Evaluation of a modified method of extraction, purification, and characterization of lipopolysaccharide (O antigen) from Salmonella Typhimurium

• Published in: Veterinary World Vol. 13; no. 11; pp. 2338 - 2345
• Main Authors: Hassan, Heba M, Fadel, Mai A, Soliman, Mohamed A
• Format: Journal Article
• Language: English
• Published: India Veterinary World 01-11-2020
• Subjects: Cell membranes; Contamination; extraction; Gene expression; Gram-negative bacteria; High-performance liquid chromatography; IL-1β; Immune response; Injection; interferon-γ and interleukin 1β genes expression; Juveniles; Lipopolysaccharides; lps (o antigen); O antigen; Polymerase chain reaction; Propanol; Public health; Purification; quantitative polymerase chain reaction; Ribonucleic acid; RNA; Salmonella; Salmonella Typhimurium; Serotyping; Sodium hydroxide; Spleen; γ-Interferon; interferon-γ and interleukin 1β genes expression; purification; Salmonella Typhimurium; quantitative polymerase chain reaction; high-performance liquid chromatography; LPS (O antigen); extraction
• ISSN: 0972-8988; 2231-0916
• DOI: 10.14202/VETWORLD.2020.2338-2345

Lipopolysaccharide (LPS) is an integral part of the outer cell membrane complex of Gram-negative bacteria. It plays an important role in the induction and stimulation of the immune system. Various LPS purification protocols have been developed. However, analysis of their efficacy is limited by contamination during downstream applications or the public health hazard of LPS. The aim of this study was to evaluate a modified method for extracting LPS as well as assess the purity of the extracted LPS by high-performance liquid chromatography (HPLC) analysis. Further, we evaluated its immunopotentiating function by measuring the relative RNA expression levels of splenic immune-related genes such as interleukin 1β (IL-1β) and interferon-γ (IFN-γ), after intramuscular injection of increasing concentrations of the extracted LPS in specific pathogen-free (SPF) chick.
Isolation, identification, and serotyping of
Typhimurium were performed using chicken flocks. We then performed molecular typing of
isolates using conventional polymerase chain reaction (PCR). A new protocol for purification of LPS from
isolate (
. Typhimurium) was conducted. HPLC analysis of the extracted LPS in the current study was compared to existing methods. An
study was performed to evaluate the ability of LPS to induce an immune response by measuring relative IFN-γ and IL-1β gene expression after injecting increasing concentrations of the extracted LPS into SPF chicks.
Isolation and serotyping revealed that
was of the serovar Typhimurium. Confirmation was conducted by molecular typing through conventional PCR. Fractionation of the LPS extract by HPLC revealed a high degree of purity comparable with standard commercial LPS. These results demonstrate the high purity of extracted LPS based on our modified method using propanol and sodium hydroxide mixture. Intramuscular injection of the extracted LPS in 22 day-old SPF chicks, compared to the negative control, revealed significant upregulation of IFN-γ and slight downregulation of IL-1β.
The new modified method can be used for high purity LPS extraction and demonstrates effective immunopotentiating activity.