Immunogenicity of potassium thiocyanate extracted and electrofocused Pasteurella multocida X-73I antigens in chickens...

The immunogenicity of antigenic fractions obtained by the extraction of Pasteurella multocida strain X-73 (serotype 1) with potassium thiocyanate (KSCN) was determined in chickens and mice. The initial KSCN extract was centrifuged at 105 000 X g, and the antigens were separated into a particulate fraction (40p) and a soluble supernatant fraction (40s). The ultracentrifuged fractions were further resolved by preparative electrofocusing. The 40p fraction was resolved into two subgroups having isoelectric points of 3.5-3.9 and 5.5-6.0; the 40s fraction was resolved into five subgroups ranging in isoelectric points from 4.4 to 9.0. The 40p fractions were antigenically similar and contained lipopolysaccharide (LPS) and protein. The 40s fractions were antigenically distinct from the 40p fractions and from each other; they contained proteins and polysaccharides but no LPS. The 40p antigens were strongly immunogenic in mice and chickens, whereas the 40s antigens were weakly immunogenic in chickens and not immunogenic in mice. The incorporation of Freund's complete adjuvant increased the immunogenicity of the 40s antigens in chickens. The 40p antigens induced greater frequencies of serological responses in chickens than the 40s antigens as detected by counterimmunoelectrophoresis and immunodiffusion. This suggested that the increased protection associated with the 40p antigens may have been the result of better antibody response. The toxicity of all the fractions was evaluated by determination of lethality for 10-day-old chicken embryos because of the sensitivity and reliability of the test. The 40p fraction had an LD50 = 0.38 micrograms, and the 40s fraction had an LD50 = 2.5 micrograms. Since the 40s fraction contained no detectable LPS, it is likely that two toxins are present, one which contains LPS and one which does not.