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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
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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

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