Abstract
Background:
Salmonella infections impose considerable global health challenges, mostly due to the variable serovar efficacy of existing vaccines. Salmonella’s lipopolysaccharide (LPS), an integral surface component that potently stimulates and activates the innate immune system, is a prime candidate for the development of universal vaccine formulations. Evaluating the immunogenic potential of LPS requires meticulous and complete molecular typing and characterization of the Salmonella subspecies.

Aim:
This study aimed to isolate, purify, and characterize LPS from Salmonella spp. isolates, confirm the bacterial strains through molecular phylogenetic analysis, evaluate the immune responses induced by LPS in host models, and assess their potential as a candidate for vaccine development.

Methods:
We identified clinical isolates using biochemical and molecular (sequence-based) tools. LPS was extracted and purified using the hot phenol-water method. The molecular integrity of LPS and the presence of a ladder O-antigen were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Experimental animals were immunized with lipopolysaccharide (LPS), and their immune responses were evaluated by estimating serum cytokine (TNF-α, IL-6, IL-1β, IFN-γ) levels, IgM and IgG antibody responses, and challenge protection rate. Descriptive statistics were performed, and p < 0.05 was considered statistically significant.

Results:
Sequence alignment and phylogenetic analyses confirmed that the isolates were Salmonella spp. with close genetic distances to the reference strains. SDS-PAGE indicated a crystal clear and smooth-type LPS profile with clear O-antigens. LPS immunization effectively provoked significant levels of TNF-α, IL-6, IL-1β, and IFN-γ (p < 0.01). Particular to LPS, anti-IgM and IgG antibody levels were vividly increased (p < 0.001). Challenge experiments demonstrated that prolonged survival and decreased clinical signs were evident in LPS-immunized groups.

Conclusion:
The extracted LPS was predictably immunogenic, exhibiting robust innate and adaptive immune responses, and thus could be envisioned as a novel candidate for the development of Salmonella vaccines.

Key words: Cytokines; Immunogenicity; Lipopolysaccharide; Phylogeny; Salmonella.