Hostname: page-component-78c5997874-xbtfd Total loading time: 0 Render date: 2024-11-10T07:10:54.715Z Has data issue: false hasContentIssue false
  • English
  • Français

Detection of Echinococcus multilocularis DNA in fox faeces using DNA amplification

Published online by Cambridge University Press:  06 April 2009

S. Bretagne ,
J. P. Guillou ,
M. Morand  and
R. Houin
S. Bretagne
Affiliation:
Laboratoire de Parasitologie, Faculté de Médecine, 8 avenue du Général Sarrail, 94010 Créteil, France
J. P. Guillou
Affiliation:
CNEVA, 22 rue P. Curie, BP 67, 94703 Maisons-Alfort, France
M. Morand
Affiliation:
Laboratoire Vétérinaire du Jura, Bd Th. Vernier, BP 376, 39016 Lons-le-Saunier, France
R. Houin
Affiliation:
Laboratoire de Parasitologie, Faculté de Médecine, 8 avenue du Général Sarrail, 94010 Créteil, France
Get access Rights & Permissions [Opens in a new window]

Summary

In order to identify Echinococcus multilocularis DNA in fox faeces for epidemiological purposes, we have developed a new method to prepare DNA suitable for PCR amplification. DNA isolation from fox excrement was performed according to a novel procedure involving lysis in KOH, phenol–chloroform extraction and a purification step on a matrix (Prep-A-Gene®). The target sequence for amplification was the E. multilocularis U1 snRNA gene. PCR products were indistinguishable for 32 different E. multilocularis isolates and no signal was observed after ethidium bromide staining with DNAs from other tapeworm species, including E. granulosus. The sensitivity of amplification was monitored by the addition of E. multilocularis DNA or eggs to faeces free of E. multilocularis and was estimated to be 1 egg per 4 g of faeces. PCR products were blotted onto nylon membranes and hybridized with an internal oligonucleotide probe in order to confirm the results. Twenty nine faecal samples from foxes shot in Franche-Comté (East France) were tested. Out of 10 samples from foxes in which no E. multilocularis adult worms could be observed after necropsy, 7 were PCR positive, showing that the PCR test is more sensitive than microscopical examination. Out of 19 samples from foxes harbouring E. multilocularis adult worms, 18 were PCR positive. The remaining PCR-negative sample could be due either to the misidentification of the species of adult worm (E. granulosus and E. multilocularis), or to DNA variation between different isolates of E. multilocularis. Further work in the field should be initiated in order to confirm these results.

Keywords

Echinococcus multilocularisDNA extractionfoxfaecespolymerase chain reaction.
Type
Research Article
Information
Parasitology , Volume 106 , Issue 2 , February 1993 , pp. 193 - 199
Copyright
Copyright © Cambridge University Press 1993

