"Hairiness" is a Facsimile of Reorganized Cytoskeletons: A Cytopathic Effect of Coxiella burnetii.

In 1993, I reported that Coxiella burnetii transforms human B cells into hairy cells (cbHCs), the first hairy cell reported outside of hairy cell leukemia (HCL). Over last few decades, advances in molecular biology have provided evidence supporting that C. burnetii induces hairiness and inhibits the apoptosis of host cells. The present review summarizes new information in support of cbHC. C. burnetii was shown to induce reorganization of the cytoskeleton and to inhibit apoptosis in host cells. Peritoneal B1a cells were found to be permissive for virulent C. burnetii Nine Mile phase I (NMI) strains in mice. C. burnetii severely impaired E-cad expression in circulating cells of Q fever patients. B-cell non-Hodgkin lymphoma was linked to C. burnetii. Mutation of BRAF V600E was pronounced in HCL, but "hairiness" was not linked to the mutation. Risk factors shared among coxiellosis and HCL in humans and animals were reported in patients with Q-fever. Accordingly, I propose that C. burnetii induces reorganization of the cytoskeleton and inhibits apoptosis as cytopathic effects that are not target cell specific. The observed hairiness in cbHC appears to be a fixed image of dynamic nature, and hairy cells in HCL are distinct among lymphoid cells in circulation. As the cytoskeleton plays key roles in maintaining cell structural integrity in health and disease, the pathophysiology of similar cytopathic effects should be addressed in other diseases, such as myopathies, B-cell dyscrasias, and autoimmune syndromes.

Reorganization of actin cytoskeleton in L929 cells infected with Coxiella burnetii strains isolated from placenta and foetal brain of sheep (Sardinia, Italy).

Coxiella burnetii, the etiological agent of Q Fever, is a zoonotic pathogen distributed worldwide. It has been reported that virulent strains of C. burnetii are poorly internalized by monocytes compared to avirulent variants. Virulence is also associated to the formation of pseudopodal extensions and transient reorganization of filamentous actin. In this article, we investigated the ability of 2 Coxiella strains isolated from ovine aborted samples to induce reorganization of the actin cytoskeleton in mouse fibroblast cells. Cells were exposed for 24 and 48 hours to ovine placenta and foetal brain tissue homogenates and then analysed by polymerase chain reaction (PCR) in order to detect Coxiella infection. The formation of pseudopodal extensions, the polarized distribution of F­actin, and the involvement of C. burnetii strain in cytoskeleton reorganization have been assessed using a laser scanning confocal fluorescence microscope. Results indicate that similarly to the virulent reference strain, strains of C. burnetii isolated from foetal brain induced morphological changes - modification in F­actin distribution and in the localization of bacteria. By contrast, C. burnetii strain isolated from ovine placenta did not induce any significant change in L929 cell morphology. In conclusion, both C. burnetii strains isolated from ovine placenta and foetal brain were pathogenic causing ovine abortion, but in vitro the C. burnetii strain isolated from brain only was able to induce F­actin reorganization in L929 infected cells.

Creating standards for absolute quantification of Coxiella burnetii in real-time PCR--a comparative study based on transmission electron microscopy.

Quantitative standards are a prerequisite for quality control and quantification of pathogens. In this study the creation of quantitative standards for use in qPCR is described using the pathogen Coxiella burnetii. Quantification of Coxiella burnetii particles by transmission electron microscopy (TEM) was used as primary standard and compared with data obtained by light microscopy as well as genome equivalents (GE) and plasmid units (recombinant plasmid). Based on pathogen quantification using TEM and light microscopy, pathogen detection limits of 6 and 2 C. burnetii particles could be determined per com1 qPCR reaction, respectively. In comparison, the detection limits were 17 and 13 pathogen units using GE and plasmid units, respectively. The standard generated by TEM can be used as gold standard for universal application due to high accuracy, quantitative control of the producing process and supplying intact pathogen particles.

Isolation from animal tissue and genetic transformation of Coxiella burnetii are facilitated by an improved axenic growth medium.

