John W. Cberwonogrodzky, Gerard Dubray, Edgardo Moreno, and Hubert Mayer*

E. Brucella Polysaccharide Used to Discriminate Infected from Vaccinated Cattle ......................................................... 24

20 Animal Brucellosis

extensive cross-reaction between the species of Brucella, 1-3 distinct antigens between the abortus and melitensis strains were recognized.4 •5 In 1932, Wilson and Miles designated the A antigen as that which predominates on strains of B. abortus, and theM antigen as that which predominates on strains of B. melitensis. 6 Two years later, Topping reported that B. abortus and B. melitensis extracts, treated with heat and dilute acetic acid, yielded polysaccharides which were antigenic. 7 However, she was unable to show any chemical difference between the isolated components of these bacteria. Miles and Pirie8 later reported that the antigen of B. melitensis was a formyl derivative of an amino-polyhydroxy compound (AP) with a specific optical rotation of [ aW + 43°. This material was released from a larger complex, which contained phospholipids and proteins, by a treatment with acetic acid. Although AP was stable in dilute acetic acid, it lost its antigenicity when treated with 0.1 M HCl (100°C/5 min).9 More recently, Redfearn10 demonstrated that the phenol-extracted smooth-lipopolysaccharide (SLPS) of B. abortus biotype 1 contained the A antigen, whereas Diaz et al. 11 suggested that both the A and M antigens were contained in lipopolysaccharideprotein complexes. Rabbit monospecific anti-A antiserum, but not anti-M nor anti-R antiserum, agglutinated both B. abortus and Yersinia enterocolitica 0:9, 12•13 the cross-reactivity of these bacteria being attributed to 0-polysaccharide similarities. 14

B. COMPOSITION OF THE 0-POLYSACCHARIDE OF BRUCELLA It is now well recognized that SLPS, from Brucella species as well as from many other

organisms of the Enterobacteriaceae, consists of an 0-polysaccharide attached via the core oligosaccharide to the lipid A which anchors the molecule to the outer membrane. The major antigenic epitopes of SLPS and, indeed, of the cell reside in this 0-polysaccharide. Within the core region, one of the 3-deoxy-2-octulosonate (KDO) residues has a ketosidic linkage that is acid labile, and under conditions of mild acid hydrolysis (e.g., 2% acetic acid, 100°C), it is cleaved to release the 0-polysaccharide along with attached core sugars. Lipid A, which is insoluble under these conditions, precipitates. 15

Serological cross-reactivity was confirmed by the use of murine monoclonal antibodies specific for both these antigens. 18 Murine monoclonal antibodies have also been used to show that antibodies specific for the 0-polysaccharide are protective in passive immunity to B. abortus infection. 19

The 0-polysaccharide structure of B. melitensis was found to be related to that of B. abortus in that both were homopolymers of 4,6-dideoxy-4-formamido-a-o-mannopyranosyl units. 20•21 Although this similarity in structure provided a rationale for the cross-reaction between these organisms, it did not account for the fact that the A antigen is serologically distinct from the M antigen. Also, the optical rotations of the purified A and M 0-polysaccharides differed ([a]0 +38° [water] for B. abortus 1119-3 0-polysaccharide; [a]0 +49°

These similarities and differences may account for the cross-reactivity and different antigenicity of the A and M antigens. That the 0-polysaccharides of B. abortus (e.g., strain 1119-3) and B. melitensis (e.g., strain 16M) are the classic A and M antigens, respectively, is based on the following:

1. Rabbit cross-absorbed serotype-specific antisera for either A or M Brucella antigens (from G. M. Brown, National Veterinary Services, Ames, lA; or L. B. Forbes, Health of Animals Laboratory, Saskatoon, Saskatchewan) will selectively precipitate purified B. abortus or B. melitensis SLPS and will selectively bind to the homologous SLPS coated on polystyrene plates. The latter reaction is inhibited by preincubating the antisera with purified homologous 0-polysaccharide. 28

2. Murine monoclonal antibodies, with specificities for B. abortus or B. melitensis apolysaccharides, show similar selectivity to that noted for rabbit antisera. 29 Indeed, monoclonal antibodies can substitute for rabbit cross-absorbed serotype-specific antisera when testing biovars of Brucella spp. for the A or M antigen. 261

Heterologous 0-polysaccharide inhibits rabbit anti-A and anti-M antisera from binding to B. abortus and B. melitensis SLPS coated onto polystyrene plates. 28 Some murine monoclonal antibodies can precipitate the 0-polysaccharides and SLPS of B. abortus 1119-3, Y. enterocolitica 0:9, and B. melitensis 16M, probably because these antibodies have affinity for common structural entities within the O-polysaccharides. 23

With regard to the production and use of synthetic antigens, Bundle et al. 30 have linked the aliphatic chain of a fatty acid ester to the 0-polysaccharide of either B. abortus of Y. enterocolitica 0:9. This synthetic antigen is comparable to the SLPS of either organism in serodiagnostic tests and has the advantage of giving fewer nonspecific reactions with cattle sera tested in an enzyme-linked immunosorbent assay (ELISA)Y Peters and Bundle also reported the synthesis of oligosaccharides of 1 ,2-linked 4,6-dideoxy-4-formamido-o:-o-mannopyranosyl residues (i.e., the A antigen) as well as structures containing the o:-1 ,3 linkages (i.e., the M antigen)32 which should now provide specific diagnostic reagents.