Characterization and growth of polymorphic Rickettsia felis in a tick cell line

Morphological differentiation in some arthropod-borne bacteria is correlated with increased bacterial virulence, transmission potential, and/or as a response to environmental stress. In the current study, we utilized an in vitro model to examine Rickettsia felis morphology and growth under various culture conditions and bacterial densities to identify potential factors that contribute to polymorphism in rickettsiae. We utilized microscopy (electron microscopy and immunofluorescence), genomic (PCR amplification and DNA sequencing of rickettsial genes), and proteomic (Western blotting and liquid chromatography-tandem mass spectrometry) techniques to identify and characterize morphologically distinct, long-form R. felis. Without exchange of host cell growth medium, polymorphic R. felis was detected at 12 days postinoculation when rickettsiae were seeded at a multiplicity of infection (MOI) of 5 and 50. Compared to short-form R. felis organisms, no change in membrane ultrastructure in long-form polymorphic rickettsiae was observed, and rickettsiae were up to six times the length of typical short-form rickettsiae. In vitro assays demonstrated that short-form R. felis entered into and replicated in host cells faster than long-form R. felis. However, when both short- and long-form R. felis organisms were maintained in cell-free medium for 12 days, the infectivity of short-form R. felis was decreased compared to long-form R. felis organisms, which were capable of entering host cells, suggesting that long-form R. felis is more stable outside the host cell. The relationship between rickettsial polymorphism and rickettsial survivorship should be examined further as the yet undetermined route of horizontal transmission of R. felis may utilize metabolically and morphologically distinct forms for successful transmission.     

Ultrastructure of erythrocytes parasitized by Haemobartonella felis.

Experimental Haemobartonella felis infections were studied in 3 mature, intact cats by examining peripheral blood, lung, and spleen by electron microscopy. Coccoid, rod, or ring forms of the organism were found on or close to the erythrocytic membrane, and adjacent parasitized erythrocytes often were attached. Intracytoplasmic crystalloid inclusions occupying most of erythrocytic cytoplasm were seen in the 3 infected cats. The cat with the highest parasitemia had inclusions in about 10% of the erythrocytes. Less than 0.01% of the erythrocytes of a control cat contained inclusions. Parasitized erythrocytes, with and without inclusions, were seen in capillaries of the lung and spleen of infected cats. Macrophages in the lung and spleen of infected cats contained parasitized erythrocytes, either with or without inclusions. Some macrophages contained erythrocyte-free organisms in phagocytic vacuoles.     

Ultrastructure of an extremely thermophilic acidophilic micro-organism.

A thermoacidophilic micro-organism, isolated from volcanic hot springs near Naples, was cultivated in vitro, and examined by electron microscopy in sections and after negative staining. The cells were almost spherical, with a diameter of about 0.7 to 1.0 mum. Their morphology was very primitive: the protoplasm was composed only of ground cytoplasm, ribosomes, and randomly distributed DNA strands. They were surrounded by a plasma membrane and by an extracellular coat about 20 nm thick which displayed a regular hexagonal pattern. Cell replication occurred by binary fission with median constriction during which a bipolar localization of nuclear material was observable. The morphology is compared with that of other known micro-organisms living in similar habitats.     

Ultrastructal morphology of some prokaryotic microorganisms associated with the hindgut of cockroaches.

Scanning electron microscopy and transmission electron microscopy have been used to visualize the morphology and ultrastructure of two types of microorganisms in the hindgut of the cockroach Blaberus posticus. Both organisms, designated as either short or long rods, are attached to chitinous projections from the gut wall. Micrographs suggest that the organisms are prokaryotic with a cell wall complex characteristic of gram-negative bacteria. However, certain differences were noted between the cell wall complex of the two types. Two forms of the long-rod type were noted, with one form appearing to be a "degenerate" or "transitional" cell. In the degenerate cells, vesicles are observed that often are contiguous with the cytoplasmic membrane. There are indications that the long-rod type may divide by longitudinal fission. Neither the short- nor long-rod type has been cultivated in its respective recognizable form.     

