Immediately after death, tissue samples were taken from three anatomical regions in the upper respiratory tract, i.e., septum, middle meatus and ventral nasal conchae and from two sites of the digestive tract, i.e., abomasum (fundic

selleck products region) and small intestine (1 m from the pylorus) for counting of mucosal mast cells, eosinophils and globule leucocytes. All tissue samples were fixed in 10% buffered formaldehyde for 48 h. The samples were then dehydrated with alcohol and embedded in paraffin wax. Sections, 2 μm thick, were stained with 1% toluidine blue or haematoxylin and eosin (H&E). Mast cells were counted in sections stained with toluidine blue and eosinophils and globule leucocytes in sections were stained with H&E. Cells were enumerated under a 10× eye piece containing a calibrated graticule and 100× objective lens viewing an area of 0.01 mm2. Thirty fields, which were randomly selected, were observed per animal for each histological region and the mean numbers of cell/surface were calculated and compared between the groups. The counts were

expressed as number of cells per mm2 of mucosa. Mucus was taken from the nasal cavities, abomasum Veliparib solubility dmso and small intestine mucosas to determine the levels of immunoglobulin A (IgA). While the larvae of O. ovis were collected, mucus from nasal mucosa was extracted by lightly scraping the mucosal surface with a glass slide and mucus was stored in a falcon tube at −20 °C until processing. A 5 cm piece of abomasum and small intestine were sampled for the extraction of mucus and stored at −20 °C until processing. Tissues were thawed and mucus was scraped off with a glass slide. The scrapings were collected in a falcon tube on ice. Three millilitres of ice cold PBS supplemented with protease inhibitors (Complete®, Roche) was added to each

Mannose-binding protein-associated serine protease sample. The samples were shaken for 1 h at 4 °C and centrifuged for 30 min at 4 °C and 3000 × g. The supernatant was collected and centrifuged again for 30 min at 4 °C and 15 000 × g ( Kanobana et al., 2002). Protein concentrations were determined using a kit (Protal método colorimétrico® – Laborlab, Brazil) and the samples of abomasum mucus were adjusted to a protein concentration of 0.4 g/dl; small intestine to 0.1 g/dl and nasal mucus to 0.7 g/dl using PBS supplemented with protease inhibitors. IgG levels in serum samples were determined against excretory and secretory products (ESP) and crude extract (CE) antigens from second instar (L2) O. ovis larvae; and against third stage larvae (L3) and adults (L5) of H. contortus and T. colubriformis antigens. IgA levels in nasal mucus were tested against excretory and secretory products (ESP) and crude extract (CE) from L2 O. ovis larvae; abomasal mucus was tested against L3 and L5 of H. contortus and small intestine mucus against L3 and L5 of T. colubriformis. Second instar (L2) of O. ovis were collected from naturally infected sheep heads and were washed several times in phosphate-buffered saline (PBS pH 7.