sepharose and Leukocytosis

Pertussis toxin (PT) and filamentous hemagglutinin (FHA) were purified from culture supernatant of Bordetella pertussis Saadet and Tohama strains, using CM-Sepharose CL-6B cation-exchange chromatography. By the rapid purification method described here, both proteins were separately eluted from the same column in pure forms. The PT and FHA in the extract of culture supernatant were bounded to CM-Sepharose CL-6B cation-exchange column in 50 mM phosphate buffer containing 2 M urea (Buffer A), pH 6.0. Then the PT was eluted from the column with Buffer A (pH 7.4) and after elution of the PT, the FHA was eluted with 0.5 M NaCl in 50 mM phosphate buffer. Pertussis toxin and filamentous haemagglutinin purified by this procedure were electrophoretically and immunologically identical to the reference preparations.

Topics: Animals; Bordetella pertussis; Chickens; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Erythrocytes; Hemagglutination Inhibition Tests; Hemagglutinins; Histamine; In Vitro Techniques; Leukocytosis; Mice; Pertussis Toxin; Sepharose

Adult Sprague-Dawley rats infected intrabronchially with Bordetella pertussis strain 18-323 encased in agarose beads (BP-beads), developed a paroxysmal cough and leucocytosis, both of which peaked at around day 10. When animals were exposed to ether for 2 min after delivery of the beads, there was an enhancement of the number of subsequent coughing episodes. Inclusion of carrageenan in the beads also enhanced coughing. Control rats, given sterile beads or left untreated, showed only a low level of coughing or no coughing, depending upon their source. Rats challenged by the same route with heat-killed B. pertussis in beads, or with live organisms in suspension (without beads) showed no cough induction or leucocytosis. However, intranasal delivery of B. pertussis suspension gave rise to a moderate amount of coughing and leucocytosis. Serum IgG responses to B. pertussis antigens were greatest in rats infected with BP-beads and antibodies against both pertussis toxin and filamentous haemagglutinin were detected. Since the rat is the only conveniently accessible laboratory animal species in which B. pertussis induces an intermittent paroxysmal cough, as in man, it merits further study for determining the mechanisms of pathogenesis and immunity in pertussis.

Topics: Animals; Antibodies, Bacterial; Bordetella pertussis; Carrageenan; Cough; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Immunoglobulin G; Leukocytosis; Male; Microspheres; Rats; Rats, Sprague-Dawley; Sepharose; Virulence; Whooping Cough

Topics: Arylsulfatases; Cell Adhesion; Complement C3; Complement System Proteins; Eosinophils; Glucuronidase; Humans; Leukocytosis; Muramidase; Phagocytosis; Sepharose; Sulfatases