Electron Microscopic Observations on the Effects of Penicillin on the Morphology of Chlamydia psittaci

L cells were infected at high multiplicity with meningopneumonitis organisms and incubated in medium containing 200 units per ml of penicillin. At intervals up to 48 hr after infection, cells were removed and thin sections were prepared for electron microscopic studies on the morphology of the developing organism. Penicillin had no effect on the initial reorganization of the infecting elementary body to form the developmental reticulate body (RB), and, up to 12 hr after infection, the treated and untreated cultures were identical. After that time, however, penicillin-treated organisms showed striking differences in that binary fission was prevented, large abnormal RB forms were seen in great numbers, masses of RB cytoplasmic membranes and envelopes were formed within and outside the RB itself, and large numbers of empty or partially filled small vesicles were pinched off the RB. After 36 hr immature nucleoids were formed within the RB. Throughout all of this period, both the outer cell envelope and the cytoplasmic membrane of these RB were recognized. When infected cells were transferred into penicillin-free medium, the abnormal RB showed recovery to form normal RB both by a budding-like process and by internal fragmentation or subdivision rather like endosporulation. We have concluded that penicillin inhibits binary fission and prevents the synthesis of certain components essential for the formation of the elementary body envelope.     

Development of a rickettsia isolated from an aborted bovine fetus.

An obligate intracellular rickettsial organism isolated from an aborted bovine fetus was studied in bovine turbinate and mouse macrophage cell cultures with light and electron microscopy. Development of the organism was similar in both cell types. The organism replicated within cytoplasmic vacuoles in a developmental cycle that resembled that of both the ehrlichiae and chlamydiae. The inoculum contained only electron-dense forms, which infected cells within 2 h postinoculation by adhering to cell membranes at thickened areas that appeared to be coated pits and then being endocytosed. A striking feature occurred next as the organisms became surrounded by host cell mitochondria and, by light microscopy, appeared to have halos. During this intimate association with mitochondria, the electron-dense organisms changed into large reticulated forms that began to divide by binary fission. These large forms were often in direct contact with mitochondrial membranes. The organisms continued to divide by binary fission, and host cells contained large cytoplasmic inclusions of reticulated organisms. The reticulated organisms gradually changed into electron-dense forms that were released from degenerated host cells.     

Multiple infection in bovines from the tropics: observation of blood parasites by scanning and transmission electron microscope

Blood samples from a splenectomized bovine, experimentally inoculated with blood from a field cow living in southwestern Venezuela, were processed for transmission and scanning electron microscopy. The blood sample showed multiple infection with hemoparasites of the genera Anaplasma marginale, Eperythrozoon wenyonii and Trypanosoma vivax. Scanning electron microscope showed that the blood from bovines with multiple infection had profound deformation in knob-like protruding structures with reduced cellular volume similar to echinocyte red blood cells. E. wenyonii parasites appear associated with the membrane, grouped in shallow to severe invaginations at the surface of the erythrocytes. The morphology of the parasites is predominantly rod-like; they also appear as coccoid-shaped and bifurcate or triskelion-shaped organisms. The organisms are present in pairs or clusters. T. vivax appeared with double flagella, which indicates active cellular division and infection processes. Transmission electron microscope study showed erythrocytes infected with intracytoplasmic bodies of A. marginale and with E. wenyonii embedded in the external membrane cell, with mature, juvenile and dividing forms present.     

Electron microscopy of stages of Isospora felis of the cat in the mesenteric lymph node of the mouse.

Stages of Isospora felis of the cat in the mesenteric lymph node of the mouse 25 days after oral inoculation with oocysts, have been described at the ultrastructural level. The organisms occurred singly within parasitophorous vacuoles in host cell cytoplasm and were sporozoite-like, having a large crystalloid body up to 5.5 mum in length posterior to the nucleus. The size and appearance of the parasitophorous vacuole varied. Some vacuoles contained numerous, small, electron dense granules about 30 nm in diameter. Because of the aggregation of granules and their arrangement within the parasitophorous vacuole, the impression was sometimes gained by light microscopy that parasites were surrounded by a sheath or cyst wall. However, a cyst wall was not present. In host cells, spherical, membrane-bound bodies with a homogeneous, electron dense core and a maximum diameter of 0.25 mum were filed along the limiting membrane of the parasitophorous vacuole. These extra-intestinal parasites were considered to be waiting stages, with a biological function similar to that of the tissue cyst stage of other general of isosporan coccidia.     

