Protection of Mice from Tularemia Infection with Ultra-Low, Serial Agitated Dilutions Prepared from Francisella tularensis -Infected Tissue 1

Abstract

Reports of immunomodulation with serial agitated dilutions (SADs) of cytokines, hormones, minerals, and whole tissue led to this inquiry as to whether exposure to a complex SAD preparation produced from Fran- cisella tularensis- infected mice could alter the immune response and the ef- fects of subsequent challenge with this pathogen in vivo. Six SAD prepara- tions of reticuloendothelial tissue from F. tularensis- infected C3H/HeN mice were produced through a process of serial log 10 and log100 dilutions in 70% ethanol interspersed with 30-second agitation. SAD preparations were ana- lyzed for protein content and for contamination with 1 H-NMR spectroscopy. Three preparations contained detectable protein by Lowry and NMR analy- sis, and three were diluted beyond detection of protein. These preparations were administered orally for 1 month to 147 animals randomly assigned to SAD or dilutent control groups. All animals were then challenged with a lethal dose (LD 50 or LD75) of F. tularensis and evaluated for time to death and total mortality. In a series of 15 trials, the SAD preparations consistently produced increased mean times to death (MTD; MTD SAD = 18.6 days (range, 12.9- 25.6); MTD controls = 13.7 days (range, 11.6- 15.6)), and de- creased mortality (SAD: 53%; control: 75%) when compared with matched control groups given the dilutent only. Protection was not related to the level of dilution, the number of times vortexed, or the presence or absence of orig- inal substance from the tissue. Active and inactive solutions could be distin- guished from one another using 1 H NMR-spectroscopy. Two preparations in- duced specific anti- tularemia IgG antibody production before challenge. This anomalous finding needs independent repetition and further investigation.