Localization and visualization of a coxiella-type symbiont within the lone star tick, Amblyomma americanum.

A Coxiella-type microbe occurs at 100% frequency in all Amblyomma americanum ticks thus far tested. Using laboratory-reared ticks free of other microbes, we identified the Amblyomma-associated Coxiella microbe in several types of tissue and at various stages of the life cycle of A. americanum by 16S rRNA gene sequencing and diagnostic PCR. We visualized Amblyomma-associated Coxiella through the use of a diagnostic fluorescence in situ hybridization (FISH) assay supplemented with PCR-based detection, nucleic acid fluorescent staining, wide-field epifluorescence and confocal microscopy, and transmission electron microscopy (TEM). Specific fluorescent foci were observed in several tick tissues, including the midgut and the Malpighian tubules, but particularly bright signals were observed in the granular acini of salivary gland clusters and in both small and large oocytes. TEM confirmed intracellular bacterial structures in the same tissues. The presence of Amblyomma-associated Coxiella within oocytes is consistent with the vertical transmission of these endosymbionts. Further, the presence of the Amblyomma-associated Coxiella symbiont in other tissues such as salivary glands could potentially lead to interactions with horizontally acquired pathogens.

Antigenic differences between Coxiella burnetii cells revealed by postembedding immunoelectron microscopy and immunoblotting.

The aim of this study was to investigate the antigenic structures of the morphologically distinct cells of the Coxiella burnetii developmental cycle. Postembedding immunoelectron microscopy with polyclonal antibodies produced in rabbits to (i) phase I cells, (ii) a chloroform-methanol residue fraction of cells, (iii) the cell walls (CW) of large and small cells and small dense cells (SDC), and (iv) the peptidoglycan-protein complexes of small cells and SDC labelled the continuum of morphologically distinct cells. But these antibodies did not distinguish between the antigenic structures of the various cells. Monoclonal antibodies to the phase I lipopolysaccharide labelled the CW of a majority of the smaller cells, but there was diminished reactivity to the larger cells. Although monoclonal antibodies to a 29.5-kDa outer membrane protein labelled the CW of the large mother cells, the large cells, and the small cells, a minority of the SDC with compact CW were not labelled. The endogenous spore within the mother cell was not labelled by the polyclonal or monoclonal antibodies to cellular components. A selected population of SDC was prepared by osmotic lysis of large cells, differential centrifugation, Renografin step-gradient fractionation, and breakage of the small cells in a French press at 20,000 lb/in2. The pressure-resistant SDC collected as fraction CL did not contain the 29.5-kDa protein, as evidenced by the lack of (i) Coomassie brilliant blue staining of protein in the 29.5-kDa region of sodium dodecyl sulfate-polyacrylamide gels and (ii) reactivity of the 29.5-kDa protein antigenic epitopes in immunoblotting with monoclonal antibodies to the protein. In contrast, CW of the pressure-sensitive small cells contained the 29.5-kDa protein. Therefore, the observed ultrastructural differences between large and small cells and SDC reflect differences in sensitivity to breakage by pressure treatment and in cell-associated antigens. Although the process of differentiation in C. burnetii remains an enigma, we have taken steps toward identifying cellular antigens as markers of differentiation. The pressure-resistant SDC in fraction CL that are devoid of the 29.5-kDa protein may be useful for answering questions about the physiological events required for triggering outgrowth and sequential regulation of the Coxiella developmental cycle.

Endocytobionts in Dermacentor reticulatus ticks (Ixodidae): an electron microscope study.

Endocytobionts (ECBs) were detected in the ovaries of Dermacentor reticulatus. Their developmental cycle is directly related to the developmental stages of tick oocytes. Two basic forms of ECBs occur in tick cells, i.e. dense forms, occurring singly or in aggregates situated free in the cytoplasm of host cells, and the light forms which are larger, pleomorphic and always situated inside vacuoles of host cells. The light forms occur together with dense forms in all oocytes. The dense forms occur freely and independently in funicular cells of the oocyte, the epithelial cells of oviducts, and additionally in the cells of the Malpighian tubules. A probable function of ECBs in the tick host is discussed.

Inactivation of Coxiella burnetii by gamma irradiation.

The gamma radiation inactivation kinetics for Coxiella burnetii at -79 degrees C were exponential. The radiation dose needed to reduce the number of infective C. burnetii by 90% varied from 0.64 to 1.2 kGy depending on the phase of the micro-organism, purity of the culture and composition of suspending menstruum. The viability of preparations containing 10(11) C. burnetii ml-1 was completely abolished by 10 kGy without diminishing antigenicity or ability to elicit a protective immune response in vaccinated mice. Immunocytochemical examinations using monoclonal antibodies and electron microscopy demonstrated that radiation doses of 20 kGy did not alter cell-wall morphology or cell-surface antigenic epitopes.