Genetic transformation of pilation and virulence into Neisseria gonorrhoeae T4.

Genetic transformation of nonpilated strains of Neisserai gonorrhoeae to pilated forms is described. The transformants displayed phenotypic T1 and T2 colonial morphology on agar and possessed pili visualized by electron microscopy. When T1 or T2 transformant cells were injected into 11-day-old chicken embryos, they exhibited virulence characteristics only slightly less than the parental donor strains, though the parental recipient strains were avirulent. Competence was maximal in the late log phase of growth, and the frequency of transformation of clonal T4s to pilation and virulence approached 2%. DNA extracted from transformants could be used to transform other T4 cells. In the course of this work, a shift to a novel colonial type, designated T2-T3 wrinkled, was observed as a consequence of growth of T4 in presence of enzymatic digests of either DNA or RNA, nucleases or individual deoxy- or ribonucleosides. In sharp distinction to the parental T4, these novel organisms were very pilated; however, they were only minimally virulent. Various nucleic acid analogs could neither induce nor inhibit this population shift. Additionally, DNA extracted from this T2-T3 wrinkled variant could be used to transform genetically both T1 and T4 gonococci to the new morphology.     

Morphology,morphometry and electron microscopy of HeLa cells infected with bovine Mycoplasma

Summary The host-parasite relationship of HeLa M cells artificially infected with a bovine species of Mycoplasma was studied by light microscopy, transmission electron microscopy and scanning electron microscopy. The use of morphometry to quantitate some of the findings was explored. The parasites were seen in locations extracellular to the cell surface. The detection of small numbers of organisms by light microscopy was well demonstrated by use of the fluorescent antibody technique. Scanning electron microscopy proved to be an excellent method for revealing the surface details of cell-parasite morphology. Ultra-thin sections showed that the parasites are aligned mostly parallel to the plasma membrane of the host cell but separated by a gap of 10 nm. Morphometry indicated an average of 69 organisms per cell surface occupying 1.7% of the surface area. An increase of 26% in diameter of the HeLa cells, possibly as a result of infection, was observed.The authors wish to thank Christiana Ulness and Andrea Erickson for expert technical assistance and Arnold Schmidt for the operation of the scanning electron microscope. This work was supported by grants from the U.S.P.H.S.: AI 09586, AI 10743, and AI 06720     

Vaccinia Virus DNA Replication Occurs in Endoplasmic Reticulum-enclosed Cytoplasmic Mini-Nuclei

Vaccinia virus (vv), a member of the poxvirus family, is unique among most DNA viruses in that its replication occurs in the cytoplasm of the infected host cell. Although this viral process is known to occur in distinct cytoplasmic sites, little is known about its organization and in particular its relation with cellular membranes. The present study shows by electron microscopy (EM) that soon after initial vv DNA synthesis at 2 h postinfection, the sites become entirely surrounded by membranes of the endoplasmic reticulum (ER). Complete wrapping requires ~45 min and persists until virion assembly is initiated at 6 h postinfection, and the ER dissociates from the replication sites. [(3)H]Thymidine incorporation at different infection times shows that efficient vv DNA synthesis coincides with complete ER wrapping, suggesting that the ER facilitates viral replication. Proteins known to be associated with the nuclear envelope in interphase cells are not targeted to these DNA-surrounding ER membranes, ruling out a role for these molecules in the wrapping process. By random green fluorescent protein-tagging of vv early genes of unknown function with a putative transmembrane domain, a novel vv protein, the gene product of E8R, was identified that is targeted to the ER around the DNA sites. Antibodies raised against this vv early membrane protein showed, by immunofluorescence microscopy, a characteristic ring-like pattern around the replication site. By electron microscopy quantitation the protein concentrated in the ER surrounding the DNA site and was preferentially targeted to membrane facing the inside of this site. These combined data are discussed in relation to nuclear envelope assembly/disassembly as it occurs during the cell cycle.     

