Presence of Trichobilharzia szidati in Lymnaea stagnalis and T. franki in Radix auricularia in northeastern France: molecular evidence
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- Ferté, H., Depaquit, J., Carré, S. et al. Parasitol Res (2005) 95: 150. doi:10.1007/s00436-004-1273-7
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A molecular approach was used to analyse a focus of cercarial dermatitis in northeastern France (Lake Der-Chantecoq), including both cercariae and snails,by sequencing the internal transcribed spacers (ITS1 for ocellate furcocercariae and ITS2 for snails). Lymnaea stagnalis were found infected with the furcocercariae of Trichobilharzia szidati, and T. franki furcocercariae were found in Radix auricularia. The record of these two visceral parasites of birds in northern France confirms strong host-parasite relationships. The use of these standardised markers will be of the highest significance for our understanding of the epidemiology of cercarial dermatitis in this recreational lake.
Cercarial dermatitis caused by different species of bird schistosomes is now considered an emergent disease (Gentile et al. 1996; Horák et al. 2002; Verbrugge et al. 2004). Several foci are well known in Europe: Italy (Golo et al. 1998), Germany (Allgöver and Matuschka 1993; Pilz et al. 1995), Austria (Allerberger et al. 1994, Cerroni et al. 1995), Switzerland (Eklu-Natey et al. 1985; Chamot et al. 1998), the Netherlands (Leenen and de Roda Husman 2004; Sluiters 2004), Iceland (Kolářová et al. 1999), Belarus (Beer et al. 1995), Poland (Zbikowska 2003, 2004), and the Czech Republic (Kolářová et al. 1989). In France, the most active focus is located in Annecy (Léger and Martin-Loehr 1999; Caumes et al. 2003) where a negative impact has been observed on tourism. At this site important mechanisms were put forward to fight against and successfully control the disease.
In September 2000, several cases of macular skin eruption with intense itching were observed in children and fishermen coming from the Lake Der-Chantecoq area. We were in charge of the epidemiological study of this new focus.
Materials and methods
Located in northeastern France (4°45′E, 48°35′N), Lake Der-Chantecoq is the most important artificial lake in Western Europe. Although tourism (yachting, swimming) is the predominant activity in the summer season, the main aim of this lake is to regulate the flow of the Seine river throughout the year. Listed as a natural area by the RAMSAR International convention on wetlands, it is a famous place for bird watching (migrant and sedentary species).
From September to November 2001, snails were collected weekly from and around five beaches. From June to September 2002, snails were collected daily by hand, early in the morning. In light of the large number of snails caught, they were pooled by tens in Petri dishes and cercarial emergence was stimulated by lighting over 30 min to 1 h in the laboratory. Snails from positive batches were separated into individual Petri dishes for a second assay. Screening of cercariae was performed using the morphological features proposed by Combes et al. (1980). Furcocercariae with pigmented eyes spot were preserved in 95% ethanol and frozen (−20°C) until DNA analysis. Positive snail hosts were also frozen directly at −20°C in individual sterile bags for storage. Their identification was performed at the generic level according to Glöer and Meier-Brook (1998). Sequencing of the internal transcribed spacers (ITS1 and ITS2) was used for the specific identification of naturally infected snails and furcocercariae.
