Parasitology Research

, Volume 111, Issue 3, pp 1103–1111 | Cite as

Molecular, biochemical, and morphometric characterization of Fasciola species potentially causing zoonotic disease in Egypt

  • Hoda H. El-Rahimy
  • Abeer M. A. Mahgoub
  • Naglaa Saad M. El-Gebaly
  • Wahid M. A. Mousa
  • Abeer S. A. E. Antably
Original Paper

Abstract

Fascioliasis is an important disease caused by Fasciola hepatica and Fasciola gigantica. The distributions of both species overlap in many areas of Asia and Africa including Egypt. Fifty adult Fasciola worms were collected from livers of cattle and sheep slaughtered in abattoirs, Cairo, Egypt. They were subjected to morphological and metric assessment of external features of fresh adults, morphological and metric assessment of internal anatomy of stained mounted worms, determination of electrophorezed bands of crude adult homogenates using SDS-PAGE, and molecular characterization of species-specific DNA segments using RFLP-PCR. It was found that the correlation between conventional morphology and its morphotype was statistically significant (P value = 0.00). Using SDS-PAGE, 13 bands were detected among both genotypes of Fasciola (35.7, 33.6, 32.4, 29.3, 27.5, 26, 24.4, 23, 21.45, 19, 16.75, 12.5, and 9.1 kDa).The most prevalent bands were that with a molecular weight of 29.3, 26, and 19 kDa. Bands detected were common for both species, but protein bands could not distinguish between F. hepatica and F. gigantica. The result of PCR for the amplification of the selected 28S rDNA fragment with the designed primer set yielded 618 bp long PCR products for F. hepatica and F. gigantica. Different band patterns generated after digestion of the 618 bp segment by the enzyme AvaII obtained with F. hepatica showed segments of the length 529, 62, 27 bp, while with F. gigantica 322, 269, 27 bp bands were obtained. Genotyping revealed no equivocal results. The conventional morphological parameters for species determination of Fasciola spp. endemic in Egypt were evaluated versus protein bands characterization and genotyping. It was concluded that conventional morphological and metric assessments were not useful for differentiation between F. gigantica and F. hepatica due to extensive overlap in the relative ranges. Similar conclusion was reached concerning protein band characterization where the patterns of protein banding were mostly similar. In contrast, genotyping using RFLP-PCR gave consistent results and clear differentiation between the two species. Considering the implications of proper speciation of endemic parasites on clinical evaluation, therapy, epidemiology, and control measures, speciation of parasites is currently revised on molecular basis. The presently used molecular tool is therefore recommended for further study to help draw a proper map for geographical distribution of Fasciola species.

