Outer membrane extracts of Treponema hyodysenteriae were used to evaluate the antibody responses in immunized or convalescent pigs. Western blot (immunoblot) analysis identified antibodies in sera reactive with 14to 19-kilodalton (kDa) antigens. Reactivity against these antigens could be removed only by absorption of sera with butanol-water-extracted endotoxin from the homologous strain of T. hyodysenteriae. Treatment of the outer membrane extracts with 0.1 M sodium meta-periodate, but not with proteinase K, abolished reactivity with both outer membrane and endotoxin antigens (14 and 19 kDa). These results indicate that swine vaccinated with the outer membrane extract of T. hyodysenteriae develop antibody responses to outer membrane antigens qualitatively similar to those of swine convalescing from active infection, especially antibodies against low-molecular-mass antigens. The nature of the 14to 19-kDa antigens appears consistent with that of treponemal endotoxin and lipopolysaccharide.

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The chemical composition and classical biologic activities of lipopolysaccharide (LPS; phenol-water) and endotoxin (butanol-water) preparations from virulent Treponema hyodysenterinae and avirulent Treponema innocens were examined. The LPS and endotoxin preparations from T. hyodysenteriae B204 contained approximately 80.9 and 35.2% hexose, 0.12 and 0.45% thiobarbituric acid-reactive compound, and <1 and 11.3% protein, respectively. The LPS and endotoxin preparations of T. innocens B1555a contained approximately 56.3 and 37.8% hexose, 0.45 and 0.4% thiobarbituric acid-reactive compound, and <1 and 26% protein, respectively. A silver-stained 7.5 to 15% sodium dodecyl sulfate-polyacrylamide gel showed four bands for the T. hyodysenteriae preparations, while the T. innocens preparations failed to resolve into discrete bands on electrophoresis. We determined by the Limulus amebocyte lysate assay that the treponemal preparations had comparable amounts of endotoxin activity when Eschenichia coli LPS was used as a standard. The 50% lethal doses of LPS and endotoxin from T. hyodysenteriae for BALB/cByJ mice were 380 and 80 ,ug, respectively. The treponemal preparations were poor adjuvants, failed to induce a dermal Shwartzman reaction, and were not pyrogenic. The treponemal LPS preparations, unlike the endotoxin preparations, were not mitogenic for murine spleen cells. Differences in virulence between the two treponemal species could not be associated with the biologic activities of the respective LPS or endotoxin moieties, but the endotoxin preparations were consistently more active than the purified LPS preparations. Lipopolysaccharide (LPS) is a molecule that is found in the outer membrane of gram-negative bacteria and is associated with numerous biologic effects on the mammalian immune system (25). These responses include B-cell mitogenicity, polyclonal antibody induction, adjuvanticity, macrophage activation, immunogenicity, pyrogenicity, lethality, induction of tolerance, and inflammatory reactions (25, 33, 34, 39). Different extraction methods for LPS have been reported (5, 8, 24, 46). LPS, when free of contaminating protein, is extracted by the hot phenol-water method of Westphal and Jann (46). Endotoxin preparations, which consist of LPS as well as lipid A-associated protein(s), is extracted by either the trichloroacetic acid methods of Boivin and Mesrobeanu (5) and Staub (36) or the butanol-water method of Morrison and Leive (24). Endotoxin possesses all of the aforementioned biologic activities and the ability to stimulate lymphoreticular cells from LPS-hyporesponsive C3H/HeJ mice (10, 45). This stimulation has been shown to reside with the protein moieties that are associated with endotoxin (10, 23, 25). In comparison with the LPS from Escherichia coli, there are numerous gram-negative organisms which have LPS with various chemical and biologic characteristics (12, 31, 35). These differences include the inability to detect the presence of 2-keto-3-deoxyoctonate (KDO) (9), differences in fatty acid composition ...

Two monoclonal antibodies (mAbs), SM3G11 and SM6C10, can be used to discriminate between functionally distinct murine CD4+ T cell subsets. In this study we use high-performance thin-layer chromatography and immunostaining techniques to show that the 3G11 mAb reacts with two bands of a ganglioside fraction from murine spleen and thymus, and rat spleen. The 6C10 antibody shows no evidence of glycolipid reactivity. The 3G11+ bands have a mobility between those of the reference gangliosides GD1a and GD1b from human brain. The 3G11+ reactive bands were eluted in the disialyl fraction of rat spleen gangliosides using DEAE anion-exchange chromatography. Treatment of spleen gangliosides with endoglycoceramidase eliminates 3G11 antibody binding over time, indicating that the antigen contains a Glc beta 1-1'ceramide linkage, characteristic of a glycosphingolipid. Treatment of thymus or spleen gangliosides with sialidase eliminates binding of 3G11, thus indicating that the 3G11 epitope is dependent on the expression of one or more sialic acid residues. Immunostaining studies with a variety of reagents indicate that the 3G11+ gangliosides: (i) probably do not contain either the asialo-GM1 or the GM1 core structures; (ii) are not recognized by mAbs specific for the oligosaccharides of asialo-GM2, GM2, GD2 and GD3 gangliosides; and (iii) are also not recognized by antibodies or reagents that are specific for several structures representative of other major glycosphingolipid classes. Overall, these studies strongly suggest that the 3G11+ gangliosides have structures that have not been previously recognized in murine lymphoid tissue. Structures that could account for the known properties of the 3G11+ molecules are described. Finally, ways in which the selective expression of 3G11+ gangliosides might be linked to functionally distinct T-cell behaviours are discussed.