Skip to main content
SpringerLink
Log in

A tool for diagnosis of Dicrocoelium dendriticum infection: hatching eggs and molecular identification of the miracidium

  • Original Paper
  • Published:
Parasitology Research Aims and scope Submit manuscript

Abstract

DNA primers were designed from the 18S rRNA sequence from the relevant digenean trematode Dicrocoelium dendriticum to evaluate a polymerase chain reaction-based diagnostic method of this parasite from its eggs in faeces of naturally and experimentally infected sheep. In order to get DNA from D. dendriticum eggs, several hatching mechanisms were studied. Successful results were obtained when the eggs were frozen to −80 °C and/or in liquid nitrogen and then defrosted. This method allowed the opening of the egg operculum and the liberation of the miracidium. DNA from D. dendriticum adults and from hatching egg miracidia was obtained and an amplification single band of 1.95 kb was observed using primers designed for the total 18S rRNA sequence in both cases as well as when the template DNA was from adults of the closely related parasite Fasciola hepatica; in addition, a single and specific 0.8-kb band was obtained when primers based on an internal partial 18S rRNA sequence were used. The method showed to be useful not only in samples coming from adults, but in eggs from gall bladder and faeces as well. F. hepatica internal 18S rRNA primers were also designed and used as a negative control to prove that the eggs in faeces came from D. dendriticum and not from F. hepatica. A molecular tool able to detect a minimum of about 40 D. dendriticum eggs in one of the definitive host faeces has been developed for the first time and could provide a useful molecular tool to improve the conventional coprological diagnosis for detecting D. dendriticum eggs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Alunda JM, Rojo-Vázquez FA (1983) Survival and infectivity of Dicrocoelium dendriticum eggs under field conditions in NW Spain. Vet Parasitol 13:245–249

    Article  PubMed  CAS  Google Scholar 

  • Alzieu JP, Ducos de Lahitte A (1991) Dicrocoeliasis in cattle. Bull Group Tech 6(B-402):135 (in French)

    Google Scholar 

  • Bazsalovicsová E, Králová-Hromadová I, Spakulová M, Reblánová M, Oberhauserová K (2010) Determination of ribosomal internal transcribed spacer 2 (ITS2) interspecific markers in Fasciola hepatica, Fascioloides magna, Dicrocoelium dendriticum and Paramphistomum cervi (Trematoda), parasites of wild and domestic ruminants. Helminthologia 47:76–82. doi:10.2478/s11687-010-0011-1

    Article  Google Scholar 

  • Boray JC (1985) Flukes of domestic animals. In: Gafaar SM, Howard WE, Marsh RE (eds) Parasites, pests and predators. Elsevier, Amsterdam, pp 179–218

    Google Scholar 

  • Bretagne S, Guillou Morand JP, Houin R (1993) Detection of Echinococcus multilocularis DNA in fox faeces using DNA amplification. Parasitology 106:193–199

    Article  PubMed  CAS  Google Scholar 

  • Campo R, Manga-González MY, González-Lanza C, Rollinson D, Sandoval H (1998) Characterization of adult Dicrocoelium dendriticum by isoelectric focusing. J Helminthol 72:109–116

    Article  PubMed  CAS  Google Scholar 

  • Campo R, Manga-González MY, González-Lanza C (2000) Relationship between egg output and parasitic burden in lambs experimentally infected with different doses of Dicrocoelium dendriticum (Digenea). Vet Parasitol 87:139–149

    Article  PubMed  CAS  Google Scholar 

  • Fagbemi BO, Obarisiagbon IO (1991) Common antigens of Fasciola gigantica, Dicrocoelium hospes and Schistosoma bovis and their relevance to serology. Vet Quarterly 13:81–87

    Article  CAS  Google Scholar 

  • Ferreras MC, Campo R, González-Lanza C, Pérez V, García-Marín JF, Manga-González MY (2007) Immunohistochemical study of the local immune response in lambs experimentally infected with Dicrocoelium dendriticum (Digenea). Parasitol Res 101:547–555

    Article  Google Scholar 

  • González-Lanza C, Manga-González MY, Campo R, Del-Pozo MP, Sandoval H, Oleaga A, Ramajo V (2000) IgG antibody response to ES or somatic antigens of Dicrocoelium dendriticum (Trematoda) in experimental infected sheep. Parasitol Res 86:472–479

