What's new in Rocky Mountain spotted fever?

Rocky Mountain spotted fever (RMSF) remains an important illness despite an effective therapy because it is difficult to diagnose and is capable of producing a fatal outcome. The pathogenesis of RMSF remains, in large part, an enigma. However, recent research has helped shed light on this mystery. Importantly, the diagnosis of RMSF must be considered in all febrile patients who have known or possible exposure to ticks, especially if they live in or have traveled to endemic regions during warmer months. Decisions about giving empiric therapy to such patients are difficult and require skill and careful judgement.

Changes in the adherens junctions of human endothelial cells infected with spotted fever group rickettsiae.

Rickettsiae of the spotted fever group are obligately intracellular bacteria that primarily infect the vascular endothelium, invade adjacent cells propelled by actin polymerization, and cause severe systemic diseases. Endothelial dysfunction and vascular leakage develop as a consequence; this effect is the pathophysiological mechanism that explains most clinical manifestations. Here we report that rickettsial infection of cultured primary human endothelial cells is associated with the formation of gaps in the interendothelial adherens junctions, occurring late during the course of in vitro infections but not early, even when rickettsial loads are significant.

Ultrastructural study of the response of cells infected in vitro with causative agent of spotted fever group rickettsiosis in Japan.

The response of host cells L929 infected with causative agent of spotted fever group (SFG) rickettsiosis in Japan, the Katayama strain, was studied by electron microscopy. The rickettsiae penetrated the cytoplasm and multiplied here and after prolonged incubation progressed into the dilated cisternae of rough endoplasmic reticulum (rER), the perinuclear space, the deep invaginated nuclear membrane, and then the nucleoplasm of the host cells. The intranuclear rickettsiae showed different states: one type was enclosed by the double membrane of the host cell and the other type was free in the nucleoplasm. In addition to these double membrane-bound and membrane-free intranuclear rickettsiae, various membrane structures, including rER-like structures, were also found in the nucleus. The cells infected with the rickettsiae underwent distinctive morphological alterations which occurred mainly within intracellular membranes of the host cells. These findings indicate the possibility that the intracellular membranes are characteristic cytopathological sites in rickettsia-host cell interaction, and that these alterations may be related to a possible route of rickettsial penetration into the nucleus: passage through vesicles formed from invaginations in the nuclear membrane.

Rickettsia rickettsii growth and temperature-inducible protein expression in embryonic tick cell lines.

Rickettsia rickettsii has limited adverse effects on its arthropod vector, but causes severe disease in man. To model differences in host-parasite interaction, R. rickettsii growth and protein expression were examined at temperatures reflective of host environment in the tick cell lines DALBE3 and IDE2, the human endothelial cell line ECV304, and the African green monkey kidney cell line Vero76. At low multiplicities of infection, rickettsial titres increased 10(2)-10(3)-fold in all cell lines after incubation for 3 days at 34 degrees C. At higher multiplicities and with extended incubation, R. rickettsii showed enhanced survival in tick versus mammalian cells. No difference in rickettsial ultrastructure or protein profiles was detected between different host cell types. Rickettsial proteins of 42, 43, 48, 75 and 100 kDa are induced in tick cells shifted from 28 degrees to 34 degrees C, but not in cells maintained at 28 degrees C. This temperature response may be associated with expression of rickettsial determinants that are pathogenic to mammalian hosts.

Scanning and transmission electron microscopy of Rickettsia rickettsii propagated in cell culture.

Scanning electron microscopy utilizing critical-point drying and transmission electron microscopy employing air-dried agar pseudoreplicas and critical-point dried carbon replicas were used to study the surface of Rickettsia rickettsii propagated in cell culture.

Ultrastructural changes observed in Rickettsia rickettsii incubated at different temperatures in cell-free medium.

When cells of Rickettsia rickettsii were suspended at different temperatures in growth medium free of host cells, ultrastructural changes were observed in some of these organisms. Depending upon the temperature and length of incubation, loosely organized cells developed into compact, intensely stained, rod-shaped organisms. These compact cells closely resembled the morphology of the original culture of Rickettsia used for inoculation. Morphological changes were primarily noted in cells maintained at 21 degrees C. The viability of the cells was also affected by the temperature of incubation.

