Internal structures of rickettsiae seen with phase contrast microscopy and in the electron microscope contain desoxyribonucleic acid and are therefore nuclear structures similar to those found in bacteria. They are minute spherical bodies, either single as in spherical rickettsiae or varying in number from 2 to 4 in rod-shaped forms. Occasional dumbbell-shaped chromatinic bodies are thought to represent these structures in the process of division. The presence of ribonucleic acid in the cytoplasm of rickettsiae was demonstrated with the use of ribonuclease and basic dyes. Rickettsiae therefore have a cellular organization similar to that of certain bacteria, with a clear differentiation into nuclear structure and cytoplasm.

Suspensions of yolk sac infected with rickettsiae of murine or of epidemic typhus, and indeed suspensions of liver or peritoneal washings from cotton rats infected with these organisms, when the suspensions contain a sufficient concentration of living rickettsiae, possess the capacity to hemolyze, in vitro , red cells of rabbit or sheep origin. Under the same conditions such material did not cause the hemolysis of cells from mice, cotton rats, or guinea pigs. Yolk sac and tissue suspensions of R. orientalis (Karp, Raub, and Gilliam strains) failed to hemolyze rabbit cells. The hemolytic principle was destroyed by formol (0.5 per cent) and by heating to 56°C. for 1 hour. The close association of the "hemolysin" with the rickettsiae was indicated by their parallel movements on centrifugation and by the close parallel demonstrated between the effects produced on hemolytic activity and on such manifestations of the presence of living rickettsiae as toxicity for mice and lethality or total infectivity for cotton rats when infected yolk sac suspensions were exposed to elevated temperature or were concentrated by high-speed centrifugation. The mechanism of hemolysis remains ill defined. It progresses slowly, but never to completion ( i.e . hemolysis of all the cells in the substrate) even when the number of such cells is small. This may be because hemolysis progresses best at temperatures (±35°C.) which are unfavorable to the hemolytic principle and because erythrocytes vary in their susceptibility to the injurious agent. The concentration of red cells also may be a determining factor, either by affecting the rate at which the reaction proceeds or by aiding in the preservation of hemolytic activity. Although it was impossible to separate the "hemolysin" from the rickettsiae, examination of smear preparations of hemolyzing mixtures showed no consistent contact relation between cells and rickettsiae. Adsorption experiments revealed that, while removal of the cells from the hemolytic system did not reduce the hemolyzing capacity of the system, the cells carried away a significant tendency to hemolyze. It is suggested that the rate at which injury is inflicted on the red cells is dependent on the concentration of "hemolysin" present and that the process may proceed in the relative absence of rickettsiae once sufficient damage has been inflicted. The toxicity of richly infected yolk sac suspensions for intravenously inoculated mice was found to be related, not to in vivo hemolysis, but apparently to a serious alteration in vascular permeability which caused a marked reduction in blood volume. This was evidenced by an increased concentration of red cells but not of plasma proteins. It is possible, but has not been demonstrated, that the factor responsible for altering vascular permeability of mice may be identical with that which causes lysis of rabbit and sheep cells. Two applications of the phenomenon of in vitro hemolysis are indicated. Following the technique described in the text for the optimum demonstration of hemolysis, the determination of the degree of hemolytic activity may serve as a rapid, roughly quantitative in vitro method for assessing the infectivity of a newly made rickettsial preparation. The phenomenon also is the basis for an additional, and potentially useful in vitro serologic technique which differs from other techniques in that it depends on a property of living rickettsiae. It has been shown that homologous antisera are capable of inhibiting hemolysis, and optimum conditions for demonstrating this inhibitory effect are described.

Methylene blue (MB) and toluidine blue (TB) when administered in maximum tolerated oral doses to mice and to cotton rats are highly effective in preventing the usual lethal outcome of intraperitoneally induced infections with R. orientalis . This activity is manifest even when dye administration is delayed until a systemic infection has been well established. Methylene blue is also effective in cerebral infections in mice. The toxicity of MB, however, limits parenteral (subcutaneous) administration of the dye to dosage levels which are much less effective than the maximum tolerated oral levels. The inability of mice to tolerate an adequately effective parenteral dose of MB suggests that the properties of the dye responsible for its toxicity may be separated from those upon which its antirickettsial effect depends. The relationship between the response of mice to oral treatment with MB and such factors as the size of the infecting dose and the times of initiation and of withdrawal of treatment may be summarized as follows: 1. With a constant infecting dose, the time of initiation of treatment largely determines the degree of therapeutic effect. 2. The interval after infection beyond which further treatment does not increase the survival rate depends not upon the previous duration of treatment but upon the size of the infecting dose. Paradoxically, treatment can be discontinued sooner after a massive infecting dose than after a smaller one.

