Amblyomma americanum: physiochemical isolation of a protein derived from the tick salivary gland that is capable of inducing immune resistance in guinea pigs |
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| Authors: | S J Brown P W Askenase |
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| Affiliation: | 1. Institute of Vertebrate Biology, v.v.i., Academy of Sciences, Kvetna 8, 603 65, Brno, Czech Republic;2. Masaryk University, Department of Experimental Biology, Kotlarska 2, 611 37 Brno, Czech Republic;3. Institute of Macromolecular Chemistry, v.v.i., Academy of Sciences, 162 06 Prague, Czech Republic;4. Aix Marseille University, CNRS 7278, URMITE, INSERM 1095, UM63,IRD Dakar 198, 27 Bd Jean Moulin, F-13385 Marseille 5, France;1. Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brazil;2. Laboratório de Hantaviroses e Rickettsioses, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brazil;3. Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, São Bernardo do Campo, SP, SP, Brazil;4. Medicina Veterinária e Bem estar animal, Universidade de Santo Amaro, SP, Brazil;5. Instituto Nacional de Tecnología Agropecuaria, Rafaela, Santa Fe, Argentina;6. Facultad de Ciencias Veterinarias, Universidad de Concepción, Chillán, Chile;7. Departamento de Patología Animal, Facultad de Veterinaria, Universidad de Zaragoza, Spain;8. Laboratorio de Vectores y enfermedades transmitidas, Universidad de la República, CENUR Litoral Norte, Salto, Uruguay;1. Department of Tropical Medicine, Infectious Diseases and Nephrology, University of Rostock Medical School, 18055 Rostock, Germany;2. State Institute for Agriculture, Food Safety and Fisheries Mecklenburg-Western Pomerania, D-18059 Rostock, Germany;3. Tick Information Center, D-07646 Lippersdorf, Germany;1. Department of Tropical Medicine, Infectious Diseases and Nephrology, University of Rostock Medical School, 18055 Rostock, Germany;2. State Institute for Agriculture, Food Safety and Fisheries Mecklenburg-Western Pomerania, D-18059 Rostock, Germany;3. Center for Virology, Medical University of Vienna, A-1090 Vienna, Austria;4. Brehm Memorial Center Renthendorf, 07646 Renthendorf, Germany;1. School of Physics and Astronomy, Tel Aviv University, Tel Aviv 69978, Israel;2. Jožef Stefan Institute, 1000 Ljubljana, Slovenia;3. Institut für Kernphysik, Johannes Gutenberg-Universität, 55099 Mainz, Germany;4. Department of Physics, University of Zagreb, HR-10002 Zagreb, Croatia;5. Rutgers, The State University of New Jersey, Piscataway, NJ 08855, USA;6. Dipartimento di Fisica, Università degli Studi di Pavia and INFN, Sezione di Pavia, via A. Bassi 6, I-27100 Pavia, Italy;7. Universität Konstanz, Fachbereich Physik, Universitätsstraße 10, 78464 Konstanz, Germany;8. Department of Physics, NRCN, P.O. Box 9001, Beer-Sheva 84190, Israel;9. Soreq NRC, Yavne 81800, Israel;10. Racah Institute of Physics, Hebrew University of Jerusalem, Jerusalem 91904, Israel;11. Faculty of Mathematics and Physics, University of Ljubljana, 1000 Ljubljana, Slovenia;12. University of South Carolina, Columbia, SC 29208, USA;1. Texas A & M University AgriLife Research, Department of Entomology, 2475 TAMU, College Station, TX 77843, United States;2. Centro de Biotecnologia and Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9500, Prédio 43421, Porto Alegre 91501-970, RS, Brazil;3. Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro – UFRJ, Rio de Janeiro, Brazil |
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| Abstract: | Crude salivary gland derived proteins from Amblyomma americanum ticks were analyzed by physiochemical (gel filtration and ion exchange chromatography) and immunochemical guinea pig IgG1 (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 0.5% Evans blue dye. Salivary gland antigen (200 ng) eluted from the immunoaffinity column by 0.2 M Na2CO3, pH 11.3, and emulsified with incomplete Freund's adjuvant conferred a significant level of tick rejection (24%, P less than 0.001) on naive guinea pigs compared with that seen in controls, but less than (P less than 0.01) the level of immunity conferred by crude salivary gland antigen (380 micrograms). 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 less than 0.001) in weight compared with ticks from naive animals. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 125I labeled proteins in the Na2CO3 eluate and the skin reactive fraction from gel filtration and ion-exchange chromatography, after immunoprecipitation with a guinea pig IgG1 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 induces skin reactions in tick sensitized guinea pigs, is recognized by antibody to the tick, and most importantly, is capable of immunizing naive guinea pigs against tick challenge. |
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