    Article  PubMed  Google Scholar 

  • Heussler V, Kaufmann H, Glaser I, Ducommun D, Müller C, Dobbelaere D (1998) A DNA probe for the detection of Dicrocoelium dendriticum in ants of Formica spp. and Lasius spp. Parasitol Res 84:505–508

    Article  PubMed  CAS  Google Scholar 

  • Jithendran KP, Vaid J, Krishna L (1996) Comparative evaluation of agar gel precipitation, counterimmunoelectrophoresis and passive haemagglutination tests for the diagnosis of Dicrocoelium dendriticum infection in sheep and goats. Vet Parasitol 61:151–156

    Article  PubMed  CAS  Google Scholar 

  • Johnston DA, Kane RA, Rollinson D (1993) Small subunit (18S) ribosomal RNA gene divergence in the genus Schistosoma. Parasitology 107:147–156

    Article  PubMed  CAS  Google Scholar 

  • Kassim O, Gibertson DE (1976) Hatching of Schistosoma mansoni eggs and observations on motility of miracidia. J Parasitol 62:715–720

    Article  PubMed  CAS  Google Scholar 

  • Manga-González MY, González-Lanza C, Cabanas E, Campo R (2001) Contributions to and review of dicrocoeliosis, with special reference to the intermediate hosts of Dicrocoelium dendriticum. Parasitology 123:S91–S114

    Article  PubMed  Google Scholar 

  • Manga-González MY, Ferreras MC, Campo R, González-Lanza C, Pérez V, García-Marín JF (2004) Hepatic marker enzymes, biochemical parameters and pathological effects in lambs experimentally infected with Dicrocoelium dendriticum. Parasitol Res 93:344–355

    Article  PubMed  Google Scholar 

  • Manga-González MY, González-Lanza C (2005) Field and experimental studies on Dicrocoelium dendriticum and dicrocoeliasis in northern Spain. J Helminthol 79:291–302

    Article  PubMed  Google Scholar 

  • Manga-González MY, Quiroz-Romero H, González-Lanza C, Miñambres B, Ochoa P (2010) Strategic control of Dicrocoelium dendriticum (Digenea) egg excretion by naturally infected sheep. Veterinarni Medicina 55:19–29

    Google Scholar 

  • Martínez-Ibeas AM, Martínez-Valladares M, González-Lanza C, Miñambres B, Manga-González MY (2011) Detection of Dicrocoelium dendriticum larval stages in mollusc and ant intermediate hosts by PCR, using mitochondrial and ribosomal internal transcribed spacer (ITS-2) sequences. Parasitology 138:1916–1923

    Google Scholar 

  • Martínez-Pérez JM, Robles-Pérez D, Rojo-Vázquez FA, Martínez-Valladares M (2012) Comparison of three different techniques to diagnose Fasciola hepatica infection in experimentally and naturally infected sheep. Vet Parasitol 190:80–86

    Article  PubMed  Google Scholar 

  • Maurelli MP, Rinaldi L, Capuano F, Perugini AG, Veneziano G, Cringoli G (2007) Characterization of the 28S and the second internal transcribed spacer of ribosomal DNA of Dicrocoelium dendriticum and Dicrocoelium hospes. Parasitol Res 101:1251–1255. doi:10.1007/s00436-007-0629-1

    Article  PubMed  CAS  Google Scholar 

  • Mitterer KE (1975a) Untersuchungen zum Schlüpfen der Miracidien des Kleinen Leberegels Dicrocoelium dendriticum. Z Parasitenkd 48:35–45

    Article  PubMed  CAS  Google Scholar 

  • Mitterer KE (1975b) Das Schlüpfen der Miracidien der Großen Leberegels Fasciola hepatica L. in Abhängigkeit verschiedener CO2-Konzentrationen. Z Parasitenkd 47:35–43

    Article  PubMed  CAS  Google Scholar 

  • Mitterer KE (2008) Oxygen inhibits—even through in traces—the hatching of the eggs of the liver Dicrocoelium dendriticum. R Parasitol Res 102:927–929

    Article  Google Scholar 

  • Monnier P, Cliquet F, Aubert M, Bretagne S (1996) Improvement of a polymerase chain reaction assay of the detection of Echinococcus multilocularis DNA in faecal samples of foxes. Vet Parasitol 67:185–195

