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.

The prevalence and aetiology of natural paramphistomosis was investigated in cattle slaughtered in the Castilla y León region (Spain) over a 3 year-period. The overall prevalence of positive animals was 6.20%. The parasite burden per animal ranged from 8 to 8005 (median=144) and the ruminal atrium had the highest parasite burden whereas the ruminal dorsal sac the lowest. The prevalence and parasite burden increased with age while these parameters were lower in cattle under intensive management. Calicophoron daubneyi was the only Paramphistomidae species identified using morphoanatomical, histological and molecular methods in the studied animals.

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Hepatic enzymes and biochemical parameter values were analysed in the sera of 32 lambs: 12 infected with 1,000 Dicrocoelium dendriticum metacercariae, 12 with 3,000 and eight controls. Blood samples were collected at intervals of 30 days from day 0 post-infection (p.i.). Half the lambs in each group were slaughtered on days 60 and 180 p.i, respectively. A histopathological and ultrastructural study was carried out on the liver, gall bladder and hepatic lymph nodes of all animals. A minute increase was detected in the bilirubin (7%) and albumin (3%) values in the infected lambs (with 30-2,063 worms) in comparison with those of the controls. The hepatic enzyme values did increase, above all aspartate aminotransferase (19%) and alanine aminotransferase (22%), mainly in the lambs tested with 3,000 metacercariae (38% and 48%, respectively). The increase was significant 60 days p.i. The severity of the lesions was closely associated with the parasite burden.

The transmission, control and the relationship between Dicrocoelium dendriticum and its definitive (sheep and cattle) and intermediate (molluscs and ants) hosts under natural and experimental conditions are described. Eleven species of molluscs and four of ants were found infected with larval

Paramphistomosis and Fasciolosis caused by Calicophoron daubneyi and Fasciola hepatica, respectively, are frequent and important trematodoses in ruminant livestock worldwide. Both parasites use the same snail, Galba truncatula, as intermediate host. The aim of this study was to develop and validate an analytical method based on a mitochondrial DNA (mtDNA) multiplex PCR technique which would allow the early and specific identification, in one step, of C. daubneyi and F. hepatica infection in G. truncatula. First of all, a 1035 bp fragment of mtDNA from adult C. daubneyi worms was obtained. Then two pairs of specific mtDNA primers, which amplified a DNA fragment of 885 pb in the case of C. daubneyi, and of 425 pb in that of F. hepatica, were designed. By means of the multiplex PCR technique developed, there was always a specific amplification in samples from adult F. hepatica and C. daubneyi, but not from Calicophoron calicophorum, Cotylophoron cotylophorum, Cotylophoron batycotyle or Dicrocoelium dendriticum. Likewise, specific amplifications of the expected DNA fragments happened in all samples from snails harbouring larval stages of C. daubneyi or F. hepatica, previously detected by microscopy. However, amplifications were not seen when DNA from snails harbouring other Digenea (Plagiorchiidae, Notocotylidae and furcocercous cercariae) was analysed. Moreover, DNA from G. truncatula molluscs free from infection was not amplified. The multiplex PCR assay permitted infection in the snails experimentally infected with 4 miracidia to be detected as early as day 1 p.i. in the case of F. hepatica and with only 2 miracidia from day 2 p.i. in both, C. daubneyi and F. hepatica. Nevertheless it was necessary to wait until days 29 and 33 p.i. to see C. daubneyi and F. hepatica immature redia, respectively, using microscope techniques. The detection limit of the PCR technique was very low: 0.1 ng of DNA from C. daubneyi and 0.001 ng of DNA from F. hepatica. This allowed infection by either F. hepatica or C. daubneyi to be detected even when pools made up with only 1 μl (60 ng of DNA) from infected snail plus 99 μl from non-infected ones were analyzed. Moreover, simultaneous detection of both parasites was experimentally possible in pools made up with uninfected (98 μl), C. daubneyi infected (1 μl) and F. hepatica infected (1 μl) snails. The most precise and early diagnosis of the infections using the multiplex PCR technique designed will allow more realistic epidemiological models of both infections to be established and consequently a better strategic control.

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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.