Several saccharides representative of the O-antigenic polysaccharide chain of Salmonella typhimurium (O antigens 4 and 12) were used as haptenic groups covalently linked to bovine serum albumin. The disaccharide abequose 1 → 3 D-mannose was synthesized, and the [Formula: see text] tetra-, octa- and dodecasaccharides were isolated after cleavage of isolated S. typhimurium O-polysaccharide chains by using bacteriophage endo -glycosidases. Rabbits immunized with the saccharide-protein conjugates suspended in Freund complete adjuvant readily responded with O4 antibody titers as high, or almost as high, as those elicited by heat-killed bacteria. The octa- and dodecasaccharide conjugates also gave rise to O12 antibody titers. The antibody response in mice differed in two ways from that seen in rabbits: mice did not respond with measurable antibody production against the disaccharide haptens, and the highest S. typhimurium lipopolysaccharide antibody response elicited by the saccharide haptens was still approximately 50-fold lower than that elicited by heat-killed bacteria. The latter difference may be a consequence of the fact that the mouse preferentially produces antibodies against the galactose residue which is terminal in the hapten but not in the native O-antigenic polysaccharide chain. Antibodies elicited in rabbits against all saccharide-protein conjugates protected passively transferred mice against intraperitoneal challenge with 100 times the 50% lethal dose of S. typhimurium SH 2201 (O4, 12) but not against challenge with S. enteritidis SH 2204 (O9, 12). The antibodies elicited by the saccharide-protein conjugates protected as well as antibodies elicited by heat-killed bacteria.
Lipoarabinomannan (LAM) is a major and structurally important outer cell wall component of all mycobacteria. LAM is also generally regarded as an important immunomodulating substance affecting several immunologic networks and hence important in the pathogenesis of mycobacterial infections. We here describe a new method for large-scale purification of mycobacterial LAM. A crude cell wall preparation was prepared from batch-grown Mycobacterium tuberculosis H37Rv. From this cell wall preparation LAM was purified by sequential extractions and chromatographic steps. From 20 g dry weight cell wall preparation 313 mg of highly purified (> 98%) LAM was obtained in only 3 days. The LAM content of the final purification step was quantified by ELISA using reference LAM as standard. The identity and purity of the LAM preparation was further confirmed by comparison with reference LAM preparation from M. tuberculosis strain Erdman in polyacrylamide gel electrophoresis and Western blots, using reference anti-LAM monoclonals CS-35 and CS-40.
To further our understanding of the mechanisms by which ethambutol potentiates the effect of other antimycobacterial drugs on mycobacteria we have studied the initial physico-chemical interaction between ethambutol and the Mycobacterium avium cell envelope using batch reaction microcalorimetry. When strains of M. avium were exposed to ethambutol an immediate endothermic reaction was recorded. When the M. avium cells were pre-treated with ethambutol this strongly affected the initial interaction between streptomycin and the bacterial cell surface. When the M. avium cells were simultaneously exposed to a combination of ethambutol and streptomycin an altered initial interaction with streptomycin was seen. These data suggest that ethambutol may potentiate the effect of other antibacterial drugs on M. avium by increasing cell wall permeability.
Tuberculosis has been diagnosed in wild boar (Sus scrofa) in several European countries during the last decade; however, almost no information has been reported to date for Portugal.This study aimed to investigate tuberculosis in wild boar in Portugal through characterization of Mycobacterium bovis infection and identification of disease risk factors.Tissue samples were obtained from hunted wild boar during the 2005 and 2006 hunting seasons.Samples were inspected for gross lesions and processed for culture.Acid-fast bacterial isolates were identified by polymerase chain reaction and spoligotyping.Associations between tuberculosis in wild boar and several variables linked to wild ungulate diversity and relative abundance, livestock density, and cattle tuberculosis incidence were investigated.Mycobacterium bovis isolates were identified in 18 of 162 wild boars from three of eight study areas.Infection rates ranged from 6% (95% confidence interval [CI P95% ]51-21%) to 46% (CI P95% 527-67%) in the three infected study areas; females in our sample were at greater risk of being infected than males (odds ratio54.33;CI P95% 53.31-5.68).Spoligotyping grouped the M. bovis isolates in three clusters and one isolate was a novel spoligotype not previously reported in international databases.Detection of M. bovis was most consistently associated with variables linked to wild ungulate relative abundance, suggesting that these species, particularly the wild boar, might act as maintenance hosts in Portugal.
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