om the Dicisioii o f V i w s n?id 1;icX.r ttsicil I ) i~r t i . s~.~, -l rnzy Medical School, Washiiqton, D . C. A \ substance toxic for mice occurs in yolk sac tissues infected with R. iitooseril and K.prowazcki. ' These related toxins are neutralized by typhus antisera,l*' but are not immunologically identical."* The present report describes the occurrence of a specific toxic material in yolk sacs infected with the Gilliam strain of R . orientalis.Materials and il4ctlzods. The Gilliain strain," received from Dr. S. H. Topping, was niaintained by serial passage in yolk sacs of 7-dayembryonated eggs which were incubated at 35°C after inoculation. 1-olk sacs infected with the 25th to 36th egg passage yielded toxic material. The methods of preparation and testing were as follows: Sacs of embryos moribund on the sixth to ninth day after inoculation were harvested, and smears, stained by a modified Rlacchiavello's technic, were examined microscopically. Those containing 25-100 recognizable rickettsiae per field were pooled and a 2070 suspension prepared in sterile skimmed milk. Following low speed centrifugation, 55, : / 1 and I, $, dilutions of the supernatant fluid were prepared in 0.9% NaCl solution containing 10% normal rabbit serum. 0.5 cc of each dilution was injected intravenously into each of 5 mice. Deaths were recorded up to 18 hours when survivors were discarded. Tosin-neu tral iza-tion tests were done as follows: A dilution of a 2Oc/o suspension of freshly harvested infectious sacs was tested for toxin in 5 mice; if 4 died within 2 hours, the material was considered suitable. Serial 2-fold dilutions of sera to be tested were then prepared in 10% normal serum-NaCI solution. These vere mixed with equal volumes of the stock 20% suspension of sacs which had been stored meanwhile at 0°C. Intravenous injection of mice with 0.5 cc amounts of the mixtures was begun immediately and usually completed within an hour. A control titration of the toxic suspension was performed at the end of the test. Toxin-neutralization tests were done with pooled sera of guinea pigs and serum of one patient recovered from infection with the Gilliam strain, and with pooled sera of rabbits hyperimmunized with splenic tissue of mice dead from infection with this strain. Antisera against the K a r~,~ Kostival,6 Host 2 1 ,ci Seerangayee? Volner: Imphal: Wild Rat No. 2,t and CBI Rat+ strains of scrub typhus were also tested for antitoxin. These hiid been shown in other experimentdo to be capable of protecting mice from death caused hy infectionll with the homologous strain.Results. Mice injected intravenously with toxic preparations appeared normal for 1 to 1)5 hours, then developed dyspnea and weak-___ *Member of United States of America Typhus Corn mission. 1 Gildemeister, E., and Hangon, E., D m t . .Mcd Wchnschr., 1940, 06, 878. 2 (a) Bcngtson, I. A, Topping, S. II., and Hcnderson, R. G.,
