Specific lipopolysaccharides of Bordetella.

In nmany species of Granm-negative bacteria one of the surface components of the bacterial cell is a material composed of polysaccharide, protein and phospholipid residues (Boivin, Mesrobeanu & Mesrobeanu, 1933; Morgan, 1937, 1949; Miles & Pirie, 1939 a; Morgan & Partridge, 1940, 1941, 1942; Freeman, 1943; Davies, Morgan & Mosimann, 1954). These substances are antigenic, toxic and pyrogenic and are thermostable with respect to these properties; the dominant immunological specificity is determined by the polysaccharide moiety. They have been referred to as endotoxins or Boivin antigens or, in special cases, as 0 somatic antigens, but these terms, especially endotoxin, are also used with other meanings. Although little is known either of the chemical composition or the biological properties of the endotoxins of Bordetella pertussis (Haemophilus pertussis), B. parapertussis and B. bronchisepticus, there is already ample evidence for the occurrence of biologically active polysaccharides in organisms of these species. Cruickshank & Freeman (1937) digested phase I B. pertussis cells with trypsin and precipitated with ethanol from the soluble digest a material containing polysaccharide. This preparation also gave positive reactions in the Millon's, ninhydrin and biuret tests. It had an immunizing potency for mice equal to that of whole cells when immunity was measured by intraperitoneal challenge. Eldering (1941, 1942) isolated lipopolysaccharides from strains of the three Bordetella spp. and obtained grossly similar products whether she used a modification of the trichloroacetic acid method (Boivin et al. 1933) or the hydrochloric acid or tryptic-digestion methods of Felton & Kauffmann (1938). The preparations were soluble in water, giving opalescent solutions; the M6lisch test was positive and reducing sugars were detected with Benedict's solution after acid hydrolysis. Tests for protein were negative. The B. bronchiseptious lipopolysaccharide was toxic for mice and an insoluble toxic component could be obtained from it by 0-2N-acetic acid hydrolysis. The three substances appeared to be serologically distinct but immunization of mice with any one of them conferred substantial protection against intraperitoneal challenge with B. bronchisepticus. It has been suggested more recently, from the results of agglutination and agglutinin-absorption tests, that the three Bordetella spp. have a common thermiiostable 0 antigen (Andersen. 1952; Eldering, Hornbeck & Baker, 1957). The detailed chemical composition of these polysaccharide materials is not known although the substances described by Eldering (1941, 1942) are clearly of an 'endotoxin nature.' However, the presence of mannose, galactose and a 'fructoselike' substance in a polysaccharide extracted with '50% pyridine' from defatted cells of B. pertussis has been reported (Akiya, Takahashi, Kuriyama & Ogawa, 1951). Although this method of extraction is known to yield endotoxin when applied to certain other bacteria (Goebel, Binkley & Perlman, 1945), the absence of any reference to biological or serological properties in this instance leaves the precise nature of the material in doubt. In the present study the chemical composition and biological properties of the lipopolysaccharide components of the Bordetella endotoxins are described. A preliminary report has already appeared (MacLennan, 1957). These toxic substances are distinct from the well-known thermolabile toxin produced by Bordetella spp. (A. P. MacLennan, unpublished work).