Mycoplasma and Salmonella are serious pathogens threaten the poultry industry. This study aimed to prepare and evaluate an inactivated pentavalent vaccine targeting bacteria, including Salmonella enterica serovar Typhimurium (ST), Salmonella enterica serovar Enteritidis (SE), Salmonella enterica serovar Kentucky (SK), Mycoplasma gallisepticum (MG), and Mycoplasma synoviae (MS), from locally isolated strains. The prepared vaccine was adjuvanted with Montanide ISA70 oil and then tested for safety, sterility, and potency. The vaccine efficacy was evaluated in 110 specific pathogen-free, 1-day-old chicks, which were divided into three groups as follows: 1) vaccinated group (50 birds), which was subdivided into five subgroups of ten birds each; 2) control positive (challenged) group (50 birds), which was subdivided into five subgroups of ten birds each; and 3) control negative (blank) group, which included ten birds. Chicks in group 1 were administered the first dose of vaccine at 7 d of age followed by a booster dose after 3 wk. At 3 wk after booster vaccination, the chicks who were administered the booster dose were challenged and kept under observation until the end of the experiment when the chicks were approximately 10 wk. Details of clinical symptoms, daily mortality, weights, and postmortem lesions; serum samples; cloacal swabs; and nasal swabs were collected during the experiment. The humoral immune response to the prepared pentavalent vaccine was assessed using enzyme-linked immunosorbent assay. Our findings revealed that the prepared vaccine showed high protective antibody titers against Salmonella and Mycoplasma with 100% efficacy and no mortalities (100% survival rate) were recorded in vaccinated and challenged birds. The vaccine reduced both clinical signs and bacterial shedding post challenge in vaccinated birds in comparison with control positive group. The prepared vaccine did not affect the body weight gain of the vaccinated birds in comparison with control negative birds. The current study concluded that locally manufactured inactivated pentavalent vaccine offers protection to birds and could be employed as an effective tool along with biosecurity measures to overcome mycoplasmosis and salmonellosis in layer and breeder chicken farms in Egypt
