Upon entry into the cytoplasm of irradiated chicken embryo cells in slide chamber cultures infected over a 2-h period, yolk sac-grown virulent (Breinl strain) and attenuated (E strain) Rickettsia prowazeki underwent indistinguishable reproducible intracellular growth cycles. They promptly entered an exponential growth phase, without detectable lag and without microscopic evidence for any unusual early replicative phase. The generation time for both strains was 8.8 to 8.9 h at 34 C. During most of this period, the state of the organisms and growth were very similar from one cell to another. The exponential-growth phase continued for at least 36 to 48 h, when the rickettsiae became too numerous to count by microscopic examination. Between about 36 and 48 h, cells packed with rickettsiae began irregularly to break down and release organisms. These began to initiate new infection cycles in previously uninfected cells over many hours, as demonstrated by the rise in percentage of cells infected, yielding a highly disordered infected culture with different cells containing rickettsiae in diverse stages of growth. The organisms underwent regular minor changes in morphology, similar to those seen in bacterial cultures, in the first infection cycle. As the cells became packed with rickettsiae, the microorganisms regularly diminished in size to become minute coccobacillary to coccoid forms. However, the rickettsiae in the second and subsequent infection cycles in aging cultures often assumed filamentous or swollen bizarre forms. Only the first infection cycle conformed closely to the concept of a one-step growth cycle. A set of terms is proposed and defined for the infection cycle.

Unique features of the primary site of rickettsial replication in typhus fevers, i.e., within the endothelial cells of small blood vessels in tissues, suggest that effector mechanisms, other than those dependent on phagocytosis by activated macrophages with enhanced microbicidal properties, most likely are necessary to explain the cell-mediated immune control of intracellular rickettsial replication in these sites. Theoretically, such mechanisms might involve contact between infected endothelial cells and activated T lymphocyte subpopulations or macrophages or immunologically induced soluble factors or lymphokines. Support for the existence of at least one of these alternative effector mechanisms is presented here for Rickettsia prowazekii. Cultures of human blood leukocytes, upon immunologically specific stimulation with R. prowazekii antigen or nonspecific stimulation with the mitogen phytohemagglutinin, produce soluble factor(s) in the supernatant fluid which, in culture, have (a) an intracellular antirickettsial action on R. prowazekii-infected human endothelial cells, fibroblasts, and macrophages, and (b) a specific cytolytic action on R. prowazekii-infected, but not uninfected bystander, human fibroblasts. Neither action is demonstrable in R. prowazekii-infected chicken embryo fibroblasts. The factor(s) has no direct antimicrobial action on extracellular rickettsiae and is inactivated by heating at 56 degree C for 1 h or by acid treatment at pH 2. Expression of the antirickettsial action requires new host cell messenger transcription and protein synthesis, whereas the cytolytic action does not. The circumstances of production and action and the properties of the factor(s) responsible for the intracellular antirickettsial, and perhaps also the cytolytic action are consistent with those of immune interferon (IFN-gamma).

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The infection cycle of Rickettsia rickettsii, studied in slide chamber cultures of chicken embryo and L-929 cells, was found to be complex and did not conform to a one-step growth cycle. Initial uptake kinetics resembled those established for Rickettsia prowazekii, but subsequent events showed very marked differences. Intracytoplasmic growth commenced exponentially without measurable lag. However, very soon after infection, intracytoplasmic rickettsiae began to escape from the host cell into the medium in large numbers, resulting in (i) failure of large numbers of rickettsiae to accumulate in the cytoplasm, (ii) sustained rapid division of the organisms in the cytoplasm, (iii) substantial accumulation of extracellular rickettsiae, and (iv) rapidly spreading infection in the culture, with most cells infected in 48 to 72 h. In the occasional cell, rickettsiae were found in the nucleus, where they multiplied to form compact masses. Thus, analysis of the growth characteristics of R. rickettsii must consider the entire culture as a unit in which the rickettsiae are distributed among three compartments in which they behave in different ways: (i) intranuclear, (ii) intracytoplasmic, and (iii) extracellular. The rickettsial traffic is bidirectional across the host cell plasma membrane and dominantly monodirectional across the nuclear membranes. The implications of this behavior with respect to location and range of receptors and substrates involved in membrane penetration are discussed. In older cultures, unique intracytoplasmic ring or doughnut colonies were common, indicating a change in the intracytoplasmic environment. The possible significance of the growth characteristics in cell culture to the characteristics of infection in humans and animals is discussed.

Two Rickettsia prowazeki seeds, an "early" seed in the logarithmic or exponential growth phase and a "late" seed in the stationary or possibly early decline phase, were prepared in chicken embryo (CE) cell cultures and compared with respect to morphology and infection cycle in CE cells in culture. Differences in size and ultrastructure of the organisms in the two seeds were similar to those seen in other gram-negative bacteria at comparable stages of growth. Vacuolar structures, rare in log-phase organisms, were common in stationary-phase organisms. Minute spherical forms reminiscent of minicells were seen in the stationary-phase preparations. In quantitative uptake experiments, organisms, typical in size and morphology of each preparation, had comparable capacity per plaque-forming unit to penetrate into CE cells in suspension when the seeds had been depleted of host cell membrane fragments and other debris. This suggests that host cell fragments, presumably of membrane origin, competitively inhibit rickettsial uptake by intact CE cells. Organisms of the log-phase seed, upon entry into a host cell, entered the logarithmic or exponential phase of intracellular growth without a measurable lag phase, whereas stationary-phase organisms displayed a lag phase of about 7.5 h, during which they enlarged and increased in intensity of staining, before entering the log phase of growth. R This equation is in fact a modification of the one given by Lodge and Hinshelwood (9), simply making use of the information contained in the equation for INFECT. IMMUN. on July 15, 2020 by guest http://iai.asm.org/ Downloaded from