Twenty-four yearling Angus-dairy cross heifers were housed in an outdoor facility at the National Animal Disease Center (NADC) in Ames, Iowa. Heifers were vaccinated subcutaneously with one of three treatments: 1 mL containing 106 colony forming units (CFU) of  Bacillus Calmette-Guérin (BCG) Danish strain 1331 (n = 8), 2 mL containing 1010 CFU of Brucella abortus strain RB51 (RB51) (n = 8), or 1 mL of 106 CFU of BCG Danish 1331 and 2 mL of 1010 CFU of RB51 mixed together in the same syringe (n = 8). Routine protocols to maintain the health and wellbeing of animals during the study were implemented. All animal procedures received prior approval from the NADC Animal Care and Use committee (ARS-21-0990, approval date 02/11/2022). Whole blood was collected from all heifers at the time of vaccination and at four-week intervals until 24 weeks post-vaccination to assess peripheral immune responses. Thirty mL of blood were collected via venipuncture of the jugular vein and added to 3 mL of acid citrate dextrose (ACD) anticoagulant. Peripheral blood mononuclear cells (PBMCs) were isolated as previously described (Boggiatto et al., 2020). Live cell count was determined for each sample using the Muse® Count and Viability Kit on the Guava® Muse® Cell Analyzer (Luminex). Cell suspensions were adjusted to a final concentration of 1x107 cells per mL in complete 1640 RPMI (cRPMI) media containing 20% heat-inactivated FBS, 1% HEPES, 1% non-essential amino acids, 1% essential amino acids, 1% sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, 2nM glutamine, and 50 µM 2-beta mercaptoethanol. PBMCs were labeled with the CellTrace™ Violet (CTV) proliferation kit (Cat. No. C34557, Thermo Fisher Scientific) to track in vitro proliferation. PBMC labeling was conducted according to the manufacturer’s recommendations with minor modifications. Briefly, CTV dye was reconstituted in 20 µL dimethyl sulfoxide (DMSO) provided by the manufacturer, suspended in 780 µL PBS, and further diluted 1:10 in sterile Dulbecco’s phosphate buffered saline (DPBS). PBMCs were washed in DPBS and centrifuged at 300x g for 10 minutes at room temperature (RT). Supernatants were discarded and cell pellets were resuspended in diluted CTV, then vortexed and incubated for 20 minutes at RT with occasional vortexing. Cells were then washed in DPBS and again centrifuged at 300x g for 10 minutes at RT. Supernatants were discarded, and PBMC were resuspended to a final concentration of 1x107 cells per mL in cRPMI. To assess in vitro recall responses, 100 µL of CTV-labeled cRPMI PBMC suspensions (1x106 cells) were plated per well in 96 well flat bottom plates and treated with various stimulation conditions. PBMCs were then left unstimulated (media only) or stimulated with g-irradiated Brucella abortus strain RB51 antigen (107 CFU/well), purified protein derivative of Mycobacterium bovis (PPDb), or Concanavalin A (ConA). All stimulation conditions were plated in duplicate. Plates were incubated for 7 days at 37°C with 5% CO2. Sixteen hours prior to beginning cell staining procedure on day 7, all cells were either treated with eBioscience™ protein transport inhibitor (Cat. No. 00-4980-93, Thermo Fisher Scientific) or restimulated with eBioscience™ cell stimulation cocktail plus protein transport inhibitors (Cat. No. 00-4975-93, Thermo Fisher Scientific) for assessment of intracellular cytokine production. PBMCs were harvested on day 7 and washed twice in DPBS at 300x g for 5 minutes at RT. PBMCs were then incubated with eBioscience eFluor™ 780 fixable viability dye (Cat. No. 65-0865-14, Thermo Fisher Scientific) for 20 minutes at 4°C and subsequently washed via centrifugation as previously described, once with DPBS and once with FACS buffer (PBS + 0.5% fetal bovine serum (FBS)). PBMCs were incubated with a primary anti-bovine CD45RO antibody (clone IL-A116, Cat. No. MCA2434GA, BioRad) for 15 minutes at room temperature and then incubated with a secondary anti-mouse IgG3 BUV395-labeled antibody (clone R40-82, Cat. No. 744138, BD Biosciences) for 15 minutes at RT. FACS buffer was used to wash cells twice, and cells were then incubated with FITC labeled anti-bovine CD4 (clone CC8, Cat. No. MCA1653F, BioRad), BV650 labeled anti-human CD62L (clone DREG-56, Cat. No. 304832, BioLegend), and PE-Cy7 labeled anti-human CCR7 (clone 3D12, Cat. No. 557648, BD Biosciences) antibodies for 15 minutes at RT. Following incubation and two washes in FACS buffer, cells were fixed and permeabilized using the BD Cytofix/Cytoperm kit (Cat. No. 554714, BD Biosciences) according to manufacturer’s recommendations. Intracellular staining was then carried out by incubating cells with PE labeled anti-bovine IFN-g antibody (clone CC302, Cat. No. MCA1783PE, BioRad) for 30 minutes at room temperature. Cells were washed once with 1X wash/perm buffer and once with FACS buffer. Cells were then resuspended in 200 µL FACS buffer and proliferation, cytokine production, and surface markers were concurrently analyzed using a BD FACSymphony A5 flow cytometer (BD Biosciences). Resulting data was analyzed using FlowJo software (version 10.8). File includes flow cytometry data used to report results in multiple manuscripts. All data is provided as cell counts. Relevant populations are named by column. Gating strategy will be described in related manuscripts.