We recently described acidified citrate cysteine medium (ACCM), which supports host cell-free (axenic) growth of Coxiella burnetii. After 6 days of incubation, greater than 3 logs of growth was achieved with the avirulent Nine Mile phase II (NMII) strain. Here, we describe modified ACCM and culture conditions that support improved growth of C. burnetii and their use in genetic transformation and pathogen isolation from tissue samples. ACCM was modified by replacing fetal bovine serum with methyl-ß-cyclodextrin to generate ACCM-2. Cultivation of NMII in ACCM-2 with moderate shaking and in 2.5% oxygen yielded 4 to 5 logs of growth over 7 days. Similar growth was achieved with the virulent Nine Mile phase I and G isolates of C. burnetii. Colonies that developed after 6 days of growth in ACCM-2 agarose were approximately 0.5 mm in diameter, roughly 5-fold larger than those formed in ACCM agarose. By electron microscopy, colonies consisted primarily of the C. burnetii small cell variant morphological form. NMII was successfully cultured in ACCM-2 when medium was inoculated with as little as 10 genome equivalents contained in tissue homogenates from infected SCID mice. A completely axenic C. burnetii genetic transformation system was developed using ACCM-2 that allowed isolation of transformants in about 2 1/2 weeks. Transformation experiments demonstrated clonal populations in colonies and a transformation frequency of approximately 5 × 10(-5). Cultivation in ACCM-2 will accelerate development of C. burnetii genetic tools and provide a sensitive means of primary isolation of the pathogen from Q fever patients.

Characterization of a Coxiella burnetii ftsZ mutant generated by Himar1 transposon mutagenesis.

Coxiella burnetii is a gram-negative obligate intracellular bacterium and the causative agent of human Q fever. The lack of methods to genetically manipulate C. burnetii significantly impedes the study of this organism. We describe here the cloning and characterization of a C. burnetii ftsZ mutant generated by mariner-based Himar1 transposon (Tn) mutagenesis. C. burnetii was coelectroporated with a plasmid encoding the Himar1 C9 transposase variant and a plasmid containing a Himar1 transposon encoding chloramphenicol acetyltransferase, mCherry fluorescent protein, and a ColE1 origin of replication. Vero cells were infected with electroporated C. burnetii and transformants scored as organisms replicating in the presence of chloramphenicol and expressing mCherry. Southern blot analysis revealed multiple transpositions in the C. burnetii genome and rescue cloning identified 30 and 5 insertions in coding and noncoding regions, respectively. Using micromanipulation, a C. burnetii clone was isolated containing a Tn insertion within the C terminus of the cell division gene ftsZ. The ftsZ mutant had a significantly lower growth rate than wild-type bacteria and frequently appeared as filamentous forms displaying incomplete cell division septa. The latter phenotype correlated with a deficiency in generating infectious foci on a per-genome basis compared to wild-type organisms. The mutant FtsZ protein was also unable to bind the essential cell division protein FtsA. This is the first description of C. burnetii harboring a defined gene mutation generated by genetic transformation.

A method for purifying obligate intracellular Coxiella burnetii that employs digitonin lysis of host cells.

Purification of the obligate intracellular bacterium Coxiella burnetii requires physical disruption of infected cells. Here we describe a gentle and safe digitonin lysis procedure to release C. burnetii from infected cells. The purity, yield, and infectivity of digitonin-prepped organisms are comparable to that of organisms purified using cell lysis by sonication.

Autophagosomes: a fast-food joint for unexpected guests.

Coxiella burnetii is a Gram-negative obligate intracellular bacterium that infects a wide range of hosts including humans, causing Q fever, a disease characterized by high fever and flu-like symptoms. After its internalization the Coxiella-containing phagosomes interact with intracellular compartments and generate a large replicative vacuole that displays certain characteristics of a phagolysosome. We have shown that this bacterially-customized replicative vacuole also has the hallmarks of an autophagosomal compartment. Furthermore, in a recent publication we have reported that induction of autophagy is beneficial for the replication and survival of Coxiella. Different morphological forms of this bacterium have been described during its developmental cycle. Here we present additional data and discuss a model indicating that induction of autophagy favors the differentiation of the Coxiella small cell variants to the metabolically active large cells variants. We postulate that nutrient acquisition, likely by fusion with the nutrient-rich autophagic vacuoles, triggers the development of the large cell variants which actively multiply in the host cell.

Temporal analysis of Coxiella burnetii morphological differentiation.

