Triterpenoid saponins containing an acetylated branched D-fucosyl residue from Quillaja saponaria Molina.

Seven novel saponins were isolated from a bark extract of Quillaja saponaria Molina. the compounds were characterized, using mainly NMR spectroscopy, mass spectrometry and chemical methods, as quillaic acid substituted at C-3 with oligosaccharides consisting of various compositions of D-glucuronic acid D-galactose, D-xylose, and L-rhamnose and at C-28 with complex oligosaccharide structures consisting of various compositions of D-xylose, L-rhamnose, D-apiose and a branched 4-O-acetyl-D-fucose residue.

Separation and structural analysis of some saponins from Quillaja saponaria Molina.

A fraction of saponins from Quillaja saponaria Molina, QH-B, was fractionated by consecutive separations on three different reverse-phase HPLC systems. Eight compounds were isolated and the structures of these were elucidated mainly by sugar analysis and NMR spectroscopy. The structures consisted of a quillaic acid substituted with two different trisaccharides at C-3, beta-D-Galp-(1-->2)-[alpha-L-Rhap-(1-->3)]-beta-D-GlcpA and beta-D-Galp-(1-->2)-[beta-D-Xylp-(1-->3)]-beta-D-GlcpA, and a tetra- or pentasaccharide at C-28, beta-D-Xylp-(1-->4)-[beta-D-Glcp-(1-->3)]-alpha-L-Rhap-(1--> 2)-beta-D-Fucp and beta-D-Apif-(1-->3)-beta-D-Xylp-(1-->4)-[beta-D-Glcp-(1-->3) ]-alpha-L- Rhap-(1-->2)-beta-D-Fucp. These compounds were further substituted with an acyl group either at O-3 or O-4 of the fucose residue, which is the sugar linked to C-28 of the quillaic acid.

Protective effect of two Mycoplasma gallisepticum protein fractions affinity purified with monoclonal antibodies.

Two protein fractions of Mycoplasma gallisepticum (Mg) were affinity purified with monoclonal antibodies A3 and B3, and tested for protective capacity in chickens. One fraction, designated MgP1, appeared as a doublet of 64 and 62kDa bands in sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels, while MgP2 consisted of five polypeptides (64, 56, 47, 45 and 43 kDa). The molecular mass, haemagglutination activity and matching amino acid sequence of MgP1 suggest that it is identical to pMGA1.2, the putative haemagglutinin of Mg. Groups of Mg-free chickens were immunized once or twice with 1 or 5 mug MgP1 or MgP2, or a combination of the two, and adjuvanted with immunostimulating complexes. Except for the group given 1 mug MgP1, all vaccinated groups showed a significant (P < 0.01) reduction in air sac lesions after challenge compared with unvaccinated controls. MgP2 appeared more protective than MgP1. Vaccination twice with MgP2 was the only regime that produced a detectable serum antibody response.

Triterpenoid saponins from Quillaja saponaria.

Three new saponins were isolated from a commercial bark extract of Quillaja saponaria Molina. These compounds were also obtained as degradation products from larger saponins in this extract when treated with strong alkali. The compounds were characterized, using mainly NMR spectroscopy, mass spectrometry and chemical methods, as quillaic acid 3-O-¿beta-D-galactopyranosyl-(1-->2)-beta-D-glucopyranosiduronic acid¿, 3-O-¿alpha-L-rhamnopyranosyl-(1-->3)-[beta-D-galactopyranosyl-(1-->2)] -beta-D-glucopyranosiduronic acid¿ and 3-O-¿beta-D-xylopyranosyl-(1-->3)-[beta-D-galactopyranosyl -(1-->2)]-beta-D-glucopyranosiduronic acid¿, respectively.

Protective immunity induced in chicken by a single immunization with Mycoplasma gallisepticum immunostimulating complexes (ISCOMS).

An experimental immunostimulating complex vaccine has been prepared from detergent (Mega-10) solubilized Mycoplasma gallisepticum (MG) antigens. Sucrose gradient centrifugation, SDS-PAGE and immunoblotting studies demonstrated that the ISCOM vaccine contained virtually all of the immunodominant MG membrane proteins, including p64 and p56. Protective immunity generated by the experimental MG ISCOM vaccine was demonstrated in challenge experiments. Chickens immunized with a single dose containing between 1 and 50 micrograms of MG ISCOMs had significantly reduced lesion scores in the air sac after challenge. The reisolation of the challenge MG strain was significantly less frequent from the chickens in vaccinated groups than from the unvaccinated control group. Presence of humoral antibodies in chickens vaccinated with 1-25 micrograms MG ISCOMs was not detectable by blocking ELISA or haemagglutination-inhibition before challenge. Chickens vaccinated once or twice with a 50 micrograms dose were transitory positive by blocking ELISA and rapid plate agglutination before challenge.

Monoclonal antibodies to Mycoplasma gallisepticum membrane proteins.

Monoclonal antibodies (MAbs) were prepared to study the immunogenesis of Mycoplasma gallisepticum. Balb/c mice were immunized with M. gallisepticum immunostimulating complexes and the supernatant of heterokaryotes screened with M. gallisepticum and closely related M. synoviae as antigens in indirect enzyme-linked immunosorbent assay. All selected MAbs proved to be M. gallisepticum species-specific when they were tested against 10 different avian Mycoplasma species. After immunoblotting analysis, five polypeptides were identified with estimated molecular weights of 110,000, 66,000, 64,000, 56,000, and 50,000. Cell membrane localization of the recognized polypeptides was studied by immunoelectron microscopy. None of the MAbs inhibited the hemagglutinating activity of freshly prepared M. gallisepticum. However, one MAb (B3) specific for p56 agglutinated the stained M. gallisepticum antigen in the slide agglutination test. Results seemed to correlate with published information on the protein composition and agglutinating activity of Mycoplasma gallisepticum.