scholarly journals Novel Parachlamydia acanthamoebae Quantification Method Based on Coculture with Amoebae

2008 ◽  
Vol 74 (20) ◽  
pp. 6397-6404 ◽  
Author(s):  
Junji Matsuo ◽  
Yasuhiro Hayashi ◽  
Shinji Nakamura ◽  
Marie Sato ◽  
Yoshihiko Mizutani ◽  
...  
Keyword(s):  
Fluorescent In Situ Hybridization ◽  
Vero Cells ◽  
Host Cells ◽  
Human Pathogens ◽  
Free Living ◽  
Infectious Dose ◽  
Quantification Method ◽  
Transmission Electron ◽  
Dilution Curves

ABSTRACT Parachlamydia acanthamoebae, belonging to the order Chlamydiales, is an obligately intracellular bacterium that infects free-living amoebae and is a potential human pathogen. However, no method exists to accurately quantify viable bacterial numbers. We present a novel quantification method for P. acanthamoebae based on coculture with amoebae. P. acanthamoebae was cultured either with Acanthamoeba spp. or with mammalian epithelial HEp-2 or Vero cells. The infection rate of P. acanthamoebae (amoeba-infectious dose [AID]) was determined by DAPI (4′,6-diamidino-2-phenylindole) staining and was confirmed by fluorescent in situ hybridization. AIDs were plotted as logistic sigmoid dilution curves, and P. acanthamoebae numbers, defined as amoeba-infectious units (AIU), were calculated. During culture, amoeba numbers and viabilities did not change, and amoebae did not change from trophozoites to cysts. Eight amoeba strains showed similar levels of P. acanthamoebae growth, and bacterial numbers reached ca. 1,000-fold (109 AIU preculture) after 4 days. In contrast, no increase was observed for P. acanthamoebae in either mammalian cell line. However, aberrant structures in epithelial cells, implying possible persistent infection, were seen by transmission electron microscopy. Thus, our method could monitor numbers of P. acanthamoebae bacteria in host cells and may be useful for understanding chlamydiae present in the natural environment as human pathogens.

scholarly journals Unexpected host dependency of Antarctic Nanohaloarchaeota

2019 ◽  
Vol 116 (29) ◽  
pp. 14661-14670 ◽  
Author(s):  
Joshua N. Hamm ◽  
Susanne Erdmann ◽  
Emiley A. Eloe-Fadrosh ◽  
Allegra Angeloni ◽  
Ling Zhong ◽  
...  
Keyword(s):  
Fluorescent In Situ Hybridization ◽  
Single Species ◽  
Hypersaline Environments ◽  
Free Living ◽  
Metagenome Analysis ◽  
Symbiotic Relationships ◽  
Cell Structures ◽  
Transmission Electron ◽  
Globally Distributed

In hypersaline environments, Nanohaloarchaeota (Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, Nanohaloarchaeota [DPANN] superphylum) are thought to be free-living microorganisms. We report cultivation of 2 strains of Antarctic Nanohaloarchaeota and show that they require the haloarchaeon Halorubrum lacusprofundi for growth. By performing growth using enrichments and fluorescence-activated cell sorting, we demonstrated successful cultivation of Candidatus Nanohaloarchaeum antarcticus, purification of Ca. Nha. antarcticus away from other species, and growth and verification of Ca. Nha. antarcticus with Hrr. lacusprofundi; these findings are analogous to those required for fulfilling Koch’s postulates. We use fluorescent in situ hybridization and transmission electron microscopy to assess cell structures and interactions; metagenomics to characterize enrichment taxa, generate metagenome assembled genomes, and interrogate Antarctic communities; and proteomics to assess metabolic pathways and speculate about the roles of certain proteins. Metagenome analysis indicates the presence of a single species, which is endemic to Antarctic hypersaline systems that support the growth of haloarchaea. The presence of unusually large proteins predicted to function in attachment and invasion of hosts plus the absence of key biosynthetic pathways (e.g., lipids) in metagenome assembled genomes of globally distributed Nanohaloarchaeota indicate that all members of the lineage have evolved as symbionts. Our work expands the range of archaeal symbiotic lifestyles and provides a genetically tractable model system for advancing understanding of the factors controlling microbial symbiotic relationships.

scholarly journals Microsporidian Infection in a Free-Living Marine Nematode

2012 ◽  
Vol 11 (12) ◽  
pp. 1544-1551 ◽  
Author(s):  
A. M. Ardila-Garcia ◽  
N. M. Fast
Keyword(s):  
Marine Nematode ◽  
Free Living ◽  
Content Type ◽  
Infection Pattern ◽  
Transmission Electron ◽  
Muscle Tissues ◽  
Natural Hosts ◽  
In The Wild ◽  
Microsporidian Infection

