Analyses of Supernatant Fluids from Exsheathing Infective Haemonchus contortus cayugensis Larvae

Abstract

Infective Haemonchus contortus cayugensis larvae were exsheathed and the supernatant fluids from such exsheathed larvae were harvested for determinations of bacteria, leucine aminopeptidase, and exsheathing activity. Supernatant fluids harvested after increasing incubation time contained increasing amounts of total nitrogen and Lowry protein, but the bacteria counts were constant. It was concluded that our system of ecdysis was unsuitable for the multiplication of bacteria. Small traces of leucine aminopeptidase were demonstrated only inconsistently in supernatant fluids concentrated to 1 mg protein/ml and harvested after the larvae were incubated for 20 min. However, the concentrated supernatant fluids produced refractile rings in sheaths dissected from infective larvae. A 20-min-incubation concentrated supernatant fluid showed much higher exsheathing activity than a 6-hr-incubation concentrated fluid. Slocombe and Whitlock (1969, 1970b) detailed a method for rapid ecdysis (20-min) of infective larvae of Haemonchus contortus cayugensis Das and Whitlock, 1960. Slocombe and Whitlock (1971) presented some nitrogen and protein analyses of supernatant fluids harvested from such exsheathed larvae. We now present analyses for bacteria, leucine aminopeptidase in, and exsheathing activity of, the supernatant fluids from larvae which were exsheathed and were incubated for varying periods before harvest of the supernatant fluids. MATERIALS AND METHODS Feces were collected from sheep infected with H. contortus cayugensis and were prepared for culture (Slocombe, 1969). The cultures were incubated, harvested, cleaned, and stored as described by Slocombe and Whitlock (1970a). When required 1 X 106 ? 6% infective larvae were exsheathed as a unit. The larvae were prepared for exsheathment and then exsheathed with 10 ml 0.02 M Na2B407'101 H20. The exsheathment rate was determined. Then the clear supernatant fluid from incubated exsheathed or unexsheathed larvae was harvested, and the total nitrogen and Lowry protein content of the clear supernatant fluid estimated (Slocombe and Whitlock, 1971). The number of bacteria, the amount of leucine aminopeptidase, and the exsheathing activity of the supernatant fluid were estimated as described below. Received for publication 24 November 1970. * Present address: Department of Pathology, Ontario Veterinary College, University of Guelph, Guelph, Ontario. This investigation was supported by the State of New York, The NSF (GB-3697), and NIHGRS, 466-001-74-604. Bacterial counts Counts of bacteria were conducted on the supernatant fluid harvested after several different experiments. The fluid was diluted with sufficient sterile physiological saline to display 30 to 200 organisms per 0.7 ml of diluted fluid, when such an aliquot was flooded over an agar plate and was incubated for 48 hr at 38 C. The counts were subjected to a square root transformation for statistical analysis. In Experiment 1, the larval units were prepared for exsheathment by washing them either 3 or 15 times with sterile 0.05% NaCl. The larvae were then exsheathed and were incubated for 20 min. In Experiments 2 and 3, the larval units were washed 3 times and then were exsheathed and were incubated for either 20 min, 1, 3, 6, or 24 hr. Leucine aminopeptidase (LAP) determination LAP activity of the supernatant fluid was assayed using a kit, containing reagents for the test and a standard curve, supplied by Sigma Chemical Company, St. Louis, Missouri. The technique, essentially a modification of the colorimetric method of Goldbarg and Rutenburg (1958), utilized the enzyme to catalyze the following reaction: L-Leucyl-B-Naphthylamide + LAP H20 > Leucine + B-Naphthylamine The colorless B-Naphthylamine was then diazotized and combined with a dye. B-Naphthylamine + NaNO2 + Dye Base ---> Blue Dye. The optical density of the dye produced was proportional to the amount of B-Naphthylamine which in turn depended on the LAP activity. These procedures were outlined by Sigma Chemical Co., for the determination of LAP activity in serum (Sigma Technical Bulletin Number 251, 1969), and were followed with one modification for assaying the LAP activity of the supernatant fluid. Instead of