Interaction between Dermacentor reticulatus cells and Coxiella burnetii in vivo.

By electron microscopy the distribution of Coxiella burnetii was followed in females of Dermacentor reticulatus injected intracoelomally in a dose of about 10(3) EID50 per tick. The heaviest infestation with coxiellae was noticed in the cells of haemolymph, fat body, Malpighian tubulus and tracheal complex. No rickettsiae were found in Gene's organ. Unexpected was the propagation of rickettsiae in muscle fibres. C. burnetii multiplied in all organs affected. Heavy infection resulted at the marked damage of cell components. Coxiellae were evident in the haemocytes; in organs they formed small and large cell variants; endospore formations were observed free in the haemolymph.

Histologic, immunofluorescence, and electron microscopic study of infectious process in mouse lung after intranasal challenge with Coxiella burnetii.

A histologic, immunofluorescence, and electron microscopic study of the intracellular parasitism of Coxiella burnetii (the Q fever agent) in mouse lungs after intranasal challenge was undertaken. It was shown that this microorganism invades type I and, rarely, type II pneumocytes as well as pulmonary fibroblasts and histiocytes. The infectious process can be described as a focal intra-alveolar inflammation with the macrophages prevailing in the exudate. It is self-limited, with a complete resolution. The inflammation is associated with atelectases and with increased secretory activity by type II pneumocytes. Alveolar macrophages and granulocytes degrade C. burnetii. This degradation is followed by damage to and eventual disintegration of some macrophages and by damage to some bacterium-free pneumocytes and vascular endothelial cells in the vicinity of macrophages degrading organisms. The cell damage might be caused by lipopolysaccharide released from degraded organisms. The infectious process is also associated with the influx of T cells in the pneumonic foci, T-cell attachment to the macrophages degrading organisms, and fusion of some macrophages. These are considered a morphologic expression of cell-mediated immunity involved in the infectious process.

In vitro susceptibility of Coxiella burnetii to antibiotics, including several quinolones.

Antibiotic susceptibility testing of the rickettsial Q fever agent Coxiella burnetii was performed by using persistently infected L929 fibroblast cells. The efficacies of a variety of antibiotics with different metabolic targets were tested and compared. The most effective antibiotics in bringing about the elimination of the parasite from infected cells included several quinolone compounds and rifampin. Of the quinolone compounds tested, difloxacin (A-56619) was the most effective, followed by ciprofloxacin and oxolinic acid. These three quinolones were apparently rickettsiacidal. After 48 h of exposure to microgram amounts of the compounds (ranging from 2 micrograms of difloxacin per ml to 5 micrograms of the other two antibiotics per ml), the number of intracellular parasites markedly declined; after 10 days of treatment, very few intracellular rickettsiae were detected. Rifampin (1 microgram/ml) was also very effective in eliminating the parasites. Some of the 13 other antibiotics tested that were somewhat effective included chloramphenicol, doxycycline, and trimethoprim. The persistently infected L929 cells were found to provide a convenient system for the relatively rapid determination of the susceptibility of C. burnetii to antibiotics.

Experimental double infection with Coxiella burnetii and tick-borne encephalitis virus in Dermacentor reticulatus ticks.

Experimental parenteral inoculation of Dermacentor reticulatus ticks with the rickettsia Coxiella burnetii (C.b.) and tick-borne encephalitis (TBE) virus resulted in a generalized rickettsial and viral infection, irrespective of whether the agents were given simultaneously or by a 7 days interval apart. Both agents multiplied intensively in ticks, C.b. being detectable predominantly in cytoplasmic vacuoles and TBE virus mostly in the endoplasmic reticulum.

[Electron microscopic methods for the rapid diagnosis of Q fever in animals]. / Elektronnomikroskopski metodi za burza diagnostika na Ku-treskata pri zhivotnite.

Methods have been worked out for the electron-microscopic diagnosis of Q-fever. A characteristic feature with them is the use of clinical, pathological, and experimental material that is investigated through negative contrast and ultra thin sections for the detection of rickettsial cells and inclusions. It is underlined that the methods referred to are readily applicable, economic, highly effective, and hazardless. They are described for the first time and could be adopted into the diagnostic and epizootiologic practice as well as in experimental investigations.

Electron microscopic studies of lipopolysaccharides from phase I and phase II Coxiella burnetii.