ABSTRACT Microsporidia are unicellular fungi that are obligate endoparasites. Although nematodes are one of the most abundant and diverse animal groups, the only confirmed report of microsporidian infection was that of the “nematode killer” ( Nematocida parisii ). N. parisii was isolated from a wild Caenorhabditis sp. and causes an acute and lethal intestinal infection in a lab strain of Caenorhabditis elegans . We set out to characterize a microsporidian infection in a wild nematode to determine whether the infection pattern of N. parisii in the lab is typical of microsporidian infections in nematodes. We describe a novel microsporidian species named Sporanauta perivermis (marine spore of roundworms) and characterize its infection in its natural host, the free-living marine nematode Odontophora rectangula. S. perivermis is not closely related to N. parisii and differs strikingly in all aspects of infection. Examination by transmission electron microscopy (TEM) revealed that the infection was localized in the hypodermal and muscle tissues only and did not involve the intestines. Fluorescent in situ hybridization (FISH) confirmed infection in the muscle and hypodermis, and surprisingly, it also revealed that the parasite infects O. rectangula eggs, suggesting a vertical mode of transmission. Our observations highlight the importance of studying parasites in their natural hosts and indicate that not all nematode-infecting microsporidia are “nematode killers”; instead, microsporidiosis can be more versatile and chronic in the wild.

Transitions in symbiosis: evidence for environmental acquisition & social transmission within a clade of heritable symbionts

2020 ◽  
Author(s):  
Georgia C Drew ◽  
Giles E Budge ◽  
Crystal L Frost ◽  
Peter Neumann ◽  
Stefanos Siozios ◽  
...  
Keyword(s):  
Honey Bee ◽  
Honey Bees ◽  
Fluorescent In Situ Hybridization ◽  
Horizontal Transmission ◽  
Bacterial Symbionts ◽  
Free Living ◽  
Social Contacts ◽  
Obligate Mutualism ◽  
Environmental Microbes

AbstractA dynamic continuum exists from free-living environmental microbes to strict host associated symbionts that are vertically inherited. However, knowledge of the forces that drive transitions in the modes by which symbioses form is lacking. Arsenophonus is a diverse clade of bacterial symbionts, comprising reproductive parasites to coevolving obligate mutualists, in which the predominant mode of transmission is vertical. We describe a symbiosis between a member of the genus Arsenophonus and the Western honey bee. We then present multiple lines of evidence that this symbiont deviates from a heritable model of transmission. Field sampling uncovered marked spatial and seasonal dynamics in symbiont prevalence, and rapid infection loss events were observed in field colonies and individuals in the laboratory. Fluorescent in-situ hybridization showed Arsenophonus localised in the gut, and detection of the bacterium was rare in screens of early honey bee life stages. We directly show horizontal transmission of Arsenophonus between bees under varying social conditions. We conclude that honey bees acquire Arsenophonus through a combination of environmental exposure and social contacts. Together these findings uncover a key link in the Arsenophonus clades trajectory from free-living ancestral life to obligate mutualism, and provide a foundation for studying transitions in symbiotic lifestyle.

Virus-Like Particles in an Ultra-Oligotrophic Lake on Vancouver Island, British Columbia

1990 ◽  
Vol 47 (4) ◽  
pp. 725-730 ◽  
Author(s):  
M. Emilia Klut ◽  
John G. Stockner
Keyword(s):  
British Columbia ◽  
Morphological Changes ◽  
Vancouver Island ◽  
Host Cells ◽  
Variable Size ◽  
Free Living ◽  
Virus Like Particles ◽  
Transmission Electron ◽  
Dense Matrices ◽  
Virogenic Stroma

Transmission electron microscopy (TEM) seasonal studies of concentrated water samples from Sproat Lake, Vancouver Island, British Columbia, revealed numerous polygonal virus-like particles of variable size (60–200 nm). These particles (ca. 107/mL) were either free-living or associated with host picoplankters. Negative staining of living samples provides clear evidence of early stages of phage–picoplankton interactions. These phages display a six-sided head (ca. 90 nm dia.) with a distinct appendage (ca. 200 nm) or striated tail (ca. 130 nm). Viruses with dense matrices, deprived of envelopes or occurring as empty shells were found in the marginal area of invaded cells. Morphological changes such as invagination of the photosynthetic lamellae with the appearance of 'virogenic stroma' or with disruption of the cell membrane and the cell wall are described. Comments on the possible functional significance of viral agents in the biology and ecology of host cells are presented.

