Isolation, purification, and partial characterization of a lipopolysaccharide from Haemophilus pleuropneumoniae

Abstract

Lipopolysaccharide (LPS) from Haemophilus pleuropneumoniae 1536, serotype 2, was isolated and purified by a procedure designed to be equally satisfactory for both smooth- and rough-type LPS. The LPS yield was 53%. Analysis of the preparations revealed that protein, nucleic acid, and cellular phospholipid contamination was negligible (less than 0.1%). Analysis of the sugar content of the LPS by gas-liquid chromatography and colorimetric analysis revealed the presence of rhamnose, mannose, galactose, glucose, heptose, glucosamine, galactosamine, and 3-deoxy-D-manno-2-octulosonic acid. The heptose and glucose contents appeared to be unusually high. The fatty acids of the LPS consisted of a mixture of C(14:0) and C(16:0) in a ratio of about 4.5:1 (50% of the total) and 3-hydroxy C(14:0). When used as a preparatory dose for the dermal Shwartzman reaction, as little as 10 μg of the LPS injected intradermally in rabbits produced reddening and swelling. After intravenous injection of a 100-μg LPS provoking dose, necrosis was observed at all intradermal injection sites. Limulus amebocyte lysate gelation was observed with an LPS concentration as low as 0.5 ng/ml. A typical biphasic fever response was noted in rabbits injected with as little as 0.25 ng of LPS per kg of body weight.