Coxiella burnetii undergoes a poorly defined developmental cycle that generates morphologically distinct small-cell variants (SCV) and large-cell variants (LCV). We developed a model to study C. burnetii morphogenesis that uses Vero cells synchronously infected with homogeneous SCV (Nine Mile strain in phase II) harvested from aged infected cell cultures. A time course transmission electron microscopic analysis over 8 days of intracellular growth was evaluated in conjunction with one-step growth curves to correlate morphological differentiations with growth cycle phase. Lag phase occurred during the first 2 days postinfection (p.i.) and was primarily composed of SCV-to-LCV morphogenesis. LCV forms predominated over the next 4 days, during which exponential growth was observed. Calculated generation times during exponential phase were 10.2 h (by quantitative PCR assay) and 11.7 h (by replating fluorescent focus-forming unit assay). Stationary phase began at approximately 6 days p.i. and coincided with the reappearance of SCV, which increased in number at 8 days p.i. Quantitative reverse transcriptase-PCR demonstrated maximal expression of scvA, which encodes an SCV-specific protein, at 8 days p.i., while immunogold transmission electron microscopy revealed degradation of ScvA throughout lag and exponential phases, with increased expression observed at the onset of stationary phase. Collectively, these results indicate that the overall growth cycle of C. burnetii is characteristic of a closed bacterial system and that the replicative form of the organism is the LCV. The experimental model described in this report will allow a global transcriptome and proteome analysis of C. burnetii developmental forms.

Phagocytosis of apoptotic cells increases the susceptibility of macrophages to infection with Coxiella burnetii phase II through down-modulation of nitric oxide production.

Coxiella burnetii, the agent of Q fever in humans and coxiellosis in other mammals, is an obligate intracellular bacterium which is sheltered and multiplies within typically large phagolysosome-like replicative vacuoles (LRVs). We have previously shown that, compared with fibroblasts, mouse resident peritoneal macrophages control the development of LRVs and bacterial multiplication within these vacuoles. Earlier experiments with the nitric oxide (NO) synthase inhibitor aminoguanidine (AG) revealed that the control is exerted by NO induced by the bacteria. We report here that phagocytosis of apoptotic-like, but not of aldehyde-killed, lymphocytes by the macrophages reduced the production of NO induced by the bacteria and increased the development of LRVs and, therefore, the total bacterial load in the cultures. Experiments with macrophages from mice deficient for inducible NO synthase (iNOS(-)/(-)) confirmed the involvement of NO in the control of infection, since neither apoptotic lymphocytes nor AG affected the development of LRVs in these phagocytes. Since macrophages are important for the clearance of apoptotic bodies and C. burnetii is able to induce apoptosis in human monocytes, the phenomenon shown here may be biologically relevant to the development of Q fever and coxiellosis.

Molecular pathogenesis of Coxiella burnetii in a genomics era.

The agent of acute and chronic Q fever, Coxiella burnetii, occupies a unique niche among intracellular pathogens. The mechanisms the organism employs to cause disease are unclear but involve persistence in a parasitophorous vacuole and the subsequent host response. Studies designed to model molecular mechanisms of pathogenesis have relied upon indirect evidence for testing the role of virulence factors since methods for generation of defined mutations have not been developed. Evidence suggests replication involving a developmental lifecycle is critical for intra- and extracellular survival but this cycle is incompletely defined. It has been proposed that survival in the phagolysosomal-like parasitophorous vacuole requires specific iron uptake systems, secretion of enzymes to detoxify the compartment (catalase and SOD), and down-regulation of an oxidative burst (acid phosphatase). Studies to test these potential virulence mechanisms can be accelerated with the recent development of the complete genome sequence for the prototype acute disease isolate, Nine Mile. Proteins differentially expressed during the developmental cycle can more readily be identified with MALDI-TOF description of proteomic profiles. Genes encoding secreted Cu/Zn SOD, catalase, and acid phosphatase are predicted and can be tested for function and expression. An iron regulon is predicted based upon Fur-regulated open reading frames. The specific role the iron-regulated genes play in iron acquisition can be tested. Confirmation of the iron regulon and others can be tested using microarrays based upon the genomic ORF predictions. These are examples of how we are rapidly changing the experimental approaches used to investigate C. burnetii to improve our understanding of the biology of this unusual and highly adapted organism.

Coxiella burnetii exhibits morphological change and delays phagolysosomal fusion after internalization by J774A.1 cells.