The rickettsial plaque. Evidence for direct cytopathic effect of Rickettsia rickettsii.

Monolayers of primary chick embryo cells were infected with Rickettsia rickettsii in a plaque assay system with 0.5 per cent agarose overlay. Plaques appeared 5 days after inoculation and were examined on day 6 by supravital staining, immunofluorescence for R. rickettsii, and electron microscopy. In all studies maintenance of the topographic integrity of the monolayer and the plaque allowed assessment of temporospatial relationships of rickettsial infection and cytopathologic changes. Plaques comprised four zones: peripheral, marginal, necrotic, and central. The cells of the peripheral zone were viable, mildly infected, and ultrastructurally normal. Cells of the marginal necrotic, and central zones exhibited a correlation between quantity of rickettsiae and presence of cytopathology. Pathologic changes included sever dilation of rough endoplasmic reticulum and necrosis. Quantitative assessment of the distribution of rickettsiae ultrastructurally showed the overwhelming predominance of cytosol location and suggested the ultrastructural sequence of events for rickettsial release from cells via lysis of cell membrane at the end of filopodia. These studies of the plaque technic provide a predictable, quantitative model for experimental investigations into mechanisms of cell injury by rickettsiae.

Comparative ultrastructural study on the cell envelopes of Rickettsia prowazekii, Rickettsia rickettsii, and Rickettsia tsutsugamushi.

Rickettsia tsutsugamushi differs from other rickettsiae in its cell envelope organization. The differences were made evident through a comparative study of the outer envelope of R. tsutsugamushi, R. prowazekii, and R. rickettsii by electron microscopy.

Human endothelial cell culture plaques induced by Rickettsia rickettsii.

Primary cultures of human umbilical vein endothelial cells were inoculated with plaque-purified Rickettsia rickettsii. After adsorption of rickettsiae, monolayers were overlaid with medium containing 0.5% agarose. Small plaques appeared on day 4 postinoculation, and distinct 1- to 2-mm plaques were observed on day 5. Plaquing efficiency was less than that of primary chicken embryo cells in the same medium. Human endothelial cell monolayers were susceptible to infection by R. rickettsii and underwent necrosis as demonstrated by supravital staining. The topographic association of endothelial cell necrosis and rickettsial infection in the plaque model confirmed the direct cytopathic effect of R. rickettsii on human endothelium. Uninfected cells appeared normal by supravital staining and transmission electron microscopy. This model offers the possibility of investigating rickettsial pathogenesis and mechanisms of enhanced severity of Rocky Mountain spotted fever in specific genetically determined conditions.

External layers of Rickettsia prowazekii and Rickettsia rickettsii: occurrence of a slime layer.

Using a simple specific-antibody stabilization procedure on organisms gently liberated from their host cells, we have demonstrated by electron microscopy that Rickettsia prowazekii and Rickettsia rickettsii possess a coat of variable thickness, external to the outer leaflet of the cell wall and the structure designated by others as a "microcapsule," which corresponds most closely to the slime layer of certain other bacteria. Reactions in the methenamine silver and ruthenium red staining procedures and the failure to be visualized by standard procedures suggest that the slime layer is largely polysaccharide in nature. It is postulated that this slime layer accounts in large part for the large, electron-lucent, halo-like zone which is found by electron microscopy to surround organisms of the typhus and spotted fever groups in the cytoplasm of their host cells, that it may be the locus of some major group-specific antigens, and that it may function as an antiphagocytic mechanism, as an aid for attachment of rickettsiae to potential host cells, or both. Moreover, because the attenuated E strain of R. prowazekii has been shown to possess a substantial slime layer, the basis for attenuation is not likely to be a simple smooth-to-rough variation.

Detection of fibrils associated with Rickettsia rickettsii.