Simplified preparation of mycoplasmas, an acholeplasma, and a spiroplasma for scanning electron microscopy.

A simple, effective procedure was developed for scanning electron microscopic examination of mycoplasmas and similar organisms. Cultivation of several mycoplasmal species, an acholeplasma, and a spiroplasma in broth media in Leighton tubes with cover slips resulted in attachment of the organisms to the cover slips. The attached cells were easily processed for either scanning electron microscopy or light microscopy. By eliminating the need for centrifugation, which was used in previously described techniques, physical stress on the cell is minimized. The effects of different preparative procedures on the morphology of Mycoplasma gallisepticum are described.     

DnaA, the initiator of Escherichia coli chromosomal replication, is located at the cell membrane

Given the lack of a nucleus in prokaryotic cells, the significance of spatial organization in bacterial chromosome replication is only beginning to be fully appreciated. DnaA protein, the initiator of chromosomal replication in Escherichia coli, is purified as a soluble protein, and in vitro it efficiently initiates replication of minichromosomes in membrane-free DNA synthesis reactions. However, its conversion from a replicatively inactive to an active form in vitro occurs through its association with acidic phospholipids in a lipid bilayer. To determine whether the in situ residence of DnaA protein is cytoplasmic, membrane associated, or both, we examined the cellular location of DnaA using immunogold cryothin-section electron microscopy and immunofluorescence. Both of these methods revealed that DnaA is localized at the cell membrane, further suggesting that initiation of chromosomal replication in E. coli is a membrane-affiliated event.     

Unbudded G2 as well as Gj arrest in the stationary phase of the basidiomycetous yeast Cryptococcus neoformans

Abstract Stationary-phase cells of Cryptococcus neoformans displayed two morphological characteristics: virtually all the cells were unbudded even in the early stationary phase and even when grown in rich media, and average cell size increased from that of exponential-phase cells. DNA contents for small and large stationary-phase cells were determined by quantitative fluorescence microscopy after DNA staining with propidium iodide or DAPI. Small cells contained G, DNA, whereas large unbudded cells had either a G₂ or G₁ DNA content, indicating that Cr. neoformans can enter into the stationary phase from either the G₁ or G₂ period.     

Structure of the nucleoid in cells of Streptococcus faecalis

The structure of the nucleoid of Streptococcus faecalis (ATCC 9790) was examined and compared in the unfixed and fixed states by immersive refractometry and electron microscopy. It appears from these studies that the nucleoid structure is much more centralized in unfixed chloramphenicol-treated (stationary-phase) cells than it is in cells in the exponential phase of growth. The more dispersed configuration of the exponential-phase nucleoid could be preserved by fixation in glutaraldehyde, but not in Formalin or in osmium tetroxide. One important factor in explaining these differences in preservation is that glutaraldehyde (but not Formalin or osmium tetroxide) can rapidly cross-link the amino groups of macromolecules in cells. It was also observed that osmium tetroxide resulted in a preferential breakdown of nascent ribonucleic acid. These results are interpreted as indicating that glutaraldehyde is able to stabilize the exponential-phase nucleoid before it assumes the more central appearance seen in osmium tetroxide- and Formalin-fixed cells. These results are discussed in terms of the proposed organization of the exponential-phase nucleoid in unfixed cells.     

Differentiation, multiplication and control of bloodstream form Trypanosoma (Duttonella) vivax in mice

