Tryptone soya broth (tsb)

MG and MS were cultured separately using Pleuro-Pneumonia Like Organisms (PPLO) broth (Becton Dickinson, Franklin Lakes, NJ, USA) and were incubated at 37°C in a CO₂ incubator for 48 h, and the cultured bacterial colonies were adjusted to contain 1 × 10⁸ colony-forming units (CFU) mL^-1 in different groups of embryonated chicken eggs and hens. Calculations were performed using Rodwell and Whitcomb's (1983) standard techniques (Bekele and Assefa, 2018 ). ST, SK, and SE were each cultured on Salmonella Shigella (SS) agar (Lab M Limited, Lancashire, UK) at 37°C for 24 h. After being transferred into tryptone soya broth (Lab M Limited), the colonies were cultivated at 37°C for 24 h. After obtaining the total colony count, the bacterial solution was adjusted to contain 1 × 10⁸ CFU mL^-1.
All bacteria were pelleted after centrifugation at 12,000 x g for 30 min at 4°C. Using the total colony count approach, the bacterial counts of ST, SK, and SE were adjusted to 1 × 10⁸ CFU 0.5 mL^-1 of the final product by preparing separate final suspensions from ST, SK, and SE, respectively. Finally, all bacteria were inactivated using 0.3% formalin via agitation, and the bacterial suspension was combined with Montanide ISA70 (SEPPIC, Courbevoie, France) in a ratio of 70% volume of adjuvant to 30% volume of antigens according to Charles et al. (1994) (link).