PS11-6. Isolation and Characterization of Bifidobacterium spp. from the Fecal Microbiome of Neonatal Dairy Calves.

Abstract One of the main challenges in the dairy industry is the high incidence of diarrhea in calves during the first month of life. Early microbial colonizers play a critical role in gut health by competing with pathogens and modulating host immune and metabolic development. However, most commercial probiotics for cattle are derived from non-ruminant sources, potentially limiting their effectiveness in calves. Therefore, isolating and characterizing native early-life gut bacteria from neonatal calves is essential for developing host-specific probiotic strategies. In this study, fecal samples were collected from one-week-old dairy calves to isolate and characterize Bifidobacterium spp. A total of 58 bacterial isolates were obtained, including 12 Bifidobacterium isolates: 2 B. breve, 2 B. pseudolongum, and 8 B. thermophilum. Antimicrobial activity was evaluated by pre-incubating isolates on DSMZ M.58 agar plates for 24-48 h, followed by challenge with Escherichia coli and Salmonella Typhimurium. Inhibition was classified as none, partial, or total based on pathogen growth. Seven Bifidobacterium isolates showing antimicrobial activity against E. coli and S. Typhimurium were selected for further characterization. Functional assays included acid tolerance, oxygen tolerance, and autoaggregation. The capacity to grow across pH 2.0-6.3 was assessed in 96-well plates under anaerobic conditions by monitoring optical density (OD600nm) over 48 h. Oxygen tolerance was evaluated under aerobic and oxygen-limited conditions, with growth measured at 0, 24, and 48 h. Autoaggregation was determined by measuring the decrease in OD600nm of cell suspensions over time. Statistical analyses were performed in R. Mean, standard deviation, and area under the curve were calculated. Autoaggregation was analyzed using ANOVA, and acid tolerance was evaluated using a linear mixed-effects model. Results showed strain-dependent functional variability. B. pseudolongum exhibited the highest acid tolerance, growing at pH 3.0 (µ = 0.19 h-1) and surviving at pH 2.5, after plating in DSMZ M.58 medium. B. thermophilum grew at pH 4.0 and survived at pH 3.5, while B. breve showed growth at pH 5.0 and survival at pH 4.0. All selected isolates demonstrated tolerance to oxygen-limited conditions; however, B. breve did not grow under fully aerobic conditions. In autoaggregation assays, B. breve showed the highest percentage of autoaggregation (44%), whereas the remaining isolates exhibited values below 20% (p < 0.01). These findings indicate that Bifidobacterium isolates from neonatal dairy calves exhibit strain-dependent probiotic traits, including antimicrobial activity and tolerance to gastrointestinal stressors. Oxygen tolerance and antimicrobial activity are also desirable characteristics from an industrial perspective, while autoaggregation is associated with bacterial adhesion to the intestinal epithelium, an important feature for probiotic function. Together, these traits support their potential as host-adapted probiotics to improve calf health, although further in vivo validation is required.