Coxiella burnetii, the etiological agent of Q fever, is an obligate intracellular bacterium proliferating within the harsh environment of the phagolysosome. Mechanisms controlling trafficking to, and survival of pathogens within, the phagolysosome are unknown. Two distinct morphological variants have been implicated as playing a role in C. burnetii survival. The dormant small-cell variant (SCV) is resistant to extracellular stresses and the more metabolically active large-cell variant (LCV) is sensitive to environmental stresses. To document changes in the ratio of SCVs to LCVs in response to environment, a protein specific to SCV, ScvA, was quantitated. During the first 2 h after internalization of C. burnetii by J774A.1 cells, the level of ScvA decreased, indicating a change from a population containing primarily SCVs to one containing primarily LCVs. In vitro experiments showed that 2 h of incubation at pH 5.5 caused a significant decrease in ScvA in contrast to incubation at pH 4.5. Measuring in vitro internalization of [(35)S]methionine-[(35)S]cysteine in response to pH, we found the uptake to be optimal at pH 5.5. To explore the possibility that after uptake C. burnetii was able to delay phagolysosomal fusion, we used thorium dioxide and acid phosphatase to label phagolysosomes during infection of J774A.1 cells. We determined that viable C. burnetii was able to delay phagolysosomal fusion. This is the first time that a delay in phagolysosomal fusion has been shown to be a part of the infection process of this pathogenic microorganism.

Construction of chimeric phagosomes that shelter Mycobacterium avium and Coxiella burnetii (phase II) in doubly infected mouse macrophages: an ultrastructural study.

Dual infection of cells may divert pathogens to intracellular compartments different from those occupied in mono-infected cells. In the present studies, mouse bone marrow in vitro-derived macrophages were first infected with virulent Mycobacterium avium, which are normally singly lodged within tight phagosomes. These phagosomes do not mature; they undergo homotypic fusion with early endosomes and do not fuse with lysosomes. Seven days later, the cultures were superinfected with phase II (non-virulent) Coxiella burnetii, organisms sheltered in lysosome- (or prelysosome)-like, multi-occupancy phagosomes. The latter can attain large size and engage in efficient homo- and heterotypic fusion with other phagosomes. Cultures were fixed for transmission electron microscopy 6, 12, 24, and 48 h later. Other M. avium-infected cultures were superinfected with amastigotes of the trypanosomatid flagellate Leishmania amazonensis, which are also sheltered in lysosome- (or prelysosome)-like multi-occupancy vacuoles, and fixed at the same time periods. Chimeric phagosomes containing both M. avium and C. burnetii, were found already at 6 h and the proportion of M. avium that colocalized with C. burnetii in the same phagosomes reached over 90% after 48 h. In such phagosomes, both organisms were ultrastructurally well preserved. In contrast, colocalization of M. avium and L. amazonensis was rarely found. Speculative scenarios that could underlie the formation of chimeric phagosomes could involve delayed maturation of C. burnetii-containing phagosomes in presence of M. avium, which would allow for fusion of C. burnetii- and M. avium-containing phagosomes; the production, by C. burnetii, of molecules that upregulate the fusion of M. avium-containing phagosomes with those that contain C. burnetii; and the secretion of factors that could favour the survival of M. avium within chimeric vacuoles.

[The use of atomic-force microscopy for the analysis of microbiological objects]. / Ispol'zovanie atomno-silovoi mikroskopii dlia analiza mikrobiologicheskikh ob''ektov.

Atomic force microscopy (AFM) was used for the analysis of rickettsiae and viruses. The specificity of interaction was evaluated on the basis of the adsorption of the analyzed antigen on the polymer-antibody film. The film was formed and transferred onto highly oriented pyrolytic graphite (HOPG) by the method of Langmuir-Schaefer with the use of amphiphilic polymers, alkylated polyethyleneimines. According to the data of AFM, polymer-antibody film was 10-30 nm thick and had ruptures, uneven surface. AFM images of Coxiella burnetii, rotavirus and Venezuelan equine encephalitis virus, immunoadsorbed on the antibody film, were obtained. C.burnetii, due to their size equal to (700-900) x (300-500) x Z = 200 nm, were clearly visible on the underlying surface and could be directly counted. Individual virus particles (60-80 nm) cold not be identified on such surface. To analyze such preparations, the program of image analysis was developed. The program classified the registered image with a certain standard. This program determined the presence of virus antigen on the underlying affinity surface with a high degree of precision.

Developmentally regulated synthesis of an unusually small, basic peptide by Coxiella burnetii.

