No abstract

Abundant adenylate cyclase activity was found in the phase I cultures not only of Bordetella pertussis but also of B. parapertussis and B. bronchiseptica. The enzyme activity in the culture fluid increased rapidly and reached a peak during the logarithmic growth phase. B. parapertussis and B. bronchiseptica especially produced a high activity of the enzyme in the culture fluid during the logarithmic phase, but little or no activity was detected in the cells throughout the growth period. In the culture of B. pertussis, the intracellular activity was higher than that in the culture fluid. Phase III cultures of these species lacked both the extracellular and intracellular enzyme activities throughout their growth.In the culture of B. parapertussis, accumulation of cyclic AMP was parallel to that of adenylate cyclase activity through the growth periods, but in B. pertussis there was no parallelism from the stationary through the declining phases. The difference in production patterns of the enzyme activity among the species is discussed.

The heat-labile toxin (HLT) of Bordetella bronchiseptica was purified successively from sonic extracts of phase I organisms grown in Stainer-Scholte medium, by partition in hydrophobic interaction, sucrose density gradient centrifugation, gel filtration through Sepharose 4B and 6B, isoelectric precipitation and isoelectric focusing. The purified HLT was homogeneous by disc polyacrylamide gel electrophoresis and the gel diffusion-test, and free of detectable hemagglutinin and endotoxin activity. A 386-fold purification over the crude extract was obtained at a yield of about 28%, and a minimum dose of 0.9 ng was dermonecrotizing with a lesion 5 mm in diameter in guinea pigs and induced splenoatrophy. The mouse LD50 was 200 ng (intraperitoneal) or 70 ng (intravenous). The HLT was found to be a simple protein with an isoelectric point of pI 6.9. It has a molecular weight of 102,000 estimated by Sepharose 6B gel filtration and was found to consist of two different types of polypeptide by SDS-polyacrylamide gel electrophoresis, their molecular weights being 30,000 and 20,000. Amino acid analysis showed 15 common amino acid residues, and methionine, cysteine and tryptophan were undetectable. The HLT crystallized by methylpentanediol showed a block form. The HLT was inactivated at 56 C when heated for 10 min, and at above pH 9 and below pH 4.

A cell wall fraction obtained by sonic oscillation, yielded a Bordetella pertussis O antigen which possessed the ability to both adsorb O agglutinins and to produce the O agglutinins in animals. It was purified by a phenol water extraction and centrifugation method. Physico‐chemical and biological properties of the O antigen were similar to that of the so‐called endotoxic lipopolysaccharide. It contained 2.6% nitrogen, 1.2–1.5% phosphorus, 9.0–11.0% heptose, 0.3% 2‐keto‐3‐deoxyoctonate (KDO), 13.3–13.4% as total carbohydrate, 16–16.5% hexosamine and 25–32% lipid. Homogeneity of the purified O antigen was confirmed by ultracentrifugation, electronmicrograph and gel‐diffusion tests. It possessed significant pyrogenicity, adjuvant effect on antibody production, and toxicity to animals, but not protective antigenicity.