A Toxic Substance Associated with the Gilliam Strain of R. orientalis

Abstract

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.,

Scrub Typhus: The Geographic Distribution of Phenotypic and Genotypic Variants ofOrientia tsutsugamushi

“…Strain variation has also been demonstrated using a toxin neutralization assay [79,116,117]. With egg yolk sac viability as an end point, the strain-specific toxic effect of O. tsutsugamushi can be neutralized by preinoculation incubation with dilutions of homologous sera but less so with heterologous antisera.…”

Antigenic Analysis by Direct Immunofluorescence of 114 Isolates of Rickettsia Tsutsugamushi Recovered From Febrile Patients in Rural Malaysia

“…Antigenic heterogeneity among strains of Rickettsia tsutsugamushi has been documented by the use of various techniques: (1) complement fixation (Bengtson, 1945(Bengtson, , 1946Shishido, 1964;Shirai and Wisseman , 1975); (2) cross-neutralization (Bell, Bennett and Whitman, 1946;Gauld , 1947, 1949;Fox, 1949;Miesse, Diercks and Danauskus, 1950;Plotnikova and Tarasevich , 1967); (3) toxin neutralization (Smadel et al, 1946); (4) cross-vaccination (Rights , Smadel and Jackson, 1948); and (5) immunofluorescence (Iida , Kawashima and Kawamura, 1965;Shirai and Wisseman, 1975).…”

Studies on Scrub Typhus (Tsutsugamushi Disease)

“…Blood from a mouse of the sixth passage was injected into embryonated eggs (6) and yolk sac material from the 6th to 24th subcultures of this line was employed in the current work. The wild rat 235 strain (also referred to as wild rat 2 (14)), was obtained during April, 1944, by Kohls and his coworkers (7) from the pooled brains of four wild rats (Rattu, s cw~olor brown/) which were trapped in the Dobadura area of New Guinea. A pool of liver and spleen tissue from mice of the fourth passage served as challenge material throughout the present experiments.…”

“…T h e mite 21 strain (also referred to as host 21) (13,14) was isolated in mice during Novem-her, 1943, by Blake and his associates from a pool of mites (T. fletcher/) which were obtained from a bandicoot caught in the Dobadura area, New Guinea. Blood from a mouse of the sixth passage was injected into embryonated eggs (6) and yolk sac material from the 6th to 24th subcultures of this line was employed in the current work.…”