Coxiella burnetii undergoes a poorly defined developmental cycle within phagolysosomes of eukaryotic host cells. Two distinct developmental forms are part of this cycle: a small-cell variant (SCV) and large-cell variant (LCV). Ultrastructurally, the SCV is distinguished from the LCV by its smaller size and condensed chromatin. At a molecular level, little is known about morphogenesis in C. burnetii, and no proteins specific to the SCV have been identified. Preparative isoelectric focusing was conducted to purify basic proteins possibly involved in SCV chromatin structure. A predominant protein of low M(r) was present in the most basic fraction, eluting with a pH of approx. 11. Degenerate deoxyoligonucleotides corresponding to the N-terminal sequence of this protein were used to recover a cosmid clone from a C. burnetii genomic library. Nucleotide sequencing of insert DNA revealed an open reading frame designated scvA (Small-Cell-variant protein A) with coding potential for a 30 amino acid protein (ScvA) with a predicted M(r) of 3610. ScvA is 46% arginine plus 46% glutamine with a predicted pl of 12.6. SDS-PAGE and silver staining of lysates of SCV and LCV purified by caesium chloride-equilibrium density centrifugation revealed a number of proteins unique to each cell type. Immunoblot analysis with ScvA antiserum demonstrated the presence of ScvA only in the SCV. By Immunoelectron microscopy, ScvA antiserum labelled only the SCV, with the label concentrated on the condensed nucleoid. In addition, ScvA bound double-stranded DNA in gel mobility-shift assays. A 66% reduction in the mean number of gold particles per Coxiella call was observed at 12 h post-infection when compared with the starting inoculum. Collectively, these data suggest that synthesis of ScvA is developmentally regulated, and that the protein may serve a structural or functional role as an integral component of the SCV chromatin. Moreover, degradation of this protein may be a necessary prerequisite for morphogenesis from SCV to LCV.

Differential interaction with endocytic and exocytic pathways distinguish parasitophorous vacuoles of Coxiella burnetii and Chlamydia trachomatis.

Coxiella burnetii and Chlamydia trachomatis are bacterial obligate intracellular parasites that occupy distinct vacuolar niches within eucaryotic host cells. We have employed immunofluorescence, cytochemistry, fluorescent vital stains, and fluid-phase markers in conjunction with electron, confocal, and conventional microscopy to characterize the vacuolar environments of these pathogens. The acidic nature of the C. burnetii-containing vacuole was confirmed by its acquisition of the acidotropic base acridine orange (AO). The presence of the vacuolar-type (H+) ATPase (V-ATPase) within the Coxiella vacuolar membrane was demonstrated by indirect immunofluorescence, and growth of C. burnetii was inhibited by bafilomycin A1 (Baf A), a specific inhibitor of the V-ATPase. In contrast, AO did not accumulate in C. trachomatis inclusions nor was the V-ATPase found in the inclusion membrane. Moreover, chlamydial growth was not inhibited by Baf A or the lysosomotropic amines methylamine, ammonium chloride, and chloroquine. Vacuoles harboring C. burnetii incorporated the fluorescent fluid- phase markers, fluorescein isothiocyanate-dextran (FITC-dex) and Lucifer yellow (LY), indicating trafficking between that vacuole and the endocytic pathway. Neither FITC-dex nor LY was sequestered by chlamydial inclusions. The late endosomal-prelysosomal marker cation-independent mannose 6-phosphate receptor was not detectable in the vacuolar membranes encompassing either parasite. However, the lysosomal enzymes acid phosphatase and cathepsin D and the lysosomal glycoproteins LAMP-1 and LAMP-2 localized to the C. burnetii vacuole but not the chlamydial vacuole. Interaction of C. trachomatis inclusions with the Golgi-derived vesicles was demonstrated by the transport of sphingomyelin, endogenously synthesized from C6-NBD-ceramide, to the chlamydial inclusion and incorporation into the bacterial cell wall. Similar trafficking of C-NBD-ceramide was not evident in C. burnetii-infected cells. Collectively, the data indicate that C. trachomatis replicates within a nonacidified vacuole that is disconnected from endosome-lysosome trafficking but may receive lipid from exocytic vesicles derived from the trans-Golgi network. These observations are in sharp contrast to those for C. burnetii, which by all criteria resides in a typical phagolysosome.

Coxiella burnetii (Q fever) pneumonia.

Pneumonia is one manifestation of acute Q fever following infection with Coxiella burnetii. Fever, headache, and myalgia dominate the clinical picture of Q fever pneumonia. Cough is nonproductive and may be absent despite the presence of pneumonia. While in most instances pneumonia results in an illness of mild-to-moderate severity, on occasion it is rapidly progressive and results in respiratory failure. Infection occurs as a result of inhalation of contaminated aerosols. Infected cattle, sheep, and goats are the usual reservoirs for this zoonosis. In some areas, infected parturient cats serve as the reservoir, and in such instances, rounded opacities are seen on the chest radiograph. The diagnosis of C. burnetii pneumonia is usually confirmed by demonstration of a fourfold or greater rise in antibody titer. Treatment is usually with a tetracycline or rifampin for 7 to 10 days.

