Purification and immunological characterization of an outer membrane protein of Pasteurella multocida type A:3

Neuraminidase, an outer membrane protein of Pasteurella multocida type A:3, was purified to near homogeneity by a combination of open column chromatography techniques including hydrophobic interaction, ion exchange on DEAE Biogel A, and high performance liquid chromatography on TSK DEAE column. Specific activity of the purified enzyme was 35.54 units/mg and its molecular weight, determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis, was apparently 34 kilo daltons. Crude neuraminidase extract did not possess N-acetylneuraminate-lyase pyruvate activity. The enzyme hydrolized N-acetylneuraminyl lactose, fetuin and bovine colostral sialomucoid but was inactive against bovine submaxillary mucin.; Antiserum was prepared in rabbits against the partially purified enzyme. The antiserum contained anti-type 3 lipopolysaccharide (lps). Anti-lps was removed by adsorption with protease digested lps on an immunoadsorbent column. The adsorbed anti-neuraminidase reduced by 78%, the neuraminidase activity of homologous P. multocida. In a related study, neuraminidase activity of 35 heterologous strains of P. multocida was reduced to varying degrees ranging from 2.8% to 27.6%.; The protective capabilities of anti-neuraminidase was studied in mice. All mice passively immunized with adsorbed anti-neuraminidase survived homologous challenge infection. Three additional groups of mice were passively immunized with antisera that contained antibodies to both neuraminidase and type 3 lps. In these groups, 71%-78% of the mice survived. Protection in these groups was attributed to anti-neuraminidase rather than anti-lps. Control groups of mice were pre-challenge treated with pre-immune serum, normal saline or nothing. In these groups, 7%-14% of the mice were protected. It was concluded that neuraminidase may be a virulence factor of P. multocida and that the enzyme may be a prospective antigen for incorporation into a subunit vaccine for protection of animals against pasteurellosis.