Lipopolysaccharides from phase I (LPSI) Coxiella burnetii Ohio and Nine Mile strains and from phase II (LPSII) Nine Mile stain were negatively and positively and examined with the electron microscope. The ultrastructure of LPSI and LPSII positively stained with uranyl formate or uranyl acetate was ribbon-like. When negatively stained with uranyl acetate, LPSI was ribbon-like but LPSII exhibited hexagonal lattice structures. However, LPSII stained negatively with sodium phosphotungstate and ammonium molybdate exhibited hexagonal lattice ultrastructures which were not identical to those observed when negatively stained with uranyl acetate. The hexagonal lattice structures formed in vitro were due to the interactions of LPSII and the staining reagents rather than to protein-LPS interactions. The differences in the ultrastructures of LPSI and LPSII are undoubtedly based on variations in their chemical composition.

Sensitivity of Coxiella burnetii peptidoglycan to lysozyme hydrolysis and correlation of sacculus rigidity with peptidoglycan-associated proteins.

The protease-resistant proteins associated with the peptidoglycan (PG) of the phase I small-cell variant Coxiella burnetii were either partially released from the PG by boiling the PG-protein complex (PG-PC) in sodium dodecyl sulfate containing 2-mercaptoethanol and EDTA or totally released by 1 N NaOH hydrolysis at 23 degrees C. An 18,300-dalton protein was released from the PG-PC under reducing conditions, whereas 1 N NaOH treatment extracted PG-associated proteins without apparent dissolution of the PG. Purified PG was composed of muramic acid, glucosamine, glutamic acid, alanine, and meso-diaminopimelic acid in a molar ratio of 0.9:0.9:1.0:1.4:1.0. Lysozyme hydrolysis of cell walls, PG-PC, and purified PG caused an increase in reducing groups which correlated with roughly 60 to 100% digestion of disaccharides. There was no significant decrease in turbidity during lysozyme hydrolysis of cell walls and PG-PC; however, hydrolysis of purified PG caused about 90% decrease in turbidity. Approximately 60% of the meso-diaminopimelic acid groups of PG were not susceptible to dinitrophenylation, thus, demonstrating an apparent contribution of PG-associated proteins, rather than cross-linkage between peptides, to sacculus rigidity of cell wall and PG-PC. This association of PG and protease-resistant covalently bound proteins may be important structural and functional determiners of resistance to both environmental conditions and intracellular digestion of C. burnetii by eucaryotic cells.

Comparison of the ultrastructure of small dense forms of chlamydiae and Coxiella burnetii.

In the course of passaging of Coxiella burnetii (C.b.) in Alveonasus lahorensis ticks, the haemocytes contained cell forms with electrondense cytoplasm, intracytoplasmic lamellar membranes, and a peculiar limiting membrane--25 to 30 nm thick "envelope complex". Similar small forms occurred when C.b. had been cultured in the yolk sack of chick embryos. The dense forms of C.b. were similar to those of Rickettsiella cells. Dense forms (elementary bodies) surrounded by an "envelope complex" were found also in some chlamydiae cultured in yolk sacs of chick embryos.

Developmental cycle of Coxiella burnetii: structure and morphogenesis of vegetative and sporogenic differentiations.

Coxiella burnetii is a gram-variable obligate intracellular bacterium which carries out its development cycle in the phagolysosome of eucaryotic cells. Ultrastructural analysis of C. burnetii, in situ and after Renografin purification, by transmission electron microscopy of lead-stained thin sections has revealed extreme pleomorphism as demonstrated by two morphological cell types, a large cell variant (LCV) and a small cell variant (SCV). Potassium permanganate staining of purified rickettsiae revealed a number of differences in the internal structures of the cell variants. (i) The outer membrane of the sCV and LCV were comparable; however, the underlying dense layer of the SCV was much wider and more prominent than that of the LCV. The periplasmic space of the SCV was not readily visualized, whereas the periplasmic space of the LCV was apparent and resembled that of other gram-negative bacteria. (ii) Complex internal membranous intrusions which appeared to originate from the cytoplasmic membrane were observed in the SCV. The LCV did not harbor an extensive membranous system. (iii) Some LCVs contained a dense body in the periplasmic space. This endogenous structure appeared to arise in one pole of the LCV as an electrondense "cap" formation with the progressive development of a dense body approximately 130 to 170 nm in diameter which was eventually surrounded by a coat of at least four layers. Our observations suggest that the morphogenesis of C. burnetii is comparable, although not identical, to cellular differentiation of endospore formation. A developmental cycle consisting of vegetative and sporogenic differentiation is proposed.

