Immunogenic and toxic properties of a purified lipopolysaccharide-protein complex from Pasteurella multocida

Abstract

An immunogenic fraction from Pasteurella multocida was found to consist chiefly of a high-molecular-weight protein-polysaccharide complex containing 25 to 27% protein and 10.7% carbohydrate. The starting material was obtained by differential centrifugation at 105,000 X g of saline extract of P. multocida cells and further purified by gel filtration on Sepharose 2B. Three peaks were usually obtained after gel filtraion.pharose 2B. Three peaks were usually obtained after gel filtration. The component in the first peak amounted to about 10% of the starting material and eluted in the void volume. It was predominately carbohydrate, although some protein was present. Two inoculations of 10 to 20 mug of the first component induced up to 80% protection in mice against a challenge inoculation with P. multocida that killed 100% of the controls. The second, or major, component amounted to about 75 to 95% of the starting material. This fraction contained 25 to 27% protein and 10.7% carbohydrate. Small amounts, 10 to 20 mug, induced active immunity in mice and turkeys, but large amounts could be lethal; the mean lethal dose was 195 mug for mice and 5.7 mug for 10-day-old chicken embryos. The components in the third peak were primarily proteins that gave reactions of nonidentity with the antigens of peak II in gel diffusion. The components present in the third fraction were definitely less effective in the induction of protective immunity than those present in the first or second. Analyses of the protective antigen(s) by the isoelectric focusing procedure in a pH 3 to 10 gradient showed that all of the precipitinogenic activity was found in the range of pH 3 to 4, with a peak at pH 3.7.