Reference

  1. Ai L, Dong SJ, ZhangWY EHM, Mahmmod YS, Lin RQ, Yuan ZG, Shi YL, Huang WY, Zhu XQ (2010) Specific PCR-based assays for the identification of Fasciola species: their development, evaluation and potential usefulness in prevalence surveys. Ann Trop Med Parasitol 104(1):65–72PubMedCrossRefGoogle Scholar
  2. Ai L, Chen MX, Alasaad S, Elsheikha HA, Li J, Li HL, Lin RQ, Zou FC, Zhu XQ, Chen JX (2011) Genetic characterization, species differentiation and detection of Fasciola spp. by molecular approaches. Parasit Vect 4:101–106CrossRefGoogle Scholar
  3. Alasaad S, Soriguer RC, Abu-Madi M, El Behairy A, Baños PD, Píriz A, Fickel J, Zhu XQ (2011) A fluorescence-based polymerase chain reaction-linked single-strand conformation polymorphism (F-PCR-SSCP) assay for the identification of Fasciola spp. Parasitol Res 108(6):1513–1517PubMedCrossRefGoogle Scholar
  4. Allam AF, El-Agamy ESI, Helmy MH (2002) Molecular and immunological characterization of Fasciola species. Br J Biomed Sci 59:191–195PubMedGoogle Scholar
  5. Ashrafi K, Valero MA, Panova M, Periago MV, Massoud J, Mas-Coma S (2006) Phenotypic analysis of adults of Fasciola hepatica, Fasciola gigantica and intermediate forms from the endemic region of Gilan, Iran. Parasitol Int 55:249–260PubMedCrossRefGoogle Scholar
  6. Bargues MD, Mas-Coma S (1997) Phylogenic analysis of lymnaeid snails based on 18S rDNA sequences. Mol Boil Evol 14:569–577CrossRefGoogle Scholar
  7. Boray JC (1982) Fascioliasis. In: Hillyer GV, Hopla CE (eds) Handbook Series in Zoonoses, Section C. Parasitic Zoonoses, vol III. CRC Press, Boca Raton, pp 71–88Google Scholar
  8. Cheesbrough M (1991) Microscopy. In: Cheesbrough M (ed) Medical laboratory manual for tropical countries, vol 1, Chapter 4. Butterworth-Heinemann, Jordan Hill, pp 67–85Google Scholar
  9. Demerdash ZA, Diab TM, Aly IR, Mohamed SI, Mahmoud FS, Zoheiry MK, Mansour WA, Attia ME, El-Bassioun AE (2011) Diagnostic efficacy of monoclonal antibody based sandwich enzyme linked immunosorbent assay (ELISA) for detection of Fasciola gigantica excretory/secretory antigens in both serum and stool. Parasit Vect 4:176–182CrossRefGoogle Scholar
  10. El-Badry AA (2006) Serum malondialdehyde levels as a biomarker of cellular injury in human fascioliasis. JT U Med Sc 1(1):57–64Google Scholar
  11. El-Shazly AM, El-Nahas HA, Soliman M, Sultan DM, Abedl Tawab AH, Morsy TA (2006) The reflection of control programs of parasitic diseases upon gastrointestinal helminthiasis in Dakahlia Governorate Egypt. J Egypt Soc Parasitol 36(2):467–480PubMedGoogle Scholar
  12. Fredes F, Gorman T, Silva M, Alcaino H (1997) Diagnostic evaluation of chromatographic fractions of Fasciola hepatica using Western Blot and ELISA in infected animals. Archivos de Med Vet 29:283–294Google Scholar
  13. Garfin DE (2003) Gel electrophoresis of protiens. In: Davey J, Lord M (eds) Essential cell biology, vol 1, Chapter 7: cell structure, a practical approach. Oxford university press, Oxford, pp 197–268Google Scholar
  14. Ghavami MB, Rahimi P, Haniloo A, Mosavinasab SN (2009) Genotypic and phenotypic analysis of Fasciola isolates. Iran J Parasitol 4(3):61–70Google Scholar
  15. Gonenc B, Sarimehmetoglu HO, Kara M, Kircali F (2004) Comparison of crude and excretory/secretory antigens for the diagnosis of Fasciola hepatica in sheep by western blotting. Turk J Vet Anim Sci 28:943–949Google Scholar
  16. Haseeb AN, El-Shazly AM, Arafa MA, Morsy AT (2002) A review on fascioliasis in Egypt. J Egypt Soc Parasitol 32(1):317–354PubMedGoogle Scholar
  17. Huang WY, He B, Wang CR, Zhu XQ (2004) Characterisation of Fasciola species from Mainland China by ITS-2 ribosomal DNA sequence. Vet Parasitol 120(1–2):75–83PubMedCrossRefGoogle Scholar
  18. Hussein AA, Khalifa RMA (2010) Phenotypic description and prevalence of Fasciola species in Qena Governorate, Egypt with special reference to a new strain of Fasciola hepatica. Science 22:1–8Google Scholar
  19. Ichikawa M, Itagaki T (2010) Discrimination of the ITS1 types of Fasciola spp. based on a PCR-RFLP method. Parasitol Res 106(3):757–761PubMedCrossRefGoogle Scholar
  20. Laemmli UK (1970) Cleavage of structural protein during the assembly of the head bacteriophage. Nature 227:680–685PubMedCrossRefGoogle Scholar
  21. Lotfy WM, Hillyer GV (2003) Fasciola species in Egypt. Exp Path Parasit 6(11):9–22Google Scholar
