The aim of this study was to develop, perfect and validate the PCR (polymerase chain reaction) technique using mitochondrial (mt) and ribosomal (ITS-2) DNA for the accurate identification of Dicrocoelium dendriticum in molluscs and ants, the first and second intermediate hosts, and their early detection. The first primers that were designed amplified a 169 pb mtDNA fragment of D. dendriticum permitted the detection of a single D. dendriticum metacercaria from the Formica rufibarbis and Formica pratensis abdomen, as well as the detection of the brainworm in the head of the ants collected in tetania. Although these primers did not amplify Dicrocoelium chinensis DNA and permitted detected D. dendriticum in the molluscs, they did not discriminate Brachylaimidae metacercariae found in the same mollusc. The PCR that was designed to amplify a 93 bp fragment of the ITS-2 is D. dendriticum specific as it did not amplify D. chinensis, Brachylaimidae and other trematodes. This technique is very sensitive since it permitted the detection of D. dendriticum in the molluscs from the first day post-infection, the brainworm in the head of the ants and only 1 D. dendriticum metacercaria from the abdomen of the ants. Both techniques are important, mainly the latter.

An epidemiological study on dicrocoeliosis caused by Dicrocoelium dendriticum was carried out on sheep, molluscs and ants in the mountains of León province (NW Spain) between 1987–1991. The results concerning the intermediate hosts and a review of some aspects of dicrocoeliosis are summarized. Mollusc collection for the helminthological study was random throughout the study area at fortnightly intervals. Twenty-nine Gastropoda species were identified. D. dendriticum infection was only detected in 2·98% of the 2084 Helicella itala examined and in 1·06% of 852 H. corderoi. The highest infection prevalence was detected in H. itala in September and in H. corderoi in February. Daughter sporocysts with well-developed cercariae predominated in spring and autumn. Infection prevalence increased with mollusc age and size. Ants were collected from anthills or plants to which they were attached. The behaviour of ants in tetania was followed. Twenty-one Formicidae species were identified, but only the following harboured D. dendriticum: Formica cunicularia (1158 examined specimens, 0·69% infection prevalence, 2–56 metacercariae per ant); F. sanguinea (234, 1·28%, 2–63); F. nigricans (1770, 4·97%, 1–186); F. rufibarbis (288, 6·59%, 2–107). In a flat area close to León town, 95·39% of the 2085 F. rufibarbis specimens collected in tetania contained metacercariae (1–240) in the abdomen. These were used for parasite characterization by isoelectric focusing and to infect lambs and hamsters. Only one brainworm per ant was found.