Clinical and Blood Chemical Effects of Crude and Purified Ostertagia Acetylcholinesterase in Cattle and Sheep

Abstract

This investigation was supported by the Swedish Council for Agricultural Sciences. U. S. Copyright Clearance Center Code Statement: 0721-4981/83/3007-0485$02.50/0 486 AXELSSON and LUTHMANN A brief communication on the subject of which this investigation is part has been given elsewhere (AXELSSON, 1981). Material and Methods AnimalsGroup A . Eleven paresis-prone cows. Seven were of the Swedish Red Breed (SRB), two were Group B. Four SRB cows without a history of parturient paresis. Group C. Eight indoor born and kept calves, six SRBs and two SLBs, all 3 4 months old. Group D. Three parasite-free female Swedish Landrace sheep, 6 months old at the beginning of Group E. Three parasite-free female Swedish Landrace sheep, 6 months old at the beginning of Swedish Friesians (SLB) and two were of the Jerseys.immunization with crude 0-AChE. immunization with electric eel AChE. AntigensAntigens were isolated and purified as briefly described by AXELSSON (1981). The worm incubation medium was concentrated in an ultrafiltration cell (Amicon 30,000) and Hanks's balanced salt solution was at the same time changed to Tris buffer p H 7.4. The enzyme preparation was centrifuged at 20,000 g for one hour. The clear supernatant is referred to as crude 0-AChE. Pellets were discarded.The crude 0-AChE was used to provoke and immunize but was also further purified. The purification steps were chromatography on ConA Sepharose, affinity chromatography on CH Sepharose 4 B with specific ligand and gel filtration on Sephadex G 100 (all matrix from Pharmacia Fine Chemicals, Uppsala). Enzyme activity was measured by the spectrophotometric method of ELLMAN et al. (1961) and protein by the method of LOWRY et al. (1951) in order to get the specific activity of pkat/mg protein. The clinically used purified 0-AChE was the de-salted specific elution pool from the affinity column, with a 2C-25 fold purification compared with the crude 0-AChE. The most highly purified gel filtration product had limited stability even when deep-frozen, which made it difficult to use and quantify for clinical trials. This preparation gave a single band on polyacrylamide gel electrophoresis and was used to characterize the enzyme as a soluble, monomeric AChE with a molecular weight of 65,000. Antigen doses were for reasons of convenience measured as activities, and were expressed as the product of volume and activity in pkat/l. A dose of 50 represents, for example, 10 ml of an enzyme preparation with the activity of 5 pkat/l. Groups A and B were given 5C-60 and C, D and E 25-30pkadl. Relative to specific activity in peak gel filtration pool, the enzyme protein contents of these doses were 0.5-0.6 mg in groups A and B, 0.25-0.30 mg in groups C and D, and 0.28 mg in group E.All antigens were given as a single intravenous injection except in the first three cows where onethird was given subcutaneously and two-thirds intravenously.The immunization in sheep was done with at least one month and sometimes 6 months interval. Electric eel AChE was purchased from Sigma. Acetylch...

Local and General Effects of Salmonella typhimuHum Endotoxin in Calves

“…The role of PGs in endotoxin-induced hypocalcaemia deserves further studies, since it was earlier observed that infusion of PGs caused a significant decrease of serum calcium in cows and goats (LUTHMAN et aI., 1983;FREDRIKSSON, 1984). AXELssoN and LUTHMAN (1983) found that acetylcholine esterase prepared from Ostertagia ostertagi, collected from bovine abomasi, induced clinical hypocalcaemia in dairy cows. Injection of this enzyme preparation was also associated with a remarkable increase of the main PGF 2a metabolite in plasma (KINDAHL et aI., unpublished).…”