Growth of Rickettsia typhi in irradiated L cells enhanced by lysosomal stabilization.

The growth of some obligate intracellular parasites is contingent upon avoidance of lysosomal activation during growth in their host cells. This is accomplished by the various parasites by different mechanisms and with different degrees of efficiency. The possibility was tested that the lysosomal stabilizer cortisone acetate might protect and thus enhance the growth of Rickettsia typhi in mouse L cells irradiated 6 days earlier. Beginning 2 days before infection of the L cells with a multiplicity of 10 rickettsiae, 20 microgram of cortisone per ml was added in medium 199 containing 5% fetal calf serum. This concentration of cortisone was below the cytotoxic level, as determined by viability staining, but was sufficient to significantly alter the ratios of cellular and released acid phosphatase and beta-glucuronidase in uninfected and infected cells, as shown by spectrophotometric analysis. Rickettsial replication, measured by hemolytic activity at 96 h and confirmed by microscopic observations at earlier stages of infection, was increased by the cortisone. Cortisone concentrations of 10 or 40 microgram/ml were less effective, and cortisone was ineffective when the rickettsial multiplicity per L cell was 2 or lower. These results indicate that amounts of cortisone that increase lysosomal stabilization in L cells favor rickettsial multiplication when the multiplicity of infection is relatively high.

Vole agent identified as a strain of the trench fever rickettsia, Rochalimaea quintana.

The vole agent described by Baker in 1946 was studied as an example of a bacterium that has been mistakenly regarded a rickettsia. Unlike rickettsiae, the vole agent killed chicken embryos with great irregularity, multipled primarily at the surface of avian or mammalian cells and not intracellularly, produced colonies rather than plaques on chicken embryo monolayers under agar, and developed small colonies after 4 to 7 days of cultivation on blood plates. It was most conveniently cultivated on monolayers of irradiated L cells and was purified by minor modifications of the Renografin gradient procedure used for rickettsiae. It actively catabolized glutamate, glutamine, succinate, and pyruvate, but not glucose or glucose-6-phosphate. Enzymatic activities of cell extracts were consistent with above findings. The base ratio (molar percent guanine plus cytosine) of its deoxyribonucleic acid was shown to be 39, which was identical to the base ratio of the deoxyribonucleic acid of Rochalimaea quintana tested simultaneously. Serological studies indicated no cross-reactivity with Rickettsia tsutsugamushi, but strong cross-reaction with R. quintana was observed when a hyperimmune rabbit serum and a convalescent human serum were tested. We conclude that the vole agent is a strain of the trench fever rickettsia, R. quintana.

Improved chicken embryo cell culture plaque assay for scrub typhus rickettsiae.

The plaque technique for three strains of Rickettsia tsutsugamushi in chicken embryo cell cultures was greatly improved by modifying the trypsinizing procedure and employing homologous chicken serum in the overlay medium.

Biological properties of Rickettsia prowazekii strains isolated from flying squirrels.

Four strains of Rickettsia prowazekii, isolated from flying squirrels (Glaucomys volans volans) from Florida and Virginia, were compared with other strains of the typhus biotype, two previously established strains each of R. prowazekii and R. typhi and one strain of R. canada, for similarities in a number of unrelated phenotypic characteristics. R. akari served as a spotted fever biotype control. All strains produced small plaques on chicken embryo cell monolayers that were clearly recognized only after 10 days of incubation at 32 degrees C. All strains were highly susceptible to erythromycin. The Renografin density gradient centrifugation procedure of separating rickettsiae from the infected yolk sacs of surviving chicken embryos was equally satisfactory in all cases and resulted in moderate to large yields of purified rickettsiae. There was relatively small variation in specific hemolytic activity or specific CO(2) formation from glutamate. None of the strains catabolized glucose. There was some strain variation in virulence for the chicken embryo, but none of the above tests separated the three species of the typhus biotype. On the other hand, R. akari was clearly distinguished by its more rapid plaque formation and by higher resistance to erythromycin. It is concluded that by the tests conducted thus far, the biological properties of the flying squirrel strains do not differ substantially from those of other strains of the typhus biotype.

Effects of 2-deoxy-D-glucose and 3 deazauridine individually and in combination on the replication of Japanese B encephalitis virus.

We have tested the potencies of the competitors of glucose, 2-deoxy-d-glucose, and of uridine, 3-deazauridine, on the inhibition of Japanese B encephalitis virus multiplication in BHK-21 cell cultures. The relative effectiveness of the viral inhibitors were evaluated individually and in combination in relation to cytotoxicity as a measure of the selectivity of inhibition. When the drugs were administered individually, the antiviral activity was masked by the cytotoxic effect on the host. By combining the two drugs, it was possible to inhibit Japanese encephalitis virus production at noncytotoxic concentrations. The effects of 2-deoxy-d-glucose and 3-deazauridine on the growth inhibition of BHK-21 cells in cultures were only additive, while they were clearly synergistic on the inhibition of Japanese encephalitis virus production. Thus, it was possible to achieve an increased antiviral effect without a significant increase in cytotoxicity. Although the precise biochemical mechanism of the antiviral activity of these antimetabolites in combination is not known, our results indicate the potential value of this approach in viral chemotherapy.

Extension of the mean time to death of mice with a lethal infection of Venezuelan equine encephalomyelitis virus by antithymocyte serum treatment.

The mean time to death of mice infected with Venezuelan equine encephalomyelitis (VEE) virus was increased 2 days by antithymocyte serum (ATS) treatment given 1 day before and 1 day after virus inoculation. Virus assays of blood, brain, and spleen indicated that VEE virus replication was delayed by ATS. Additionally, mice treated with ATS exhibited neurological signs later than untreated mice. During the infection, the percentage of splenic B lymphocytes as determined by surface immunoglobulin staining increased. ATS treatment caused a further elevation of the percentage of splenic B lymphocytes. These results show a selective depletion of the non-immunoglobulin-bearing lymphocyte population during VEE virus infection and support the hypothesis that ATS destroys or alters an important population of cells associated with the normal course of pathogenesis and the replication of VEE virus to high titers in the mouse.