Limits of rickettsial infectivity.

Seven species of pathogenic rickettsiae were compared in five assay systems for group, species, strain, and phase differences in infectivity. The species examined include Rickettsia prowazekii (Breinl and Cairo 3 strains), R. typhi, R. canada, R. rickettsii (Sheila Smith and R strains), R. conorii, R. sibirica, and Coxiella burnetii in phases I and II. Pathogenicity was charcterized in terms of fever in guinea pigs. All comparisons of infectivity and pathogenicity were described in terms of numbers of rickettsiae in the inocula, as determined by direct rickettsial count. The data characterize the various species and strains of rickettsiae in quantitative terms, which are also estimates of the sensitivity of the assay systems used. Phase I C. burnetii was found to be the most, and R. canada the least, infective of the species examined. In general the primary chicken embryo cell culture system proved to be the most, and that of the mouse the least, sensitive assay system.

Plaque formation in tissue cultures by Rickettsia rickettsi isolated directly from whole blood and tick hemolymph.

A simple technique is described for isolation of Rickettsia rickettsi directly from tick hemolymph and whole blood of rickettsemic guinea pigs by means of the plaque assay technique in primary chicken embryo tissue cultures. Plaque-forming units per drop of hemolymph were almost 100-fold greater for partially engorged ticks than for unengorged ticks. Rickettsemia in guinea pigs fed upon by infected ticks was detected as early as 24 hr before fever. No morphological differences were noted between plaques formed by rickettsiae isolated from tick hemolymph or from whole guinea pig blood.

Effects of various suspending media on plaque formation by rickettsiae in tissue culture.

Effects of some media used for suspending rickettsiae during purification, for metabolic studies, and in titrations of infectious rickettsiae were examined with respect to the plaque-forming ability of Rickettsia rickettsi and R. typhi in primary chicken embryo tissue cultures and the infectivity of R. typhi in mice. Brain heart infusion broth (BHI) was found superior to all other media tested in preventing both a significant decrease in plaque-forming units (PFU) and a delay in plaque formation. Skim milk, egg yolk, and some metabolic media were effective in maintaining PFU at 0 C, but did not prevent a significant delay in plaque formation. However, infectivity of R. typhi for tissue culture and mice was markedly decreased when suspended in metabolic media at 26 C. Addition of BHI to the routine tissue culture overlay reversed the deleterious effects of sucrose-phosphate solutions. The effects of Mg(2+), Mn(2+), K(+), Na(+), sucrose, and glutamate were also examined. No significant differences were observed between R. rickettsi and R. typhi in their responses to different media. The results of this study suggest the necessity for a reappraisal of previous studies of metabolism and infectivity of rickettsiae in these media.

Studies of the rickettsial plaque assay technique.

A plaque assay system for pathogenic rickettsiae, which utilizes primary chick embryo tissue cultures, is described. It proved to be a highly reproducible measure of infectiousness for Rickettsia rickettsi and R. typhi, which were employed in most studies; as well as for R. canada, R. prowazeki, R. sibirica, R. akari, R. conori, and Coxiella burneti. Plaque-forming units (PFU) were compared to direct rickettsial counts and to 50% infectious dose (ID(50)) values for embryonated eggs, mice, and guinea pigs. Plaque size, appearance, and number were influenced by diluent, incubation temperature after nutrient overlay, centrifugation of inoculated tissue cultures, and number of host cells planted initially in each flask. The most critical factors in plaque formation were diluent used in making rickettsial suspensions and incubation temperature (32 C) after nutrient overlay. Brain Heart Infusion was the only diluent capable of preventing significant delay in plaque formation and decreases in PFU and mouse ID(50). Plaque formation was unaffected by genetic background of host cells, volume of inoculum, temperature and length of incubation period before nutrient overlay, and rapid freezing and thawing of rickettsial seed. Centrifugation of inoculated cultures at 600 x g resulted in 100% irreversible absorption of rickettsiae to host cells within 5 min, whereas without centrifugation at least 4 hr was required to achieve the same effect.