In vivo endogenous spore formation by Coxiella burnetii in Q fever endocarditis.

AIMS: To determine whether Coxiella burnetii, the aetiological agent of Q fever, undergoes endogenous spore-like formation, the crucial stage of the developmental cycle, in the infected cardiac valves of patients with chronic Q fever endocarditis. METHODS: Surgically removed valves from three cases of Q fever endocarditis were processed for electron microscopy. Sections were stained with potassium permanganate and uranyl acetate before being extensively examined by transmission electron microscopy. RESULTS: In all three cases endogenous spore-like formation was seen in the infected cardiac valves. CONCLUSIONS: As the factors that govern sporogenesis in C burnetii are still largely unknown, it is uncertain how important are the implications of the discovery of endogenous spore-like formation in Q fever endocarditis. However, this finding may add new dimensions to current thinking about the treatment of chronic Q fever.

Purification of Coxiella burnetii antigen by gel-chromatography for use in enzyme-linked immunosorbent assay (ELISA).

Cell wall antigens from Coxiella burnetti were extracted by heat, ultrasonication, phenol, SDS or sodium deoxycholate. These antigens were subsequently purified by gel-chromatography yielding molecular weights of 700 and 270 KD. Using gel-chromatographical purification of antigens extracted by heat, ultrasonication or phenol Coxiella antigens nearly devoid of contaminations could be obtained. Of these highly purified antigens heat extracted antigen was treated with proteases, lysozyme or metaperiodate exemplarily. These treatments did neither destroy nor change its antigenic activity, except for the treatment with metaperiodate reducing the antigenic activity significantly. In further experiments heat extracted and gel-chromatographically purified Coxiella antigen was used for testing bovine sera in ELISA.

Acid phosphatase activity in Coxiella burnetii: a possible virulence factor.

High-speed supernatant fluids derived from sonicated Coxiella burnetii contained considerable acid phosphatase activity when assayed by using 4-methylumbelliferylphosphate; they also contained a factor that blocked superoxide anion production by human neutrophils stimulated with formyl-Met-Leu-Phe. The pH optimum of the enzyme was approximately 5.0. The level of phosphatase activity detected in several isolates of C. burnetii implicated in acute (Nine Mile) and chronic (S Q217, PRS Q177, K Q154) Q fever was 25 to 60 times greater than that reported in other microorganisms, including Leishmania and Legionella spp. The enzyme was found in rickettsiae grown in different hosts (L929 cells and embryonated eggs) and, in the case of L929 cells, for both short periods (less than a month) and the long term (years). Cytochemical techniques coupled with electron microscopy localized the phosphatase activity to the periplasmic gap in the parasite. Ion-exchange chromatography revealed a major species of the enzyme and showed that the enzyme of the parasite was distinct from that of the host cell (L929 fibroblasts); its apparent molecular weight was 74,000. Phosphatase inhibitors (i.e., molybdate heteropolyanions) had differential effects on the phosphatases of the parasite and host cell. C. burnetii supernatant fluid inhibited superoxide anion production by formyl-Met-Leu-Phe-stimulated human neutrophils; molybdate inhibitors reversed the inhibition. Treatment of C. burnetii-infected L929 cells with one of the molybdate compounds (complex B') significantly reduced the level of infection and did not affect the viability or growth of the host cell. These data suggest that the acid phosphatase of the parasite may be a major virulence determinant, allowing the agent to avoid being killed during uptake by phagocytes and subsequently in the phagolysosome.

Three-dimensional reconstruction of Coxiella burnetii-infected L929 cells by high-voltage electron microscopy.

Previous examination of thin sections of L929 cells heavily infected with the Q fever Priscilla isolate by conventional transmission electron microscopy indicated that the rickettsiae resided within multiple vacuoles. The present study using high-voltage electron microscopy and three-dimensional reconstruction revealed that, in heavily infected cells, the rickettsiae, in fact, reside in one multilobed vacuole. As a result of asymmetric cell division, the multilobed vacuole containing the rickettsiae apparently segregates into one daughter cell, while the companion daughter cell emerges parasite free. This likely explains the appearance of naive uninfected cells in long-term-infected (i.e., ca. 2 years) cell populations that had not been supplemented with uninfected L929 host cells.