Fate of phase I and phase II Coxiella burnetii in several macrophage-like tumor cell lines.

Several macrophage-like tumor cell lines of murine origin were exposed to phase I and phase II Coxiella burnetii, and the subsequent fate of the parasites was determined by electron and bright-field microscopy. Phase I C. burnetii proliferated within and established a persistent infection of P388D1, J774, and PU-5-IR cell lines but not of WEHI-3 and WEHI-274 cell lines. Phase II C. burnetii, however, entered into and persistently infected all five cell lines. The parasites proliferated within vacuoles. Macrophage cell lines persistently infected with phase I and phase II C. burnetii were maintained for over 200 and 100 days, respectively. Within P388D1 cells, the phase I C. burnetii converted, in part, to phase II; phase II organisms remained in the phase II state. The differential fate of the two rickettsial phases after exposure to the WEHI-3 and WEHI-274 cells may be attributable to surface differences such as lipopolysaccharide content.

[A method for production of Coxiella burnetii antigen and cell walls by guanidiniumchloride extraction (author's transl)]. / Eine Methode zur Gewinnung von Antigen bzw. Zellwänden von Coxiella burnetii mittels Guanidiniumchlorid-Extraktion.

By use of 6-molar guanidinium chloride a potent Coxiella burnetii antigen could be produced for diagnostic purposes from infected yolk sacs, with little technical expense. This treatment did not only a cause remarkably purifying effect (Tab. 1) but also the extraction of soluble cytoplasmic substance. From guanidinium chloride-treated suspensions a highly purified and uniform suspension of cell walls could be separated by Saccharose Density Gradient centrifugation (Fig. 2). Guanidine extracted organisms retained their full antigenic potential with respect to Phase I and Phase II and lacked anticomplementary activity. Such preparations can be used for serological tests like complement fixation reaction or Enzyme Linked Immunosorbent Assay and are particularly suitable for biochemical studies of Phase antigens of Coxiella burnetii.

Comparison of ribosomes from Coxiella burnetii and Escherichia coli by gel electrophoresis, protein synthesis, and immunological techniques.

Ribosomes and postribiosomal supernatant fluid (S-100) were isolated from Coxiella burnetii. The ribosomes functioned in polyuridylic acid-directed polyphenylalanine synthesis in the presence of S-100 from either C. burnetii or Escherichia coli. C. burnetii S-100 promoted translation with E. coli ribosomes. Antisera against E. coli elongation factor G and ribosomal proteins L7/L12 cross-reacted with rickettsial S-100 and ribosomes, respectively. Ribosomal proteins were analyzed by two-dimensional gel electrophoresis.

Some ultrastructural effects of persistent infections by the rickettsia Coxiella burnetii in mouse L cells and green monkey kidney (Vero) cells.

Mouse fibroblasts (L-929) and Vero (green monkey kidney) cells were infected with the rickettsia Coxiella burnetti, and persistent infections developed and were studied over a 6- to 10-month period. Ultrastructural comparisons were made between the two infected cell types, and both were tested cytochemically for the presence of acid phosphatase, a marker enzyme of lysozymes. Rickettsiae were always observed within vacuoles, and some infected L cells showed flattened endoplasmic reticulum as compared with uninfected cells. Rickettsiae in Vero cells were most often seen in vacuoles containing whorls of membranes ("myelin configurations") which were also seen in uninfected cells. Rickettsiae in Vero cells were pleomorphic, with acid phosphatase reaction product in their periplasmic space. This suggests either rickettsial degradation by lysosomal enzymes which penetrated the cell envelope or a penetration after the rickettsiae were dead. Vacuoles of infected Vero cells showed much more reaction product than that in infected L cells, and most rickettsiae in L cells had a normal appearance and showed no reaction product in their periplasmic space.

Ultrastructural investigations on surface structures involved in Coxiella burnetii phase variation.

By using the cytochemical staining procedure with concanavalin A, horseradish peroxidase, and diaminobenzidine, no surface carbohydrates with terminal alpha-glucosyl or sterically closely related residues could be detected on the cell walls of Coxiella burnetii phases I and II. Using a polycationized ferritin derivative as a cytochemical probe, anionic binding sites were visualized in the electron microscope on cell membranes of C. burnetii phase II, but not on phase I organisms. The sites appeared to be masked in phase I particles. Anionic sites could be demonstrated on phase I organisms after treatment with NaIO4 or dimethyl sulfoxide. A number of different biological properties of C. burnetii phases I and II may depend on the presence or absence of a net negative charge on the surface of the cell walls of these organisms.