Amblyomma americanum: Physiochemical isolation of a protein derived from the tick salivary gland that is capable of inducing immune resistance in guinea pigs

Abstract

Crude salivary gland derived proteins from Amblyomma americanum ticks were analyzed by physiochemical (gel filtration and ion exchange chromatography) and immunochemical guinea pig IgG 1 (anti-tick immunoaffinity column) techniques for the presence of antigens responsible for the induction of host immune resistance responses. Gel filtration (G-75 Sephadex) and ion exchange (diethyl aminoethyl cellulose) chromatography of crude salivary gland antigen yielded multiple fractions, but only one fraction from each procedure induced significant cutaneous anaphylaxis bluing reactions when used for skin tests in tick sensitized animals treated intravenously with 0.05% Evans blue dye. Salivary gland antigen (200 ng) eluted from the immunoaffinity column by 0.2 M Na 2CO 3, pH 11.3, and emulsified with incomplete Freund's adjuvant conferred a significant level of tick rejection (24%, P < 0.001) on naive guinea pigs compared with that seen in controls, but less than ( P < 0.01) the level of immunity conferred by crude salivary gland antigen (380 μg). The immunizing dose of immunoaffinity purified salivary gland antigen was 1 1900 the dose of the crude antigen preparation representing 99.9% purification. Furthermore, engorged ticks from animals immunized with salivary gland antigen exhibited a significant decrease ( P < 0.001) in weight compared with ticks from naive animals. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 125I labeled proteins in the Na 2CO 3 eluate and the skin reactive fraction from gel filtration and ion-exchange chromatography, after immunoprecipitation with a guinea pig IgG 1 antibody to the tick that transferred resistance, revealed the presence of a 20 kDa weight protein reported previously to be the antigen responsible for the induction of host resistance. These studies present physiochemical and immunochemical procedures for the purification of an important tick protein that (1) induces skin reactions in tick sensitized guinea pigs, (2) is recognized by antibody to the tick, and most importantly, (3) is capable of immunizing naive guinea pigs against tick challenge.