasitol Res. 2008;103:1247-1255.
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