Immunogenic activity of Liposome incorporated Lipopolysaccharide Antigen of Salmonella enterica serovar Typhimurium in Mice

The study was conducted to assess the role of Salmonella enterica serovar Typhimurium as a causative pathogen in hospitalized diarrhoeal patients younger than five years old, extract and purify endotoxin (lipopolysaccharide; LPS) from isolated and identified Salmonella enterica serovar Typhimurium, determine the role of LPS-liposome conjugate as a potential vaccine against Salmonella enterica serovar Typhimurium in albino male mice, and finally evaluate humoral and cellular immune responses (total and absolute counts of leukocytes, phagocytosis, Arthus reaction; AR and delayed type hypersensitivity reaction; DTHR) of vaccinated mice, in addition to histopathological changes in liver and spleen. Ninety five patient were admitted to the Central Pediatric Hospital and Al- Kadhiymiah Pediatric Hospital in Baghdad during the period 24/10/2010 - 30/11/2010, because of severe diarrhoea and fever. Bacterial evaluation of stool
samples revealed the identification of two (2.1%) Salmonella enterica serovar Typhimurium isolates (S1 and S2). Antibiotic sensitivity test demonstrated that S1 isolate was more resistance than S2 isolate; therefore it was considered more virulent and subjected for further manipulations, which included extraction of
LPS from the bacterial outer membrane. Chemical characterization of the extracted LPS revealed that the carbohydrate content was 2.34 mg/ml, while the protein concentration was very low (0.52 μg/ml). Partial purification extracted LPS by using gel-filtration chromatography (sephacryl 200 S) showed three peaks, and after determination of protein and carbohydrate concentrations for each peak, the second peak observed to have the highest carbohydrate content (25%) and the lowest contaminated protein (0.001%). The LPS of this peak was immunologically evaluated in mice at a concentration of 100 μg/ml, alone or in
conjugation with a commercially available liposome (LIP). The mice were distributed into eight groups (negative controls(NC), positive controls( PC), complete Freund's adjuvant (CFA), heat-killed bacteria;
(HKB), formalin-killed bacteria (FKB), (LPS), (LIP) and (LPS-LIP conjugate). Each mouse was injected intraperitoneally (IP) with 0.1 ml of the respective solution in day 1, and a further dose in day 8. These mice were considered as pre-challenged groups, and they were dissected for laboratory evaluations in day 15. Further similar groups were challenged with 0.1 ml live bacteria in day 15 (5 x 104 cell/ ml), and they were issected for laboratory evaluations in day 29 (post-challenged groups). The following results were obtained: 1. The total leukocyte count in all groups (pre- and post-challenged) was almost approximated the normal leukocyte count range in mice, but mice vaccinated with HKB or FKB showed the highest count in pre-challenged animals, and such increase also contributed to increased counts of lymphocytes, eutrophils and monocytes. However, mice vaccinated with LPS showed a significant increased count of lymphocytes in post-challenged animals; an observation that suggests that LPS may be able to enhance the adaptive immunity against Salmonella enterica serovar Typhimurium. 2. The highest percentage of phagocytosis was observed in mice vaccinated with LPS+LIP in pre- and post-challenged groups. Such finding highlights the importance of conjugation between LPS and LIP in enhancing the function of phagocytic cells. 3. In most types of post-challenged vaccinated groups, AR and DTHR responses were increased, while in pre-challenged groups some variations were observed, but mice vaccinated with LPS+LIP conjugates were almost recorded the highest results. 4. Bacterial evaluation of liver and spleen in mice vaccinated with LPS-LIP conjugate in post-challenged mice showed no growth of bacteria (Salmonella enterica serovar Typhimurium for liver and a scantly growth for spleen. In addition, histological section of liver showed look-like normal hepatic tissue appearance especially near the portal area.