Abstract. In order to improve the spatial resolution of current risk maps for fasciolosis in cattle, more knowledge is needed with respect to farm-level factors that determine infection risk. In this study, we visited 39 dairy farms within a predefined low-and high-risk area for fasciolosis in Belgium and assessed their infection status by an indirect bulk tank milk enzyme-linked immunosorbent assay (ELISA). Management factors were collected and all pastured lands of the farms were visited to identify and georeference potential snail habitats. The habitats were visually characterised, investigated for the presence of the intermediate host snails of Fasciola hepatica (i.e.Galba truncatula and Radix spp.) and used in a geographical information system (GIS) to construct overlays including information on soil and hydrology. A linear regression model was used to evaluate associations between bulk tank milk ELISA results and farm level management and habitat factors. A logistic, mixed model was used to identify possible risk factors for the presence of intermediate host snails on a potential habitat. Potential snail habitats were found in 35 out of 39 farms. A total of 87 potential habitats were identified and on 29% of these, intermediate host snails were found. The number of potential habitats, the presence of snails, drainage of pastures, month of turnout of the cows, stocking rate, type of watering place and risk area were significantly associated with the bulk tank milk ELISA result and explained 85% of the observed variation. Intermediate host snails were more likely to be present with increasing surface of the potential habitat and on loamy soils. This study confirms the importance of farm management factors in the infection risk for F. hepatica in cattle and highlights that the combination of management factors with characterization of snail habitats is a powerful means to predict the infection risk with F. hepatica at the individual farm level. Further research is needed to investigate how this knowledge can be incorporated in nation-wide spatial distribution models of the parasite.

In this review, ten methods used to study digenean infections in their intermediate hosts were compared to determine which one should be used either in the field or in the lab to establish the prevalence and intensity of infections in snails. Snail crushing and snail dissection allow quick establishing of prevalence in natural or experimental infections, whereas histology is considered as the most accurate approach to assess the intensity of infection. The follow-up of cercarial shedding only gave an idea on cercarial production. Among recently developed techniques, polymerase chain reaction (PCR) brings the most accurate information and shows high sensitivity and specificity levels when compared to blotting techniques. The easiness and relatively low cost of the basic PCR protocol make it interesting to investigate the epidemiology of the liver fluke in a lab with limited financial resources. Nevertheless, if this technique allows a relatively good estimation of the prevalence, information concerning the intensity of infection is best obtained through real time PCR. However, at the time being this technique is too expensive to be used routinely in the field. The choice between classical or new techniques is usually based on a compromise, as each technique has its advantages and drawbacks.

Phenol/Chloroform/Proteinase K Multiplex PCR Epidemiology a b s t r a c t This study deals with the development and validation of an original PCR protocol to assess the presence of Fasciola hepatica in Galba truncatula its main intermediate host in Western Europe. In the present study two DNA extraction techniques are compared and a new multiplex PCR is described. The Chelex ® DNA extraction technique showed to be more appropriate than the classical Phenol/Chloroform/Proteinase K based method because of the absence of toxic organic solvent, shorter duration and lower cost, and a higher reproducibility regarding DNA concentrations and wavelength ratios. The multiplex PCR was set up to amplify the lymnaeid internal transcribed spacer 2 sequence (500-600 bp) that act as an internal control and a 124 bp Fasciola sp. sequence that is repeated more than 300,000 times in fluke whole genome. Ninety six snails were pooled and 6 snails (6.25%) found positive for Fasciola sp. The limit of detection is lower than the minimal biological infestation unit (one miracidium). DNA extracts from Paramphistomum daubneyi, Dicrocoelium lanceolatum, and Fascioloides magna did not cross react.

BackgroundThe present study aims to assess the epidemiological role of different lymnaeid snails as intermediate hosts of the liver fluke Fasciola hepatica in Belgium and Luxembourg.MethodsDuring summer 2008, 7103 lymnaeid snails were collected from 125 ponds distributed in 5 clusters each including 25 ponds. Each cluster was located in a different biogeographic area of Belgium and Luxembourg. In addition, snails were also collected in sixteen other biotopes considered as temporary wet areas. These snails were identified as Galba truncatula (n = 2474) (the main intermediate host of F. hepatica in Europe) and Radix sp. (n = 4629). Moreover, several biological and non-biological variables were also recorded from the different biotopes. DNA was extracted from each snail collected using Chelex® technique. DNA samples were screened through a multiplex PCR that amplifies lymnaeid internal transcribed spacer 2 gene sequences (500–600 bp) (acting as an internal control) and a 124 bp fragment of repetitive DNA from Fasciola sp.ResultsLymnaeid snails were found in 75 biotopes (53.2%). Thirty individuals of G. truncatula (1.31%) and 7 of Radix sp. (0.16%) were found to be positive for Fasciola sp. The seven positive Radix sp. snails all belonged to the species R. balthica (Linnaeus, 1758). Classification and regression tree analysis were performed in order to better understand links and relative importance of the different recorded factors. One of the best explanatory variables for the presence/absence of the different snail species seems to be the geographic location, whereas for the infection status of the snails no obvious relationship was linked to the presence of cattle.ConclusionsEpidemiological implications of these findings and particularly the role of R. balthica as an alternative intermediate host in Belgium and Luxembourg were discussed.

A previous study conducted in Belgium revealed that genetic material of Fasciola sp. was present in snail species belonging to the genus Radix. Here, these snails were collected and identified by DNA-based techniques as Radix labiata and Radix balthica. These two species and Galba truncatula (the major intermediate host in Europe) were experimentally infected with Fasciola hepatica. The resulting metacercariae were fed to rats and the infection was monitored using several techniques. Microscopy revealed the presence of larval stages in 78.3, 45, and 6.25% of G. truncatula, R. labiata, and R. balthica snails, respectively. These results were confirmed by a PCR that amplifies a Fasciola sp. specific sequence. Furthermore, this PCR was found to be more sensitive than microscopic examination. R. labiata shed fewer metacercariae than G. truncatula but these were as infective to rats as those shed by G. truncatula. This study demonstrates that R. labiata may act as an incidental intermediate host for F. hepatica in Belgium. #