  22. Lotfy WM, El-Morshedy HN, Abou El-Hoda M, El-Tawila MM, Omar EA, Farag HF (2002) Identification of the Egyptian species of Fasciola. Vet Parasit 103:323–332CrossRefGoogle Scholar
  23. Marcilla A, Bargues MD, Mas-Coma S (2002) A PCR-RFLP assay for the distinction between Fasciola hepatica and Fasciola gigantica. Mol Cell Probes 16:327–333PubMedCrossRefGoogle Scholar
  24. Mas-Coma S, Bargues MD, Valero MA (2005) Fascioliasis and other plant-borne trematode zoonoses. Int J Parasitol 35:1255–1278PubMedCrossRefGoogle Scholar
  25. Mas-Coma S, Valero MA, Bargues MD (2009) Fasciola, lymnaeids and human fascioliasis, with a global overview on disease transmission, epidemiology, evolutionary genetics, molecular epidemiology and control. Adv Parasitol 69:41–146PubMedCrossRefGoogle Scholar
  26. Meshgi B, Eslami A, Hemmatzadeh F (2008) Determination of somatic and excretory-secretory antigens of Fasciola hepatica and Fasciola gigantica using SDS-PAGE. Iran J Vet Res 9(1):77–80Google Scholar
  27. Moazeni M, Sharifiyazdi H, Izadpanah A (2011) Characterization of Fasciola hepatica genotypes from cattle and sheep in Iran using cytochrome C oxidase gene (CO1). Parasitol Res (in press)Google Scholar
  28. Peng M, Ichinomiya M, Ohtori M, Ichikawa M, Shibahara T, Itagaki T (2009) Molecular characterization of Fasciola hepatica, Fasciola gigantica, and aspermic Fasciola sp. in China based on nuclear and mitochondrial DNA. Parasitol Res 105:809–815PubMedCrossRefGoogle Scholar
  29. Periago MV, Valero MA, Panova M, Mas-Coma S (2006) Phenotypic comparison of allopatric populations of Fasciola hepatica and Fasciola gigantica from European and African bovines using a computer image analysis system (CIAS). Parasitol Res 1–20. The website where it is located is at: http://www.ncbi.nlm.nih.gov/pubmed/16568331
  30. Periago MV, Valero MA, El-Sayed M, Ashrafi K, El-Wakeel A, Mohamed MY, Desquesnes M, Curtale F, Mas-Coma S (2008) First phenotypic description of Fasciola hepatica/Fasciola gigantica intermediate forms from the human endemic area of the Nile Delta, Egypt. Infect Genet Evol 7:51–58CrossRefGoogle Scholar
  31. Ramadan NI, Saber LM, Abd El Latif MM, Abdalla NA, Ragab HM (2010) Molecular genetic approach by using the RAPD-PCR technique for detection of genetic variability in non-human isolates of Fasciola. NY Sci J 3(10):88–96Google Scholar
  32. Reblánová M, Spakulová M, Orosová M, Králová-Hromadová I, Bazsalovicsová E, Rajský D (2011) A comparative study of karyotypes and chromosomal location of rDNA genes in important liver flukes Fasciola hepatica and Fascioloides magna (Trematoda: Fasciolidae). Parasitol Res 109(4):1021–1028PubMedCrossRefGoogle Scholar
  33. Rokni M, Mirhendi H, Behnia M, Fasihi M, Jalalizand N (2010a) Molecular characterization of Fasciola hepatica isolates by RAPD-PCR and ribosomal ITS1 sequencing. IRCMJ 12(1):27–32Google Scholar
  34. Rokni MB, Mirhendi H, Mizani A, Mohebali M, Sharbatkhori M, Kia EB, Abdoli H, Izadi S (2010b) Identification and differentiation of Fasciola hepatica and Fasciola gigantica using a simple PCR-restriction enzyme method. Exp Parasitol 124:209–213PubMedCrossRefGoogle Scholar
  35. Sampaio-Silva ML, Da Costa JM, Da Costa AM, Pires MA, Lopes SA, Castro AM, Monjour L (1996) Antigenic components of excretory–secretory products of adult Fasciola hepatica recognized in human infections. AmJTrop Med Hyg 54:146–148Google Scholar
  36. Sarimehmetoglu HO (2002) Application of Western Blotting for the immunodiagnosis of Fasciola hepatica in cattle using excretory/secretory antigens. Turk J Vet Anim Sci 26:1061–1065Google Scholar
  37. Tantrawatpan C, Maleewong W, Wongkham C, Wongkham S, Intapan PM, Nakashima K (2003) Characterisation of Fasciola gigantica adult 27-kDa excretory–secretory antigen in human fascioliasis. Parasitol Res 91(4):325–7PubMedCrossRefGoogle Scholar
  38. World Health Organization (2007) Report of the WHO Informal Meeting on use of triclabendazole in fascioliasis control. WHO/CDS/NTD/PCT/2007.1Google Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Hoda H. El-Rahimy
    • 1
  • Abeer M. A. Mahgoub
    • 1
  • Naglaa Saad M. El-Gebaly
    • 1
  • Wahid M. A. Mousa
    • 2
  • Abeer S. A. E. Antably
    • 1
  1. 1.Department of Parasitology, Faculty of MedicineCairo UniversityGarden CityEgypt
  2. 2.Department of Parasitology, Faculty of Veterinary MedicineCairo UniversityCairoEgypt

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