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Allan, J. C., Craig, P. S., Garcia Noval, J., Mencos, F., Liu, D., Wang, Y., Wen, H., Zhou, P., Stringer, R., Rogan, M. & Zeyhle, E. (1992). Coproantigen detection for immunodiagnosis of echinococcosis and taeniasis in dogs and humans. Parasitology 104, 347–55.CrossRefGoogle ScholarPubMed
Allard, A., Girones, R., Juto, P. & Wadell, G. (1990). Polymerase chain reaction for detection of adenoviruses in stool samples. Journal of Clinical Microbiology 28, 2659–67.CrossRefGoogle ScholarPubMed
Barker, R. H. (1990). Mini review: DNA probe diagnosis of parasitic infections. Experimental Parasitology 70, 494–9.CrossRefGoogle Scholar
Bretagne, S., Beaujean, F., Uner, U., Bresson-Hadni, S., Liance, L. & Houin, R. (1990). Rapid technique for cryopreservation of Echinococcus multilocularis metacestodes. International Journal for Parasitology 20, 265–7.CrossRefGoogle ScholarPubMed
Bretagne, S., Robert, R., Vidaud, D., Goossens, M. & Houin, R. (1991). Structure of the Echinococcus multilocularis U1 snRNa gene repeat. Molecular and Biochemical Parasitology 46, 285–92.CrossRefGoogle ScholarPubMed
Craig, P. S., Macpherson, C. N. L., Watson-Jones, D. L. & Nelson, G. S. (1988). Immunodetection of Echinococcus eggs from naturally infected dogs and from environmental contamination sites in settlements in Turkana, Kenya. Transactions of the Royal Society of Tropical Medicine and Hygiene 82, 268–74.CrossRefGoogle ScholarPubMed
Cui, X., Li, H., Goradia, T. M., Lange, K., Kazazian, H. H., Galas, D. & Arnheim, N. (1989). Single-Sperm typing: determination of genetic distance between the Ggamma-globin and parathyroid hormone loci by using the polymerase chain reaction and alleli-specific oligomers. Proceedings of the National Academy of Sciences, USA 86, 9389–93.CrossRefGoogle Scholar
Eiden, J. J., Wilde, J., Firoozmand, F. & Yolden, R. (1991). Detection of animal and human group B rotaviruses in fecal specimens by polymerase chain reaction. Journal of Clinical Microbiology 29, 539–43.CrossRefGoogle Scholar
Eisenstein, B. I. (1990). The Polymerase Chain reaction: a new method of using molecular genetics for medical diagnosis. The New England Journal of Medicine 322, 178–83.Google ScholarPubMed
Ewald, D., Eckert, J., Gottstein, B., Deplazes, P., Straub, M. & Nigg, H. (1991). Parasitological and serological examinations of foxes in Switzerland for Echinococcus multilocularis. In Archives de la Hidatidosis, Vol. 30 (ed. De Rose, F.), pp. 911–914. Rome: John Libbey CIC srl.Google Scholar
Flisser, A., Reid, A., Garcia-Zepeda, E. & McManus, D. P. (1988). Specific detection of Taenia saginata eggs by DNA hybridization. Lancet 2, 1429–30.CrossRefGoogle Scholar
Frankel, G., Riley, L., Giron, J. A., Valmassoi, J., Friedmann, A., Strockbine, N., Falkow, S. & Schoolnik, G. K. (1990). Detection of Shigella in feces using DNA amplification. Journal of Infectious Diseases 161, 1252–6.CrossRefGoogle ScholarPubMed
Gemmell, M. A., Lawson, J. R. & Roberts, M. G. (1987). Towards global control of cystic and alveolar hydatid diseases. Parasitology Today 3, 144–51.CrossRefGoogle ScholarPubMed
Gottstein, B., Deplazes, P., Tanner, I. & Skaggs, J. S. (1991). Diagnostic identification of Taenia saginata with the polymerase chain reaction. Transactions of the Royal Society of Tropical Medicine and Hygiene 85, 248–9.Google Scholar
Gottstein, B. & Mowatt, M. R. (1991). Sequencing and characterization of an Echinococcus multilocularis DNA probe and its use in the polymerase chain reaction. Molecular and Biochemical Parasitology 44, 183–94.CrossRefGoogle ScholarPubMed
Grisot, L. (1990). Contribution à l'épidémiologie de l'échinococcose alvéolaire dans le Doubs. Veterinary thesis, Lyon.Google Scholar
Jansen, R. W., Siegl, G. & Lemon, S. M. (1990). Molecular epidemiology of human hepatitis A virus defined by an antigen-capture polymerase chain reaction method. Proceedings of the National Academy of Sciences, USA 87, 2867–71.Google Scholar
Perez-Esandi, M. V., Colli, C. W. & Schantz, P. M. (1974). The ovicidal effect of selected chemicals against eggs of Echinococcus granulosus. Bulletin of the World Health Organization 51, 550–1.Google ScholarPubMed
Rausch, R. L., Wilson, J. F. & Schantz, P. M. (1990). A programme to reduce the risk of infection by Echinococcus multilocularis: the use of Praziquantel to control the cestode in a village in the hyperendemic region of Alaska. Annals of Tropical Medicine and Parasitology 84, 239–50.CrossRefGoogle Scholar
Rishi, A. K. & McManus, D. P. (1987). Genomic cloning of human Echinococcus granulosus DNA: isolation of recombinant plasmids and their use as genetic markers in strain characterization. Parasitology 94, 369–83.Google Scholar
Sarker, G. & Sommer, S. S. (1990). Shedding light on PCR contamination. Nature, London 343, 27.CrossRefGoogle Scholar
Scholl, D. R., Kaufmann, C., Jollick, J. D., York, C. K., Goodrum, G. R. & Charache, P. (1990). Clinical application of novel sample processing technology for the identification of Salmonellae by using DNA probes. Journal of Clinical Microbiology 28, 237–41.CrossRefGoogle ScholarPubMed
Sidranski, D., Tokino, T., Hamilton, S. R., Kinzler, K. W., Levin, B., Frost, P. & Vogelstein, B. (1992). Identification of ras oncogene mutations in the stool of patients with curable colorectal tumors. Science 256, 102–4.CrossRefGoogle Scholar
Smyth, J. D. & McManus, D. P. (1989). The Physiology and Biochemistry of Cestodes, 1st Edn.Cambridge: Cambridge University Press.CrossRefGoogle Scholar
Thompson, R. C. A. (1986). The Biology of Echinococcus and Hydatid Disease. 1st Edn.London: George Allen & Unwin.Google Scholar
Thompson, R. C. A. & Eckert, J. (1982). The production of eggs by Echinococcus multilocularis in the laboratory following in vivo and in vitro development. Zeitschrift für Parasitenkunde 68, 227–34.Google Scholar
Wilde, J., Eiden, J. & Yolken, R. (1990). Removal of inhibitory substances from human fecal specimens for detection of group A rotaviruses by reverse transcriptase and polymerase chain reaction. Journal of Clinical Microbiology 28, 1300–7.CrossRefGoogle Scholar
Yap, K. W. & Thompson, R. C. A. (1987). CTAB precipitation of cestode DNA. Parasitology Today 3, 220–2.CrossRefGoogle ScholarPubMed