Endotoxin content and lethal toxicity on duck (Anas luzonica) embryos of crude lipopolysaccharide extracts from clinical and non clinical isolates of Escherichia coli (Migula) Castellani and Chalmers

This study was conducted to evaluate selected lipopolysaccharide (LPS) extraction protocols, semi-quantify the endotoxin content (in EU per ml) of the LPS extracts from clinical and non-clinical strains of Escherichia coli, and compare their lethal toxicity (LD¬ sub 50) on ten day old duck embryos. Three LPS extraction protocols (I, II, and III) were evaluated for use in subsequent LPS extraction experiments. Only protocol III, based on the method of Braun and Landy (1964) yielded crude E. coli LPS with a higher concentration of 0.03 to 0.06 EU/ml; the rest had 0.03 EU/ml. Protocol III was also easier to perform and used less toxic reagents. The mean LD sub 50 values (in mg/g) of the extracts obtained using the three selected methods (Protocol I: Alaubydi et al., 2012; Protocol II: Rezania et al., 2011 and Protocol III: Braun and Landy, 1964 and Roberts, 1966, respectively) were, however, not significantly different (p-value 0.05). Five clinical (MNH 1647, MNH 1648, MNH 1650, MNH 1652 and BIOTECH 1959) and five non-clinical (MNH 1646, MNH 1649, MNH 1651, BIOTECH 1878 and ATCC 25922) E. coli strains, sourced from local culture collections, were checked for typical phenotypic properties and subjected to O-serogrouping. All exhibited the expected E. coli phenotype (Gram negative reaction, rod-shaped, catalase positive and, oxidase negative reactions, indole production positive, methyl red positive and Voges-Proskauer negative reactions, citrate utilization negative, smooth, white colonies on Nutrient Agar, green, metallic colonies on Eosin Methylene Blue Agar and pink colonies on MacConkey Agar) and almost all were positive for agglutination in one of the eight polyvalent O-antisera tested. Lipopolysaccharides (LPS) from these ten strains of E. coli were extracted using Protocol III. The extracts were compared o the basis of their semi-quantified endotoxin content (EU/ml), determined using the Limulus Amebocyte Lysate (LAL) Gel-Clot Assay, and on their lethal toxic activity (based on Lethat Dose sub 50) on duck embryos. All the ten E. coli isolates had an endotoxin content of 0.03 to 0.06 EU/ml. The LD sub 50 values of the crude LPS extracts from clinical strains (mean