    Article  PubMed  CAS  Google Scholar 

  • Morozova EV, Ryskov AP, Semyenova SK (2002) RAPD variation in two Trematode species (Fasciola hepatica and Dicrocoelium dendriticum) from a single cattle population. Russian J Genetics 38:977–983

    Article  CAS  Google Scholar 

  • Otranto D, Rehbein S, Weigl S, Castacessi C, Parisi A, Lia RP, Olson PD (2007) Morphological and molecular differentiation between Dicrocoelium dendriticum (Rudolphi, 1819) and Dicrocoelium chinensis (Sudarikov and Ryjikov, 1951) Tang and Tang, 1978 (Platyhelminthes: Digenea). Acta Trop 104:91–98. doi:10.1016/j.actatropica.2007.07.008

    Article  PubMed  CAS  Google Scholar 

  • Ratcliffe LH (1968) Hatching of Dicrocoelium lanceolatum eggs. Exp Parasitol 23:67–78

    Article  Google Scholar 

  • Rojo-Vázquez FA, Cordero M, Díez P, Chaton M (1981) Relation existant entre le nombre d'oeufs dans les fèces et la charge parasitaire lors des infestations naturelles à Dicrocoelium dendriticum chez les ovins. Rev Med Vet (Toul) 132:601–607

    Google Scholar 

  • Romia SA (1991) Length and hatchability of Schistosoma mansoni eggs. J Egypt Soc Parasitol 21:439–444

    PubMed  CAS  Google Scholar 

  • Sánchez-Andrade R, Paz-Silva A, Suarez JL, Arias M, López C, Morrondo P, Scala A (2003) Serum antibodies to Dicrocoelium dendriticum in sheep from Sardinia (Italy). Prev Vet Med 57:1–5

    Article  PubMed  Google Scholar 

  • Sandoval H, Manga-González MY, Campo R, García P, Castro JM, Pérez de la Vega M (1999) Preliminary study on genetic variability of Dicrocoelium dendriticum determined by random amplified polymorphic DNA. Parasitol Int 48:21–26

    Article  PubMed  CAS  Google Scholar 

  • Steindel M, Dias Neto E, de Menezes Carla LP, Romanha AJ, Simpson AJG (1993) Random amplified polymorphic DNA analysis of Trypanosoma cruzi strains. Mol Biochem Parasitol 60:71–80

    Article  PubMed  CAS  Google Scholar 

  • Tchounwou PB, Englande AJ Jr, Malek EA, Anderson AC, Abdelghani AA (1991) The effects of ammonium sulphate and urea upon egg hatching and miracidial survival of Schistosoma mansoni. J Environ Sci Health B 26:241–257

    Article  PubMed  CAS  Google Scholar 

  • Xu YZ, Dresden MH (1986) Leucine aminopeptidase and hatching of Schistosoma mansoni eggs. J Parasitol 72:507–511

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We would like to thank Dr. R. Campo as well as C. Espiniella and M.L. Carcedo, members of the Parasitology Laboratory at the Spanish National Research Council (CSIC) in León (Spain). This work was supported by the Spanish CICYT (project no. AGF96-0416) and Castile and León Autonomy (project no. CSI5/98). H. Sandoval was supported by a Scientist Research Contract from the CSIC.

Author information

Authors and Affiliations

  1. Departamento de Sanidad Animal, Instituto de Ganadería de Montaña, Consejo Superior de Investigaciones Científicas (CSIC)- Universidad de León (ULE), Grulleros, 24346 , León, Spain

    H. Sandoval & M. Yolanda Manga-González

  2. Área de Microbiología, Departamento de Biología Molecular, Universidad de León, 24071, León, Spain

    José M. Castro

Authors
  1. H. Sandoval
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. Yolanda Manga-González
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. José M. Castro
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to José M. Castro.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sandoval, H., Manga-González, M.Y. & Castro, J.M. A tool for diagnosis of Dicrocoelium dendriticum infection: hatching eggs and molecular identification of the miracidium. Parasitol Res 112, 1589–1595 (2013). https://doi.org/10.1007/s00436-013-3313-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00436-013-3313-7

Keywords

Search

Navigation