The terrariums were moistened with tap water and the snails were fed with fresh lettuce leaves in alternate days. Samples

of randomly chosen snails were dissected to ensure that the snails were free of larval helminths. Snails free of helminthic infection were experimentally infected. The adult worms were collected from the pancreas of naturally infected bovines that were slaughtered in an industrial abattoir (Matadouro Municipal de Barra Mansa, Barra Mansa, RJ, Brazil). The adult worms were kept overnight in Petri dishes with Locke’s saline solution (Humason, 1979). Adult worms were discarded and eggs were sedimented. The eggs were washed three times in Locke’s saline solution and stored at 10 °C until their utilization. The eggs were spread on pieces selleck inhibitor of fresh lettuce leaves in Petri dishes with a moistened filter paper at the bottom, and the snails were put over the lettuce leaves. The Petri dishes were closed and the snails were maintained in contact with eggs overnight. After

this period, they were transferred to terrariums and maintained as described above. The mother and the daughter sporocysts were collected after dissections of snails 30 and 60 days after exposure, respectively. After 70 days post exposure, the snails were maintained isolated in Petri dishes, with tap water moistened filter paper at the bottom; the dishes were daily observed under a stereomicroscope to check the presence FRAX597 chemical structure of expelled sporocysts. So, the larvae obtained formed three groups: i – dissected mother sporocysts; ii – dissected daughter sporocysts; and, iii – expelled daughter sporocysts. The collected larvae were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4 for 24 h. For TEM the fixed larvae were washed in 0.1 M cacodylate buffer, post fixed in 1% osmium tetroxide and 0.8% ferrocyanide, washed again in the same buffer, dehydrated in a crescent series

of acetone, infiltrated and embedded in epoxy resin (Polybed). Semi-thin sections were stained with toluidine blue and observed under a Zeiss Axioplan light ADAMTS5 microscope; images were captured with an MRc5 AxioCam digital camera and processed with the Axiovision program. Ultrathin sections were stained with uranyl acetate and lead citrate (De Souza, 2007) and observed in a Zeiss 900 Transmission Electron Microscope at 80 kV. The images were obtained using iTEM Software. The developing larva was adhered to the intestinal wall (Fig. 1a). The transversal semithin sections of this form showed that the tegument was divided in two different regions: an external layer more electrondense and, immediately below, an internal one, electrondense, followed by a body cavity with germinal balls (Fig. 1a). By TEM the tegument showed an external surface with many projections and folds (Fig. 1b). Pieces of the intestine with the larvae adhered to it were collected and processed.