Isolated cercariae were characterised by stereomicroscopy, then placed individually into sterile microvials. DNA extraction was done using the Qiamp DNA mini kit (Qiagen, Germany) following the manufacturer’s instructions. During the first step (tissue lysis), cercariae (or a small part of the foot of each positive snail) were crushed one by one using a piston pellet (Treff, Switzerland), and the DNA eluted in 50 µl of the elution buffer provided by the manufacturer. PCR were performed in a 50 µl volume using 5 µl of extracted DNA solution and 50 pmol of each of the primers. For the cercariae, we used the primers designed by Dvořák et al. (2002): the whole region including ITS1, 5.8S and ITS2 of the ribosomal DNA was amplified using primers its5Trem (5′-GGAAGTAAAAGTCGTAACAAGG-3′) complementary to the conserved region at the 3′ end of the 18S rDNA gene and its4Trem (5′-TCCTCCGCTTATTGATATGC-3′) complementary to the conserved region at the 5′ end of the 28S rDNA gene. ITS1 alone was amplified using the primers its2Trem (5′-GCTGCACTCTTCATCGACGC-3′) and its5Trem under the same conditions as those previously used (Dvořák et al. 2002). For the amplification of the positive snails, we used universal primers C1a (5′-CCTGGTTAGTTTCTTTTCCTCCGCT-3′) and ITS3 (5′-GTCGATGAAGAACGCAGC-3′) in order to amplify the ITS2 rDNA. The PCR mix contained (final concentrations) 10 mM Tris HCl, pH 8.3, 1.5 mM MgCl2, KCl 50 mM, TritonX 100 0.01%, 200 µM dNTP each, and 0.25 µl (1.25 units) of Taq polymerase (Qiagen, Germany). For cercariae, initial denaturation at 94°C for 5 min was followed by 40 cycles of denaturation at 94°C for 1 min, annealing at 62°C for 45 s and extension at 72°C for 2 min with a final elongation time of 10 min at 72°C. For snails, the initial denaturation at 94°C for 5 min was followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 57°C for 30 s and extension at 72°C for 1 min with a final elongation time of 10 min at 72°C. Amplicons were analysed by electrophoresis in 1.5% agarose gel containing ethidium bromide. PCR products were directly sequenced in both directions (Qiagen, Hilden, Germany) using the primers used for DNA amplification. Sequence alignment was performed using the MUST software package (Philippe 1993). The identification of furcocercariae was performed by comparisons of the ITS1 sequences obtained from the studied specimens with those published by Dvořák et al. (2002). Similarly, the identification of snails was performed by comparison of the sequences obtained with those provided by Bargues et al. (2001). Sequence alignment and distance analysis using neighbour-joining (NJ) were performed using MUST. Maximum parsimony and bootstrap analyses were performed using PAUP* version 4.0 software (Swofford 2002). Outgroups were taken from sister groups, according to Bargues et al. (2001). The sequences are deposited in GenBank (accession nos. AY795570–AY795575).
Results and discussion
The prevalence of snails infected by ocellate furcocercariae in different countries
Kolářová et al.(1992)
Golo et al. (1998)
Loy and Hass (2001)
There is a strong association between the genotypes of R. auricularia and T. franki. Similar pairs were observed in France and the Czech Republic. Aquatic birds could carry both parasites and snails during their flyways.
T. franki and T. szidati are two visceral schistosomes of birds. We did not record furcocercariae of T. regenti Horák, Kolářová and Dvořák, 1998, the nasal schistosome found in Europe.
Molecular identification of these Trichobilharzia, agents of swimmer’s itch in Europe, only began a few years ago. This approach is today needed in order to understand the epidemiological features of the cycles adequately, both locally as well as for comparisons of the pathogens from different foci. Moreover, the clinical features associated with these pathogens do not seem to be limited to cercarial dermatitis as was believed for a long time (Brumpt 1931; Desportes 1945). In the recent past, experimental infections carried out on rodents showed new symptoms affecting the lungs (Hass and Pietsch 1991; Gay et al 1999; Bayssade-Dufour et al. 2002, Horák and Kolářová 2000). The migration of Trichobilharzia schistosomules and their development in man is not known (Horák and Kolářová 2001).
For the future, a molecular approach (using the markers ITS1 for cercariae and ITS2 for snails) should become a standardised method for obtaining robust and comparable data when ambiguous morphological features of molluscs and furcocercariae are present. Taking into account the molecular heterogeneity, this tool will also be useful in the evaluation of the risk of cercarial dermatitis by the detection of Trichobilharzia DNA in water, as suggested by Hertel et al. (2002).
The authors thank Mr Thierry Cherrière and the technical staff of the syndicat mixte of Lac du Der-Chantecoq, Bertrand Desanlis, Aurélie Fortin, Aurélie Urano, Julie Berthou and Grégoire Payen (students) for help in providing samples; Monique Boutry, Chantal Grimplet and Agathe for their technical assistance; Matthieu Kaltenbach for proof reading this manuscript. Financial support for this study was provided by le Syndicat mixte du lac du Der-Chantecoq.