scholarly journals Interaction of Pasteurella multocida with Free-Living Amoebae

2005 ◽  
Vol 71 (9) ◽  
pp. 5458-5464 ◽  
Author(s):  
Matthew J. Hundt ◽  
Carmel G. Ruffolo
Keyword(s):  
Pasteurella Multocida ◽  
Fluorescent Protein ◽  
Host Cells ◽  
Free Living ◽  
Fowl Cholera ◽  
Transmission Electron ◽  
Membrane Bound ◽  
Green Fluorescent ◽  
First Time

ABSTRACT Pasteurella multocida is a highly infectious, facultative intracellular bacterium which causes fowl cholera in birds. This study reports, for the first time, the observed interaction between P. multocida and free-living amoebae. Amoebal trophozoites were coinfected with fowl-cholera-causing P. multocida strain X-73 that expressed the green fluorescent protein (GFP). Using confocal fluorescence microscopy, GFP expressing X-73 was located within the trophozoite. Transmission electron microscopy of coinfection preparations revealed clusters of intact X-73 cells in membrane-bound vacuoles within the trophozoite cytoplasm. A coinfection assay employing gentamicin to kill extracellular bacteria was used to assess the survival and replication of P. multocida within amoebae. In the presence of amoebae, the number of recoverable intracellular X-73 cells increased over a 24-h period; in contrast, X-73 cultured alone in assay medium showed a consistent decline in growth. Cytotoxicity assays and microscopy showed that X-73 was able to lyse and exit the amoebal cells approximately 18 h after coinfection. The observed interaction between P. multocida and amoebae can be considered as an infective process as the bacterium was able to invade, survive, replicate, and lyse the amoebal host. This raises the possibility that similar interactions occur in vivo between P. multocida and host cells. Free-living amoebae are ubiquitous within water and soil environments, and P. multocida has been observed to survive within these same ecosystems. Thus, our findings suggest that the interaction between P. multocida and amoebae may occur within the natural environment.

scholarly journals Quantitative Use of Fluorescent In Situ Hybridization To Examine Relationships between Mycolic Acid-Containing Actinomycetes and Foaming in Activated Sludge Plants

2000 ◽  
Vol 66 (3) ◽  
pp. 1158-1166 ◽  
Author(s):  
Russell J. Davenport ◽  
Thomas P. Curtis ◽  
Michael Goodfellow ◽  
Fiona M. Stainsby ◽  
Marc Bingley
Keyword(s):  
In Situ Hybridization ◽  
Activated Sludge ◽  
Fluorescent In Situ Hybridization ◽  
Threshold Value ◽  
Mycolic Acid ◽  
Foam Formation ◽  
Quantification Method ◽  
Nested Analysis ◽  
The Relationship

ABSTRACT The formation of viscous foams on aeration basins and secondary clarifiers of activated sludge plants is a common and widespread problem. Foam formation is often attributed to the presence of mycolic acid-containing actinomycetes (mycolata). In order to examine the relationship between the number of mycolata and foam, we developed a group-specific probe targeting the 16S rRNA of the mycolata, a protocol to permeabilize mycolata, and a statistically robust quantification method. Statistical analyses showed that a lipase-based permeabilization method was quantitatively superior to previously described methods (P << 0.05). When mixed liquor and foam samples were examined, most of the mycolata present were rods or cocci, although filamentous mycolata were also observed. A nested analysis of variance showed that virtually all of the measured variance occurred between fields of view and not between samples. On this basis we determined that as few as five fields of view could be used to give a statistically meaningful sample. Quantitative fluorescent in situ hybridization (FISH) was used to examine the relationship between foaming and the concentration of mycolata in a 20-m3completely mixed activated sludge plant. Foaming occurred when the number of mycolata exceeded a certain threshold value. Baffling of the plant affected foaming without affecting the number of mycolata. We tentatively estimated that the threshold foaming concentration of mycolata was about 2 × 106 cells ml−1 or 4 × 1012 cells m−2. We concluded that quantitative use of FISH is feasible and that quantification is a prerequisite for rational investigation of foaming in activated sludge.

scholarly journals In Vitro Induction of Neospora caninum Bradyzoites in Vero Cells Reveals Differential Antigen Expression, Localization, and Host-Cell Recognition of Tachyzoites and Bradyzoites

2004 ◽  
Vol 72 (1) ◽  
pp. 576-583 ◽  
Author(s):  
Nathalie Vonlaufen ◽  
Nicole Guetg ◽  
Arunasalam Naguleswaran ◽  
Norbert Müller ◽  
Camilla Björkman ◽  
...  
Keyword(s):  
Host Cell ◽  
Cyst Wall ◽  
Neospora Caninum ◽  
Antigen Expression ◽  
Vero Cells ◽  
Dense Granule ◽  
Host Cells ◽  
Transmission Electron ◽  
Intracellular Parasites

