Detection ofEchinococcus multilocularisDNA in fox faeces using DNA amplification

Abstract

SUMMARY: In order to identifyEchinococcus multilocularisDNA 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 theE. multilocularisU1 snRNA gene. PCR products were indistinguishable for 32 differentE. multilocularisisolates and no signal was observed after ethidium bromide staining with DNAs from other tapeworm species, includingE. granulosus.The sensitivity of amplification was monitored by the addition ofE. multilocularisDNA or eggs to faeces free ofE. multilocularisand 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 noE. multilocularisadult 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 harbouringE. multilocularisadult worms, 18 were PCR positive. The remaining PCR-negative sample could be due either to the misidentification of the species of adult worm (E. granulosusandE. multilocularis), or to DNA variation between different isolates ofE. multilocularis. Further work in the field should be initiated in order to confirm these results.