Salmonella remains the major cause of food-borne diseases worldwide, with chickens known to be the main reservoir for this
zoonotic pathogen. Among the many approaches to reducing Salmonella colonization of broilers, bacteriophage offers several
advantages. In this study, three bacteriophages (UAB_Phi20, UAB_Phi78, and UAB_Phi87) obtained from our collection that
exhibited a broad host range against Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium
were characterized with respect to morphology, genome size, and restriction patterns. A cocktail composed of the three bacteriophages
was more effective in promoting the lysis of S. Enteritidis and S. Typhimurium cultures than any of the three bacteriophages
alone. In addition, the cocktail was able to lyse the Salmonella enterica serovars Virchow, Hadar, and Infantis. The effectiveness
of the bacteriophage cocktail in reducing the concentration of S. Typhimurium was tested in two animal models using
different treatment schedules. In the mouse model, 50% survival was obtained when the cocktail was administered simultaneously
with bacterial infection and again at 6, 24, and 30 h postinfection. Likewise, in the White Leghorn chicken specific-pathogen-
free (SPF) model, the best results, defined as a reduction of Salmonella concentration in the chicken cecum, were obtained
when the bacteriophage cocktail was administered 1 day before or just after bacterial infection and then again on different days
postinfection. Our results show that frequent treatment of the chickens with bacteriophage, and especially prior to colonization
of the intestinal tract by Salmonella, is required to achieve effective bacterial reduction over time.