Stefanini de Guzmán, A.M.
Universidad Nacional de San Luis, Bioquímica y Ciencias Biológicas, San Luis, Argentina.

Clostridrium chauvoei causes the fatal disease blackleg in bovines, ovines and other ruminants. Cellular antigens are important for immunization as well as toxins and other extracellular antigens. In our laboratory, 48% of C. chauvoei were isolated and identified from animals presumably killed by blackleg. In order to improve the immunogeniticy of vaccines, different antigenic preparations and culture conditions were studied using strains 5078 and two regional strains. From strain 5078, a cellular extract (CE) with veronal buffer pH 8.5, 4.5-5.2 mg/ml protein content, and a flagellar suspension (F) (1.5 mg/ml) were obtained. White mice (18-21 g b.w) were immunized with both antigens, by injecting twice subcutaneously with 0.2 ml of CE previously absorbed in AlPO4 1:1 gel (v/v) at intervals of three weeks between doses. Fourteen days after immunization the mice were challenged with spores, 10 DL50 mixed with 2.5% CaCl2. CE protected 100% of the inoculated animals, undiluted and also diluted to at least 1/500, and compared favorably to commercial vaccines. Flagellar suspension protected 90%. Experiments with guinea pigs using CE gave similar results. By SDS-PAGE and immunoblotting, CE and F shared the 42 KDa band. Lots of white rabbits weighing 2.5-3 kg b.W were intravenously inoculated with 5 doses 0.2, 0.4, 0.8, 1.6, and 3.2 ml administered at intervals of 3-4 days. Fourteen days after, the titer of agglutinant antibodies was determined. Passive immunization with antisera was performed on mice, obtaining 100 and 75% protection with CE and F, respectively. Culture conditions (pH, temperature and carbon source) were modified to increase the biomass and extracellular products yield. Partial feeding of carbon source at 37° C significantly increased biomass. For strains 8 and 17 at temperatures of 37 and 41° C there was not significant different in hemolysine production, and at 41° C there was no DNAase production. Protective immunogenicity was assessed with extracellular products. Also, the rabbit antisera were marked with fluorescein -isothiocyanate. Direct immunofluorescence (DIF) was applied to samples coming from bone marrow of animals presumably killed by the blackleg, obtaining 46.5% positive results. For the coagglutination technique (COA), IgG were fixed to Staphyloccoccus aureus Cowan I strain by their Fc fragment. COA was applied to cultures, peritoneal fluid and muscle exudate of mice inoculated with C. chauvoei, obtaining 100% positive results. Results indicate the importance of selecting highly immunogenic strains for the preparation of vaccines as well as of employing strains of recognized virulence for vaccine control.