Effect of Dermonecrotic Toxin of Bordetella pertussis on the Spleen of CFW and C57BL/10ScN Mice

Abstract

The dermonecrotic toxin (DNT), also called heat-labile toxin, was discovered by Bordet and Gengou (3) in extracts of Bordetella pertussis cells. The toxin had a marked necrotic effect on the skin of rabbits and guinea pigs and a degenerative action on the spleens of mice (5,7,12). The toxicity was inactivated at 56 C for 10 min (4,6,8). In this note we describe a pronounced difference in the response of spleens from CFW (random bred) and C57BL/IOScN (inbred) mice, raised in the animal production unit at the Rocky Mountain Laboratory, to the effects of this toxin. To our knowledge, differences among strains of mice in their response to purified DNT have not been recorded in the literature.DNT was prepared by a modification of Iida and Okonogi's method (5) and partly Nakase's method (9) from a Phase I Maeno strain of B. pertussis (agglutinogen type 1.2.3.4) grown in a modified synthetic medium of Stainer and Scholte (composition given in Ref. 1). The medium (500 ml per 4-liter Erlenmeyer flask) was inoculated with 2 ml of a cell suspension containing 25 X 10 9 cells per ml. The inoculum was prepared from 24-hr-old Bordet-Gengou agar cultures (composition given in Ref. 7). The inoculated flasks were incubated for 5 days at 35 C under stationary conditions; the cells were then collected by centrifugation (3,000 xg for 1 hr) and resuspended in cold sterile water to a concentration of 10 12 cells per ml. These cells were sonically disrupted (kept cold with iced water) in a Biosonik III apparatus (Bronwill Scientific, Rochester, N.Y.) at 20 kHz for 5 min. The disrupted cells were centrifuged at 27,000 xg for 20 min to remove the cell debris, and the supernatant containing the DNT was collected by decantation. All steps were carried out at 2 to 10 C. About 6 ml of the clear solution of DNT was passed through a DEAE-Sephacel column (2.5 X 60 cm) equilibrated in 0.01 M phosphate buffer (pH 8.0) containing 20 mM sucrose. The column was successively eluted with (i) 800 ml of0.01 Mphosphate buffer, pH 8; (ii) 450 ml of 0.05 Mbuffer, pH 7.2; and (iii) 800 ml of 0.2 M buffer, pH 7.0. All the buffers contained 20 mx sucrose as a stabilizer of the toxin. The toxin, assayed by its dermonecrotic action in the