The ultrastructural appearance of the "halozone" formed at the interface between the spotted fever agent Rickettsia rickettsii and the cytoplasm of persistently infected cultured vole cells (Microtus pennsylvanicus) was studied by transmission electron microscopy. In sections of epoxy-embedded specimens stained with uranyl acetate and lead citrate, the halozone appeared clear and devoid of ultrastructural features. However, when unembedded preparations of whole infected cells were examined at 1,000 kV, fine structural features were observed within the halozone. These features, associated with the rickettsial outer membrane, were more clearly detectable when the infected cells were extracted with the detergent Triton X-100 before fixation. Under such conditions, long extensions of the rickettsial outer membrane, microfilament-like structures attached to that membrane, and extensive attachments between adjacent rickettsiae were seen. The fine structural features within the rickettsial halozone were also seen at 75 kV when unembedded sections were prepared from polyethylene glycol-embedded specimens. Thus, epoxy-embedding medium obscures the fine structural features within the halozone surrounding the rickettsiae in infected cells.

Some contributions of electron microscopy to the study of the rickettsiae.

Electron microscopy has provided valuable insights into the study of rickettsiae as intracellular parasites from several important perspectives. This tool has allowed researchers to delineate the fine structural features of these organisms and to show that they truly resemble free-living bacteria. Furthermore, it has been shown that there are subtle, but distinct differences in the outer envelope structure of some members of the genus Rickettsia that may explain reported differences in tinctorial properties and in their sensitivity to certain antibiotics. With Coxiella burnetii, electron microscopy has helped significantly in the characterization of the pleomorphic nature of the organism including formation of terminal bodies that resemble endospores of gram-positive bacteria. Electron microsxopy has also helped to define the relationship of the rickettsiae to their host cells. For example, ultrastructural analysis can reveal whether organisms exist free within the cytoplasm or nucleus (members of the genus Rickettsia), or whether they are bound by a phagosomal or phagolysosomal membrane (Ehrlichia and Coxiella). Finally, although all rickettsiae eventually destroy their host cell, it has been shown through transmission electron microscopy that this destruction might be mediated by different mechanisms that are specific for different rickettsial species.

Origin and structure of the group-specific, complement-fixing antigen of Rickettsia rickettsii.

Rickettsia rickettsii was treated with ether and examined by negative-contrast electron microscopy. Group-specific complement-fixing antigen was seen to be originating from the cell wall. The antigen was composed predominately of round particles 10 to 60 nm in diameter. Intact R. rickettsii and antigen from ether-treated organisms were purified by density gradient centrifugation and analyzed by polyacrylamide gel electrophoresis. The whole rickettsial cell was composed of a minimum of 30 proteins which ranged in molecular weight from about 23,000 to 155,000. The "soluble" antigen contained nine proteins ranging in molecular weight from about 28,000 to 150,000.

Ultrastructure of Rickettsia rickettsii actin tails and localization of cytoskeletal proteins.

Actin-based motility (ABM) is a mechanism for intercellular spread that is utilized by vaccinia virus and the invasive bacteria within the genera Rickettsia, Listeria, and Shigella. Within the Rickettsia, ABM is confined to members of the spotted fever group (SFG), such as Rickettsia rickettsii, the agent of Rocky Mountain spotted fever. Infection by each agent induces the polymerization of host cell actin to form the typical F (filamentous)-actin comet tail. Assembly of the actin tail propels the pathogen through the host cytosol and into cell membrane protrusions that can be engulfed by neighboring cells, initiating a new infectious cycle. Little is known about the structure and morphogenesis of the Rickettsia rickettsii actin tail relative to Shigella and Listeria actin tails. In this study we examined the ultrastructure of the rickettsial actin tail by confocal, scanning electron, and transmission electron microscopy. Confocal microscopy of rhodamine phalloidin-stained infected Vero cells revealed the typhus group rickettsiae, Rickettsia prowazekii and Rickettsia typhi, to have no actin tails and short (approximately 1- to 3-micrometer) straight or hooked actin tails, respectively. The SFG rickettsia, R. rickettsii, displayed long actin tails (>10 micrometer) that were frequently comprised of multiple, distinct actin bundles, wrapping around each other in a helical fashion. Transmission electron microscopy, in conjunction with myosin S1 subfragment decoration, revealed that the individual actin filaments of R. rickettsii tails are >1 micrometer long, arranged roughly parallel to one another, and oriented with the fast-growing barbed end towards the rickettsial pole. Scanning electron microscopy of intracellular rickettsiae demonstrated R. rickettsii to have polar associations of cytoskeletal material and R. prowazekii to be devoid of cytoskeletal interactions. By indirect immunofluorescence, both R. rickettsii and Listeria monocytogenes actin tails were shown to contain the cytoskeletal proteins vasodilator-stimulated phosphoprotein profilin, vinculin, and filamin. However, rickettsial tails lacked ezrin, paxillin, and tropomyosin, proteins that were associated with actin tails of cytosolic or protrusion-bound Listeria. The unique ultrastructural and compositional characteristics of the R. rickettsii actin tail suggest that rickettsial ABM is mechanistically different from previously described microbial ABM systems.

