Factors Affecting the Growth of Rickettsias of the Spotted Fever Group in Fertile Hens' Eggs

The age of embryo, incubation temperature, period of incubation after death of embryo, dose of rickettsias, and diluent used for preparing the inoculum affected growth of Rickettsia rickettsii and R. conorii in fertile hens' eggs. Growth in 4 or 5-day-old embryonated eggs was more luxuriant than that in 6 or 7-day-old embryos, and growth of rickettsias at 32.5 C and 34.5 C was superior to that at 36.5 C. Yolk sacs regularly contained toxin if the infecting dose was large enough to cause death by 104 hours after inoculation. Eggs inoculated with rickettsias suspended in 0.1 M phosphate-buffered saline, pH 7.6, contained higher levels of toxin and antigen than did those in which modified sucrose-phosphate-glutamate medium was used. When eggs were incubated after death of the embryo, levels of toxin and antigen in yolk sacs increased until a peak was reached. After this the level of toxin decreased at a rate dependent upon temperature. Therefore, in order to obtain maximum growth of spotted fever rickettsias, 4.5 day-old embryonated eggs should be inoculated with sufficient rickettsias suspended in 0.1 M phosphate-buffered saline, pH 7.6, to kill most of the embryos within 104 hours. Inoculated eggs should be incubated at 33.5 C and candled at 12-hour intervals. Those dead at each candling should be incubated for another 48 hours before yolk sacs are harvested.