Vegetative Cells, and Free Toxin

Millipore diffusion chambers (MDC) with 0.22-j,m filters loaded with spores or vegetative cells of Clostridium botulinum were surgically implanted intraperitoneally (ip) into guinea pigs. MDC expose C. botulinum spores or vegetative cells to body fluids yet protect them from phagocytes. Guinea pigs receiving MDC containing 109 spores plus 108 polymorphonuclear (PMN) leukocytes and MDC with 109 vegetative cells died within 48 hr, indicating that toxin was released and diffused out. MDC with 109 spores alone allowed 90% of animals to survive for at least 96 hr. Microscopically, it was observed that vegetative cells in MDC were disintegrated and leukocytes plus spores were phagocytized and germinated; spores alone remained intact and phase bright. Chemotactic attraction of leukocytes to MDC walls was also observed. Apparently, body fluids do not attack spores; thus, PMN leukocyte engulfment is essential for germination and release of sporebound toxin in this type of C. botulinum pathogenesis. However, vegetative cells appear to be attacked by bacteriolytic enzymes (e.g., lysozyme) in body fluids, and leukocyte engulfment is not essential for toxin release.