Rickettsia rickettsii has proteins with cross-reacting epitopes to eukaryotic phospholipase A2 and phospholipase C.

The entry, and possibly the exit, of rickettsiae from eukaryotic cells, as well as erythrocyte lysis by some members of this group of organisms, is thought to be mediated by a phospholipase A activity even though the enzyme has not been isolated from these organisms. Evidence for phospholipase C, on the other hand, has not been reported for the genus Rickettsia. In this study, in a preliminary attempt to demonstrate the presence of phospholipase A2 and phospholipase C in the virulent Sheila Smith strain of Rickettsia rickettsii, we performed immunoblotting and immuno-gold electron microscopy using anti-phospholipase A2 and anti-phospholipase C IgG antibodies (raised against mammalian enzymes). We provide evidence for cross-reactivity of the antibodies with proteins present in R. rickettsii. Western blots showed a higher staining intensity with anti-phospholipase C antibody than with anti-phospholipase A2. According to the results obtained with the immuno-gold labeling of phospholipase A2 and phospholipase C reactive epitopes, most of the phospholipase A2 cross-reactive material appears to be associated with the membrane of the organism while the phospholipase C cross-reactive material appears to be randomly distributed throughout the cell.

Biological properties of rabbit antibodies to a surface antigen of Rickettsia rickettsii.

Because of the potential significance of external components of the obligate intracellular parasite Rickettsia rickettsii in host-parasite interactions, we have begun the first phase of a study to isolate and characterize surface antigens of this organism. An antiserum to a rickettsial surface component was obtained from rabbits inoculated with immune precipitates prepared by crossed immunoelectrophoresis of Triton X-100 extracts of R. rickettsii strain R. This antiserum (i) protected guinea pigs inoculated with 10,000 guinea pig 50% infectious doses of R. rickettsii against fever, (ii) prevented death of mice challenged with 2 50% lethal doses of R. rickettsii, and (iii) reacted in the microimmunofluorescence test with 9 of 13 spotted fever group serotypes tested. The location of this antigen on the rickettsial surface was demonstrated by immunoelectron microscopy with ferritin-labeled antibodies.

Identification of Rickettsia rickettsii in a guinea pig model by immunofluorescent and electron microscopic techniques.

Moribund guinea pigs infected with Richettsia rickettsii were examined by necropsy, histology, immunofluorescence, electron microscopy, and serology. Untreated animals died at 9 and 10 days after inoculation. Animals given saline subcutaneously survived from 1 to 4 days longer. Prolonged survival was accompanied by more severe lesions: scrotal necrosis; infarction of ears; and swollen, hemorrhagic footpads, epididymis, and cremaster muscle. Histopathologic examination demonstrated that acute, necrotizing vasculitis, perivascular hemorrhage, and focal necrosis were more extensive. Direct immunofluorescence indicated many more rickettsiae in endothelium and vascular wall of saline recipients. Ultrastructurally, typical rickettsiae were present focally in the cytoplasm of endothelial and vascular smooth muscle cells. Cytopathology in infected and adjacent cells included swelling, mitochondrial enlargement with decrease in matrix density and loss of cristae, and increased pinocytosis. In addition, treated animals had more cytonecrosis, thrombosis, extravascular fibrin deposition, prominent inflammatory cells with polymorphonuclear phagocytosis of rickettsiae, and antibody production.

Reactivation of Rickettsia rickettsii in Dermacentor andersoni ticks: an ultrastructural analysis.

