Members of the spotted fever group (SFG) of rickettsiae spread rapidly from cell to cell by an unknown mechanism(s). Staining of Rickeffsia rickettsii-infected Vero cells with rhodamine phalloidin demonstrated unique actin filaments associated with one pole of intracellular rickettsiae. F-actin tails greater than 70 ,m in length were seen extending from rickettsiae. Treatment of infected cells with chloramphenicol eliminated rickettsia-associated F-actin tails, suggesting that de novo protein synthesis of one or more rickettsial proteins is required for tail formation. Rickettsiae were coated with F-actin as early as 15 min postinfection, and tail formation was detected by 30 min. A survey of virulent and avirulent species within the SFG rickettsiae demonstrated that all formed actin tails. Typhus group rickettsiae, which do not spread directly from cell to cell, lacked F-actin tails entirely or exhibited only very short tails. Transmission electron microscopy demonstrated fibrillar material in close association with R. rickeftsii but not Rickeffsia prowazekii. Biochemical evidence that actin polymerization plays a role in movement was provided by showing that transit ofR. rickettsii from infected cells into the cell culture medium was inhibited by treatment of host cells with cytochalasin D. These data suggest that the cell-to-cell transmission of SFG rickettsiae may be aided by induction of actin polymerization in a fashion similar to that described for Shigella flexneri and Listeria monocytogenes.

Serological parameters were compared in 15 cases of Coxiella burnetii infection comprising 5 cases each of primary Q fever, chronic granulomatous hepatitis, and endocarditis. The diagnosis was made on the basis of clinical history and serology and on the isolation of C. burnetii phase I from biopsy specimens of liver and bone marrow from two patients with granulomatous hepatitis and from the aortic valve vegetations of five patients with endocarditis. The temporal sequences of immunoglobulin levels, rheumatoid factor, and specific antibody responses to phase II and phase I antigens of C. burnetii were evaluated as predictive correlates of the three Q fever entities. Serum levels of immunoglobulin classes G, M, and A were variable in all the entities of Q fever. Increased mean levels (in milligrams per deciliter) of immunoglobulin G (IgG) and IgA were noted with chronic disease in the sera of some patients, whereas IgM levels were not significantly different from normal values. Rheumatoid factor was significantly elevated in chronic disease but not in primary Q fever. The temporal sequence of C. burnetii phase II and phase I antibodies were compared by microagglutination, complement fixation, and indirect microimmunofluorescence tests. All of these serological tests were useful in distinguishing primary from chronic disease. Thus, the ratio of anti-phase II to anti-phase I antibodies was greater than 1, greater than or equal to 1, and less than or equal to 1 for primary Q fever, granulomatous hepatitis, and Q fever endocarditis, respectively. Moreover, the high phase-specific IgA antibody titers in the indirect microimmunofluorescence test were diagnostic for endocarditis.

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We isolated lipopolysaccharides (LPSs) from phase variants of Coxiella burnetii Nine Mile and compared the isolated LPS and C. burnetii cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The LPSs were found to be the predominant component which varied structurally and antigenically between virulent phase I and avirulent phase II. A comparison of techniques historically used to extract the phase I antigenic component revealed that the aqueous phase of phenol-water, trichloroacetic acid, and dimethyl sulfoxide extractions of phase I C. burnetii cells all contained phase I LPS, although the efficiency and specificity of extraction varied. Our studies provide additional evidence that phase variation in C. burnetii is analogous to the smooth-to-rough LPS variation of gram-negative enteric bacteria, with phase I LPS being equivalent to smooth LPS and phase II being equivalent to rough LPS. In addition, we identified a variant with a third LPS chemotype with appears to have a structural complexity intermediate to phase I and II LPSs. All three C. burnetii LPSs contain a 2-keto-3-deoxyoctulosonic acid-like substance, heptose, and gel Limulus amoebocyte lysates in subnanogram amounts. The C. burnetii LPSs were nontoxic to chicken embryos at doses of over 80 ,ug per embryo, in contrast to Salmonella typhimurium smooth-and rough-type LPSs, which were toxic in nanogram amounts. Coxiella burnetii, the etiologic agent of Q fever, is an obligately intracellular bacterium that multiplies within the

Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, was lethal for the majority of experimentally and transovarially infected Rocky Mountain wood ticks (Dermacentor andersoni). Overall, 94.1% of nymphs infected as larvae by feeding on rickettsemic guinea pigs died during the molt into adults and 88.3% of adult female ticks infected as nymphs died prior to feeding. In contrast, only 2.8% of uninfected larvae failed to develop into adults over two generations. Infected female ticks incubated at 4°C had a lower mortality (80.9%) than did those held at 21°C (96.8%). Rickettsiae were vertically transmitted to 39.0% of offspring, and significantly fewer larvae developed from infected ticks. The lethal effect of R. rickettsii may explain the low prevalence of infected ticks in nature and affect its enzootic maintenance.

Coxiella burnetii, phase I and II, cells cultivated in the yolk sac of chicken embryos were separated from host cell components by two cycles of isopycnic Renografin gradient centrifugation. Initial steps in the purification of viable C. burnetii involved differential centrifugation and sedimentation through an aqueous solution of 30% sucrose and 7.6% Renografin. After the first, but not the second, cycle of Renografin gradient centrifugation, the cells were passed through microfilter glass filters which facilitated the removal of host components. The integrity of morphologically different cell variants was maintained during purification procedures by suspending highly purified C. burnetii in phosphate-buffered saline-sucrose solutions. C. burnetii, phases I and II, obtained by these methods appeared to be free from host cell components by serological methods while retaining morphological integrity and infectivity for yolk sacs and experimental animals. Average yields of C. burnetii were 2.83, 1.5, and 0.84 mg (dry weight) per yolk sac of the Ohio strain (phase I), 9 Mile strain (phase I), and 9 Mile strain (phase II), respectively. Recovery of phase I cells averaged about 70%, whereas the recovery of phage II cells was approximately 40%. The temporal sequence of phase I and II antibody response was demonstrated in infected and vaccinated animals. Also, no antibody response in mice and guinea pigs to yolk sac antigens was detectable after two injections of vaccine or viable cells. Importantly, this is the first report of the separation of viable phase II cells of C. burnetii free of host components.

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 (ID5o) values for embryonated eggs, mice, and

A comparison was made of the performance of the commonly used complement fixation test and the more recently developed indirect immunofluorescence test in the early diagnosis of Q fever. The 303 sera tested were from 181 patients who contracted Q fever during an outbreak in Switzerland in 1983. Specific IgM antibodies were detected by the immunofluorescence test in 53% and 89% of sera obtained during the first and second week respectively after onset of illness. With the complement fixation test, the diagnosis could not be made until the second week of illness. The immunofluorescence test proved to be superior to the complement fixation test in the early detection of Q fever. Not only was it more specific but also faster and simpler to perform, permitting an earlier diagnosis on the basis of results obtained with a single serum specimen.