Echinococcus multilocularis in Estonia

To the Editor: Alveolar echinoco-ccosis (AE) caused by Echinococcus multilocularis is one of the most important emerging zoonosis in Europe. The fatality rate is >90% in untreated patients (1). In Europe, the distribution range of the zoonotic tapeworm E. multilocularis has expanded over the last few decades, and the parasite attracts increasing awareness as a public health issue (2–5). In 2003, AE was added to the list of zoonoses to be monitored in the member states of the European Union, according to Directive 2003/99/EC. This is the first report of E. multi-locularis in Estonia, which extends its northern distribution in Europe. Results of examinations of 17 red foxes shot in the eastern (Võnnu and Räpina) and western (Hiiumaa) districts of Estonia from February to December 2003 were included in this study. We examined the intestinal tracts by the sedimentation and counting technique as described (1). Echinococcus adult stages were found in 5 foxes (29.4%). Two foxes, infected with 3 and 5 adult worms, were from the Räpina district; 2 foxes, infected with 66 and 133 worms, were from the Võnnu district; and 1 fox, infected with the highest number of worms (927), was from the Hiiumaa District. The worms were retrieved, counted, washed, and stored in 90% ethanol until DNA purification. The parasites were identified as E. multi-locularis, based on the most important morphometric parameters of adult stages (length of worms, number of proglottids, terminal proglottids in percentage of total worm length, position of genital pore, and form of uterus) (2). To confirm the taxonomic status of the worms, polymerase chain reaction (PCR) was conducted, followed by restriction fragment length polymor-phism (RFLP) analysis and direct sequencing of a portion of the NADH dehydrogenase subunit I (ND1) gene of the mtDNA. A total of 6 specimens of E. multilocularis were used for genetic analysis. Total genomic DNA was extracted with the High Pure PCR Template Preparation Kit (Roche Molecular Biochemicals, Mannheim, Germany) according to manufactur-er's instructions. PCR-RFLP was performed as described by Gonzalez et al. (6). The RFLP pattern of E. multi-locularis isolates differed from that of E. granulosus. Diagnostic cleavage at the locus Eg9 of E. multilocularis with the enzyme CfoI is able to distinguish E. multilocularis and its closest relative E. granulosus (Figure, lanes 3 and 4 vs. lane 10). All 6 specimens of E. multilocularis produced identical results. A 426-bp fragment of the mitochondrial ND1 gene was amplified …