Virulent Rickettsia in Dermacentor andersoni lose their pathogenicity and virulence for guinea pigs when subjected to physiological stresses, such as starvation (overwintering), of its tick vector. However, incubation of infected ticks at an elevated temperature (37 degrees C) for 24 to 48 h or feeding for a time (usually greater than 10 h) induces R. rickettsii to revert to a virulent state, a phenomenon defined as "reactivation." Electron microscopy reveals that the microcapsular and slime layers of R. rickettsii undergo changes dependent upon the physiological conditions within the tick vector. In engorged ticks, the microcapsular layer is readily identified as a discrete layer, approximately 16 nm thick, composed of globular subunits that have a periodicity of approximately 10 nm. The slime layer external to the microcapsular layer forms a discrete electron-lucent zone around the rickettsia. In starved ticks, neither the microcapsular layer nor slime layer remains a discrete entity. Instead, they are shed and form stringy, shredded, and somewhat flocculent strands of low electron density without periodicity. Incubation at 37 degrees C or feeding of starved infected ticks results in the restoration of a discrete microcapsular and slime layer. These reversible structural modifications are linked to physiological changes in the tick host and correlate with reactivation, i.e., restoration of pathogenicity and virulence of R. rickettsii.

Production of antibody to and cellular localization of erythrocyte-sensitizing substance from Rickettsia rickettsii.

Antibodies to Rickettsia rickettsii erythrocyte-sensitizing substance (ESS) were raised in rabbits by using a derivatized ESS. The resulting antibodies reacted with R. rickettsii and cross-reacted with Rickettsia conorii, a member of the spotted fever group rickettsiae, but did not react with Rickettsia typhi, a member of the typhus group rickettsiae, Legionella bozemanii, or Proteus vulgaris OX19 or OX2. Immunoblot analysis indicated that ESS was present in more than one fraction and that the major haptenic fraction was proteinase resistant. Immunoelectron microscopy indicated that the antibodies to R. rickettsii were specific to components located on the cell surface and intracellularly to components between the cell wall and cytoplasmic membrane.

Rickettsia rickettsii infection of the EA.hy 926 endothelial cell line: morphological response to infection and evidence for oxidative injury.

EA.hy 926 is a permanent human cell line that expresses highly differentiated functions characteristic of human vascular endothelium. Rickettsia rickettsii can efficiently infect and cause a cytopathic effect in EA.hy 926 cells. R. rickettsii produced visible lytic plaques in EA.hy 926 cells at 10 d post-infection (p.i.) following application of a secondary agarose overlay containing 2 micrograms emetine ml-1 and 40 micrograms NaF ml-1 on day 2. Rickettsial growth in EA.hy 926 cells had a similar profile to that occurring in human umbilical vein endothelial cells (HUVEC) and rickettsiae catalysed polymerization of actin tails. Intracellular multiplication of R. rickettsii resulted in significant changes in the internal morphology of EA.hy 926 cells, most notably extensive dilatation of the membranes of the endoplasmic reticulum and outer nuclear envelope by 72 h p.i. These events correlated with significant alterations in the host-cell antioxidant system, including decreased levels of intracellular reduced glutathione and glutathione peroxidase activity and increased amounts of intracellular peroxide through to 96 h of infection. These findings are similar to the changes described previously for R. rickettsii-infected HUVEC and suggest that common mechanisms associated with rickettsia-induced oxidative injury occur in the two cell lines. EA.hy 926 cells were also used to investigate the influence of the antioxidant alpha-lipoic acid on rickettsial infection. Overnight pretreatment with 1-500 microM alpha-lipoic acid did not prevent cells from being destroyed following infection with rickettsiae. Supplementation of the culture medium with 1 and 10 microM alpha-lipoic acid 2 h after rickettsial inoculation also did not provide any protective effect. However, 100, 200 and 500 microM alpha-lipoic acid increased the viability of infected cells at 96 h to 45, 51 and 70%, respectively compared with 26% for untreated, infected samples. Thiol levels and glutathione peroxidase activity in treated, infected cells increased and peroxide content decreased proportionally to increasing alpha-lipoic acid concentrations. Furthermore, treatment with 500 microM alpha-lipoic acid for 72 h p.i. completely prevented ultrastructural changes in infected cells. In conclusion, the permanent endothelial cell line EA.hy 926 is susceptible to injury induced by R. rickettsii infection. Although the cellular changes resulting from infection are not identical in all aspects to that demonstrated previously in HUVEC, the increased reproducibility and convenience of EA.hy 926 cells make them suitable for biochemical and morphological studies.