Effect of Liposome Conjugated Lipopolysacchariede of Shigella flexneri Against Shigellosis in Mice

This study was conducted to determine the role of lipopolysaccahride-liposome conjugate (LPS-LIP) as a potential vaccine against shigellosis in mice. One hundred stool samples were collected from children and infants (100cases: 63 males and 37 females) under the age of five years (40 days – 2.5 years old) with diarrhea from different hospitals in Baghdad city during the period December 2011-May 2012. Thirty isolates were suspected to be Shigella. Four isolates of Shigella flexneri were obtained after performing some biochemical tests and Api System. Antibiotic sensitivity test was carried out and results revealed that all isolates were resist to antibiotics used in this study. One isolate was selected for LPS extraction by phenol hot water extraction method. Chemical characterization of the extracted LPS revealed that the carbohydrate content was 2.34 mg/ml, while the protein concentration was 0.52µg/ml. Partial purification of the extracted LPS was carried out by using gel- filtration chromatography on Sephacryl S-200 and results showed that three peaks were obtained and protein, carbohydrate concentration were estimated for each peak. The second peak observed to have the highest carbohydrate content (29%) and the lowest contaminated protein (0.001%). The partial purified LPS was analyzed by Sodium Dodecyl Sulphate Gel Electrophoresis (SDS-PAGE) and results reveled that two bands with molecular weight 100 KDa and 150 KDa were present and band with MW 100 kDa represented LPS. The role of LPS- liposome conjugate as a potential vaccine against S. flexneri was evaluated by determining total and absolute count of leukocyte, IgG titer and histopathological changes in liver, spleen and intestine. Twenty five Mice treated with LPS at a concentration 100µg/ml, alone or in conjugation with commercially liposome were preformed as follows:
Group1: (negative control) Group2: mice infected with S. flexneri Group3: mice treated with conjugate (LPS+Liposome) and infected with S. flexneri Group4: mice treated with LPS and liposome to infected with S. flexneri Group5: mice treated with LPS and infected with S. flexneri Each mouse was injected intramuscularly with 0.1 ml of the LPS and conjugate solution in day 1, and further dose in day 8, then these groups dosed with 0.1 ml of live bacteria in day 15, and they were dissected in the laboratory in day 30. The following results were obtained: Mice treated with conjugate and infected with S. flexneri showed a significant increase in total count of leucocytes (7300±361 ab), neutrophils (2469±107 a) and lymphocyte (4818±238 bc).A significant increase in IgG titer (0.9243±0.1094 a) was recorded in mice treated with conjugate after infection with S. flexneri. Results of histopathological study exhibited that sections of spleen, and liver of mice infected with S.flexneri. the spleen showed mild degenerative and loss its structure appearance while degeneration and necrosis of hepatocytes cells observed in the liver. While, sections of spleen, intestine and liver of mice treated with conjugate and infected with S. flexneri showed that the spleen intestinal and liver tissue near normal appearance.