Monkeys and mice surviving cerebral infection with yellow fever virus of relatively avirulent strains have been found to resist maximal intracerebral doses of yellow fever virus of a highly neurotropic strain. Such animals, however, do not resist more than very small doses of intracerebrally inoculated virus of Eastern equine encephalomyelitis. Animals immunized by extraneural routes, on the other hand, are not uniformly resistant to neural infection with neurotropic yellow fever virus. Monkeys which have undergone systemic infection with virus of the avirulent 17D strain or of several jungle strains resist only small intracerebral doses of neurotropic virus; while mice, even when possessed of very high serum-antibody levels as the result of intraperitoneal hyperimmunization, manifest only an irregular resistance to intracerebral challenge inocula. The difference in the resistance of neurally and extraneurally immunized animals is not related to similar differences in the levels of protective antibody in the sera. Indeed, the average of the serum-antibody titers of the hyperimmune mice is several times that of the intracerebral immunes. A possibly significant relation does exist, however, between the resistance of mice to neural infection and the content of protective antibody in the brain. The protective activity of suspensions of brains from mice surviving cerebral infection was found to be several times that of brain suspensions from the hyperimmunized animals. It is concluded that the superior resistance to neural infection of animals whose immunity results from a previous non-fatal infection of the nervous system is effected by a specific local mechanism which is based at least in part upon an increased concentration of antibody in the cerebral tissue.

Observations have been reported which indicate that mice inoculated intracerebrally with active yellow fever virus may develop an infection which is not only non-fatal but may also be completely inapparent. The most extensive observations were made on mice which showed signs of infection but were still alive 22 days after inoculation with virus of one or another of several 17D substrains. In such cases, the infection usually progressed no further and partial or complete recovery often ensued. Agents other than yellow fever virus were excluded as a significant cause of such nonfatal infections by the failure of repeated attempts to isolate other infective agents, by the demonstration of antibodies against yellow fever virus in the sera of the mice, and by the demonstration of a high degree of resistance on the part of such surviving mice to reinoculation with large doses of neurotropic yellow fever virus. Completely inapparent infections with 17D virus were also shown to occur. Studies of apparently normal survivors of 17D virus titrations revealed a small but significant number of animals resistant to intracerebral challenge with neurotropic yellow fever virus. Further, pooled sera from such mice were shown to contain specific protective antibodies. The occurrence of non-fatal infections with 17D virus was found related to virus dose and substrain. Small doses of virus provoked a significantly higher proportion of non-fatal infections than large doses; while different 17D substrains, tested over equivalent ranges of virus dose, varied greatly with respect to the proportion of infections which did not terminate with death. In the case of two substrains (17DD low and 17D 3 ), non-fatal infections (as demonstrated by resistance to intracerebral challenge with neurotropic virus) were sufficiently frequent to cause an increase, when included in the computation of the infective titers, of 25 per cent above the figures based on deaths alone. The demonstration of non-fatal infections, thus, may be important to the accuracy of quantitative determinations of infectivity. Limited observations with virus of the French neurotropic and the pantropic Asibi strains revealed that non-fatal infections do occur, but only rarely. Somewhat more extensive observations with unmodified virus of strains isolated from Brazilian cases of jungle yellow fever, in contrast, revealed an occurrence of non-fatal infections much greater than that observed with the most productive 17D substrains. With these jungle strains, the demonstration of non-fatal infections proved indispensable to any measure of the level of infectivity of virus preparations. The demonstration of the proportional occurrence in mice of non-fatal infections with yellow fever virus provides an additional means by which different virus strains and substrains may be characterized.

The present study comprises observations on the course and outcome of experimentally produced Type I pneumococcus lobar pneumonia in dogs. It was found that within certain limits of dosage the outcome of the disease was closely related to the amount of culture and the volume of the starch inoculum employed. The much higher mortality rate resulting from simply increasing the volume of starch in which the pneumococci were suspended, appeared to be due chiefly to the greater area of lung tissue initially involved. Certain striking relationships were observed to exist between the leucocytic response, extent of pulmonary involvement, bacteremia and outcome. Marked changes in the number of white blood cells at end of the first 24 hours of the disease provided more valuable prognostic information than either the blood culture or the size of the lesion. Dogs showing high white counts at this stage seldom succumbed, while absence of leucocytosis and leucopenia were associated with a mortality which increased with the diminishing numbers of circulating white blood cells. Over 90 per cent of the animals with counts of less than 2,000 cells per c. mm. died. The extent of the pulmonary lesion was likewise observed to bear a direct relationship to death rate. With the lesion confined to a single lobe the mortality was only 1 per cent. Spread to other lobes was accompanied by a sharply rising death rate until it reached 98 per cent in dogs with three-fourths the lung field involved. Increasing occurrence of bacteremia paralleled both the decreasing white counts and the extending pulmonary lesions and subsequent to the 24 hour stage of the disease the presence or absence of pneumococci in the blood appears to be of more significance, in relation to outcome, than either of these two other factors. Even in the presence of leucopenia and an extensive lesion the animal might survive provided the blood remained sterile.