The growth and differentiation of Trypanosoma vivax was studied in intact and irradiated C3H/He and C57Bl/6 mice. In irradiated (800 R) or intact C3H/He and irradiated (800 R) C57Bl/6 mice, T. vivax parasitaemia increased rapidly then entered a plateau phase and thereafter declined in an antibody-independent remission phase. Throughout the infection, variations were observed in parasite morphology, density, DNA content, number of organisms with 2 nuclei and 2 kinetoplasts and infectivity of parasites for mice. Parasites in exponential phase had the highest number of members in the S, G2 and M phases of the cell cycle as determined by staining with the interchalating dye Chromomycin A3 and analysis on a flow cytometer. During this phase there were numerous parasites with 2 nuclei and 2 kinetoplasts and infectivity was high for mice. As the parasitaemia approached and entered the plateau phase, the proportion of organisms in the S, G2 and M phases of the cell cycle as well as the number of those with 2 kinetoplasts decreased slightly; the number of organisms with 2 nuclei decreased rapidly; and parasites had a considerably reduced capacity to infect mice. Organisms from the remission phase contained only 1 nucleus and 1 kinetoplast and were not infective for mice. The study suggests that T. vivax organisms transit from dividing to committed non-dividing forms and that some non-diving, non-infective T. vivax organisms remain trapped in the S, G2 and M stages of the cell cycle and die without completing binary fission.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth morphology ofMycoplasma mobile 163K on solid surfaces: Reproduction,aggregation, and microcolony formation

The morphology ofMycoplasma mobile 163K grown on glass coverslips and Pioloform-coated copper grids in liquid medium was studied by dark-field, interference-contrast, scanning, and negative-contrast electron microscopy. The growth morphology was observed over a period of 5 days. Within this time, three distinct stages could be distinguished: (a) single cells; (b) reproduction, aggregation, and microcolony formation; and (c) degeneration. The single cells were typically flask-shaped. Different modes of reproduction were observed: symmetrical binary division by transverse fission, budding-like processes, and fragmentation. Symmetrical binary division occurred mainly in the first half stage of reproduction, leading to the formation of rosette-like microcolonies, whereas budding and fragmentation took place only in the second half. During degeneration, the flask-shaped cells converted into rounded forms and lost their viability.     

Lethal and mutagenic action of N-nitroso-N-methylbiuret on the producers of levorin, amphotericin and mycoheptin]

The lethal and mutagenic effect of N-nitrozo-N-methylbiuret (NMB) on the organisms producing levorin, amphotericin B and mycoheptin was studied. The mutagen effect depended on the dose, culture and physiological state of the spores. NMB had a low mutagenic effect on the levorin-producing organism characterized by high activity and genetic homogenicity with respect to the colony morphology and antibiotic production. As for the organisms producing amphotericin B and mycoheptin characterized by high genetic heterogenicity, significant variation of all the features studied was observed on their exposure to the mutagen. Inspite of diverse reaction of the organisms producing levorin, amphotericin B and mycoheptin to the effect of NMB mutants with increased antibiotic production were obtained from the three cultures. The lethal and mutagenic effect of NMB on the mycoheptin-producing organism depended on the process of the spore DNA replication. The spores during the DNA replication period were least sensitive to the lethal effect of the mutagen and most mutable with the respect to the colony morphology. For selection of highly active and stable strains exposure to NMB of the spores of the mycoheptin-producing organism during replication of DNA proved to be more effective than that of the spores during the lag-phase.     

Electron Microscopy of the Cell Wall of Rickettsia prowazeki

Purified Rickettsia prowazeki were found to undergo morphological changes resembling plasmolysis when stained with uranyl acetate, resulting in rod-like forms. Sequential electron micrographs of disintegrating organisms provide evidence for the cell wall origin of these rod-like forms. The substructure of the cell wall was discerned by using negative-contrast electron microscopy. The wall was found to be composed of repetitive subunits with a periodicity of 13 nm and was surrounded by a thin membrane.     

Analysis of replicating DNA molecules from embryos of the sea urchin, Hemicentrotus pulcherrimus, by electron microscopy

DNA was extracted from embryos of the sea urchin, Hemicentrotus pulcherrimus, at the S phase and examined by electron microscopy. We detected replication microbubbles with a mean size of 404 bases, in addition to replication macrobubbles of more than 1.0 kilobase (kb) in length. Seventy-five percent of the center-to-center distances of the microbubbles were 0.6-1.8 kb with a mean of 1.2 kb. Forty-five percent of the microbubbles were arranged as clusters of four or five microbubbles. These results suggest that at least 34% of the initiation sites for DNA replication are present on a DNA molecule in clusters in which the sites are arranged at 1.2-kb intervals.