Dr. Kassai Tibor - Dr. Murai Éva szerk.: Parasitologia Hungarica 10. (Budapest, 1977)
streptomycin and 100 ug nystatin. Plerocercoids were incubated in the medium for 48 hours at 37 C. Samples for enzyme and inhibitor assay were taken at 24 and 48 hours. Similarly treated media not containing parasites were used as control. In the second series 0.1% trypsin (BDH, 0.5 Anson U/G) or chymotrypsin (REANAL, 10 000 ATEE U/mg) was added to KRT, TS and TC 135 incubation media, and enzyme activity was assayed after 24-hour incubation of the plerocercoids at 37°C, using similarly treated media free from parasites as control. In the third series domestic ducks were infected with larvae, and plerocercoids removed from fish hosts by sectioning under aseptic conditions were cultured at 41°C in TC 135 and TC 199 media to which antibiotics, nystatin and 10% calf serum were added. Ducks were killed and autopsied 4 days after infection to collect adult worms. The nutrient medium was exchanged daily for the in vitro cultured larvae, which took 8 days to reach sexual maturity. Mature specimens were used for the preparation of extracts. Adults and freshly collected, washed larvae were homogenized in fivefold volume of distilled water, homogenate centrifuged at 19 500 g at +5°C for 60 minutes, and the supernatant was used for assays. Trypsin activity was measured on BAEE (N- (X-benzoyl-arginin-ethy lester) substrate by the method of Schwert and Takenaka (1955), chymotrypsin activity on BTEE (N-benzoyl-L-tyrosin-ethy lester) substrate as proposed by Hummel (1959). Assays were carried out at 30°C, in 3 ml final volume of the system (0.1 ml test material +2.4 ml 0. 1 m TRIS buffer, ph 7. 8 +0. 05 M CaCl2+0. 5 ml substrate, viz. 0. 1 ml test material + 0. 1 ml trypsin, viz. chymotrypsin + 2.3 ml buffer + 0. 5 ml substrate). Unit enzyme activity was expressed as the hydrolytic activity increasing the optical density (OD) of trie above 3 ml system by 1. 00 OD unit as read at 253 nm for trypsin and 256 nm for chymotripsin. Unit inhibitory activity was defined as the activity reducing by 1.00 OD unit the hydrolytic activity of the mammalian pancreatic enzymes added to the system. The values assessed in parasite extracts and nutrient medium were uniformly related to 1 g crude parasite weight. Protease activities of larvae in different culture media KRT TS TC 135 TC 199 Tyrode 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 1. 5 1. 5 0 1 0 2 0 2. 5 0 1 2 3 0 2 1. 5 1 2 2. 5 1 1 0 2 1. 5 1. 5 2 2 1 2 2 3 1 3 2 2 2 2 1 1 1. 5 2 1. 5 2 1 6 1 2 1 1. 5 2 1. 5 3 2. 5 2 2 2 2 0 2 1. 7 2 0. 5 1 1 1 1.5 1 0 1 Means: 1. 7 2. 8 1. 05 1. 85 1. 2 1.8 0. 9 1.9 0. 7 1. 7 x Each figure is the mean of three replicates
