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Microbiol. bison vaccinated with RB51+were similar to responses of bison vaccinated with RB51. Pregnant bison were intraconjunctivally challenged in midgestation with 107 CFU of strain 2308. Bison vaccinated with RB51, but not RB51+vaccinates, had greater protection from abortion, fetal/uterine, mammary, or maternal infection than nonvaccinates. Our data suggest that the RB51+strain is less efficacious as a calfhood vaccine for bison than the parental RB51 strain. Our data also suggest that the RB51 vaccine is a currently available management tool that could be utilized to help reduce brucellosis in free-ranging bison. Although several newly infected herds have been recently identified, the United States achieved a milestone in 2008 in that all states were simultaneously declared free of cattle brucellosis. However, the persistence of in bison and elk reservoirs in the greater Yellowstone area (GYA; areas adjacent to Yellowstone National Park) remains a potential threat for reintroduction of brucellosis into domestic livestock. The fact that recently identified strain RB51 (RB51) was previously evaluated as a calfhood vaccine for bison and found to be efficacious in preventing abortion and fetal/uterine infection after experimental challenge (9). However, the efficacy of RB51 as a calfhood vaccine for bison appears to be slightly reduced compared to data obtained from similar studies with DZNep cattle (4). The previous vaccine DZNep for cattle, strain 19 vaccine (S19), was reported to not be efficacious as a calfhood vaccine for DZNep bison (5). Because vaccination programs for free-ranging wildlife are likely to be difficult and expensive, vaccines that provide optimal safety and efficacy are needed. Data suggest that domestic livestock and wildlife reservoirs have DZNep diverse immunologic responses to RB51 and S19 vaccines, with differing levels of protection (3, 4, 6, 8, 10). In an effort to develop new and more efficacious vaccines, strains have been generated via recombinant DNA techniques and screened through laboratory animal models. When the protective antigen superoxide dismutase (and (RB51+cultures. A commercial vaccine derived from RB51 and an experimental vaccine containing strain RB51 overexpressing and (RB51+strain 2308 (S2308) was grown on tryptose agar for 48 h at 37C. The bacteria were harvested from the agar by aspiration using saline. Suspensions of S2308 were adjusted by using a spectrophotometer, and the concentrations of viable bacteria were determined by standard plate counts. Animals and inoculation. Twenty-four 10-month-old bison heifers were obtained from a brucellosis-free herd. After acclimation for 4 weeks, bison were randomly assigned to three groups (= 8 animals/group) for subcutaneous vaccination with saline (control), RB51, or RB51+were identified based on colony morphology (1), growth characteristics, and a were determined by tube agglutination (1) and enzyme-linked immunosorbent assay (ELISA) procedures. For the ELISA procedure, RB51 was grown on tryptose agar for 48 h at 37C and 10% CO2. Bacteria were harvested off the plates using phosphate-buffered saline (PBS) containing 0.001 M EDTA. After the bacteria were washed in PBS, the concentration was determined by using standard plate counts. Bacteria were killed by the addition of 0.5% formalin. After adjustment to 108 CFU/ml in carbonate-bicarbonate buffer, 100 l/well was added to a microtiter plate, followed by incubation at 4C overnight. Mouse monoclonal to ALCAM After a washing step with PBS, 300 l of saline containing 25 mg of casein/ml (PBS-casein) was added to each well. Plates were then incubated at room temperature for 2 h and washed three times with 300 l of PBS containing 0.05% Tween 20 (PBS-Tween). Based on previous data (S. C. Olsen, unpublished data), serum samples were diluted 1:1,600 in PBS-casein, and 100 l was added in quadruplicate to wells. After incubation at room temperature for 2 h, plates were washed three times with PBS-Tween. After the addition of 100 l of a 1:2,500 dilution of peroxidase-conjugated rabbit anti-bovine immunoglobulin G (IgG) (Jackson Immunoresearch), the plates were incubated for 2 h at room temperature. After the addition of substrate (3,3,5,5-tetramethylbenzidine and H2O2 in 0.1 M citric buffer), the plates were incubated in the DZNep dark for 30 min, the reactions were stopped with 100 l/well of 0.18 M sulfuric acid, and optical densities were read at 380 nm on a microtiter plate reader. PBMC and lymph node cells for lymphocyte proliferation assays. At all sampling times after vaccination, blood was obtained from the jugular vein of all bison and placed into an acid-citrate dextrose solution. Peripheral blood mononuclear cells (PBMC) were enriched by density centrifugation using a Ficoll-sodium diatrizoate gradient (Sigma Diagnostics, Inc., St..