ABSTRACT We report on an optimized method for the in vitro culture of tissue cyst-forming Neospora caninum bradyzoites in Vero cells and the separation of viable parasites from host cells. Treatment of tachyzoite-infected Vero cell cultures with 17 μM sodium nitroprusside for 8 days severely scaled down parasite proliferation, led to reduced expression of tachyzoite surface antigens, and induced the expression of the bradyzoite marker NcBAG1 and the cyst wall antigen recognized by the monoclonal antibody MAbCC2. Transmission electron microscopy demonstrated that intracellular parasites were located within parasitophorous vacuoles that were surrounded by a cyst wall-like structure, and the dense granule antigens NcGRA1, NcGRA2, and NcGRA7 were incorporated into the cyst wall. Adhesion-invasion assays employing purified tachyzoites and bradyzoites showed that tachyzoites adhered to, and invaded, Vero cells with higher efficiency than bradyzoites. However, removal of terminal sialic acid residues from either the host cell or the parasite surface increased the invasion of Vero cells by bradyzoites, but not tachyzoites.

The Use of Advanced Analytical Techniques for Characterization of the Chemical, Physical, and Crystallographic Nature of a Surface

1973 ◽  
Vol 31 ◽  
pp. 132-133 ◽  
Author(s):  
R. E. Herfert
Keyword(s):  
Surface Characterization ◽  
Analytical Techniques ◽  
X Ray Diffraction ◽  
Depth Of Penetration ◽  
X Ray ◽  
The Past ◽  
Transmission Electron ◽  
Scanning Electron

Studies of the nature of a surface, either metallic or nonmetallic, in the past, have been limited to the instrumentation available for these measurements. In the past, optical microscopy, replica transmission electron microscopy, electron or X-ray diffraction and optical or X-ray spectroscopy have provided the means of surface characterization. Actually, some of these techniques are not purely surface; the depth of penetration may be a few thousands of an inch. Within the last five years, instrumentation has been made available which now makes it practical for use to study the outer few 100A of layers and characterize it completely from a chemical, physical, and crystallographic standpoint. The scanning electron microscope (SEM) provides a means of viewing the surface of a material in situ to magnifications as high as 250,000X.

In situ REM imaging of surface processes on ceramic bulk crystals from 300 to 1670 K in a conventional TEM

1991 ◽  
Vol 49 ◽  
pp. 660-661
Author(s):  
Z. L. Wang ◽  
J. Bentley
Keyword(s):  
High Temperatures ◽  
Image Resolution ◽  
Atomic Processes ◽  
Crystal Surfaces ◽  
Beam Radiation ◽  
Surface Areas ◽  
Transmission Electron ◽  
Reflection Electron Microscopy ◽  
Gas Reactions

Studying the behavior of surfaces at high temperatures is of great importance for understanding the properties of ceramics and associated surface-gas reactions. Atomic processes occurring on bulk crystal surfaces at high temperatures can be recorded by reflection electron microscopy (REM) in a conventional transmission electron microscope (TEM) with relatively high resolution, because REM is especially sensitive to atomic-height steps.Improved REM image resolution with a FEG: Cleaved surfaces of a-alumina (012) exhibit atomic flatness with steps of height about 5 Å, determined by reference to a screw (or near screw) dislocation with a presumed Burgers vector of b = (1/3)<012> (see Fig. 1). Steps of heights less than about 0.8 Å can be clearly resolved only with a field emission gun (FEG) (Fig. 2). The small steps are formed by the surface oscillating between the closely packed O and Al stacking layers. The bands of dark contrast (Fig. 2b) are the result of beam radiation damage to surface areas initially terminated with O ions.

Role of boundaries in the crystallization of amorphous films

1988 ◽  
Vol 46 ◽  
pp. 626-627
Author(s):  
D. A. Smith
Keyword(s):  
Low Temperature ◽  
Amorphous State ◽  
Nucleation And Growth ◽  
Amorphous Films ◽  
Island Nucleation ◽  
Surface Steps ◽  
Transmission Electron ◽  
Component Systems ◽  
Temperature Deposition

The nucleation and growth processes which lead to the formation of a thin film are particularly amenable to investigation by transmission electron microscopy either in situ or subsequent to deposition. In situ studies have enabled the observation of island nucleation and growth, together with addition of atoms to surface steps. This paper is concerned with post-deposition crystallization of amorphous alloys. It will be argued that the processes occurring during low temperature deposition of one component systems are related but the evidence is mainly indirect. Amorphous films result when the deposition conditions such as low temperature or the presence of impurities (intentional or unintentional) preclude the atomic mobility necessary for crystallization. Representative examples of this behavior are CVD silicon grown below about 670°C, metalloids, such as antimony deposited at room temperature, binary alloys or compounds such as Cu-Ag or Cr O2, respectively. Elemental metals are not stable in the amorphous state.

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