An increase in cases of acute febrile encephalitis occurred in Western Malaysia between September 1998 and May 1999, and a similar illness was reported in Singapore in March 1999. Most cases occurred in males who had been exposed to pigs, or among abattoir workers, and at least 100 deaths were reported. The illness was characterized by fever and headache, followed by drowsiness and disorientation; patients with severe cases developed seizures and coma within 24 to 48 hours. Concurrently, there were also illnesses and deaths among pigs in the same region, although the symptoms predominantly involved the respiratory system, and only a few pigs had signs of neurologic disease. From a cerebral spinal fluid specimen from a human patient, Dr. Chua and colleagues were successful in isolating a virus that was morphologically identified as belonging to the family Paramyxoviridae, and is now known as Nipah virus.

Transmissible encephalopathy of animals (scrapie and bovine spongiform encephalopathy) and of man (Creutzfeldt-Jacob disease, new variant Creutzfeldt-Jacob disease, Kuru, Gerstmann-Straussler-Scheinker disease and familial fatal insomnia) have been well characterized as progressive neurodegenerative diseases, often associated with spongiform degeneration, neuronal loss, reactive astrocytic gliosis and variable amyloid plaques, without any sign of an immune response or inflammatory infiltrates. Prion proteins are elements that propagate variability through multiple biologically active conformers of a host encoded sialoglycoprotein (PrPc). The agent responsible for transmissible encephalopathies is composed mainly, if not exclusively, of an isoform of PrP, designated PrPSc. Extensive information of post-mortem material has been described at the light microscopic level, where the emphasis has been on spongiform tissue and amyloid plaques. Our study provides ultrastructural observations of the central nervous system, specifically of degenerating neurons and their associated changes during the course of scrapie infection in a hamster model.

The protozoan parasite Toxoplasma gondii represents a large group of Apicomplexan parasites with a highly unusual motility system that is crucial for cell locomotion and host cell invasion. Studying the motility system and interactions with host cells will increase our knowledge on how to prevent infection. Apicomplexan parasites can cause considerable health problems to animals including sheep, goats, pigs, and chicken. Eimeria is known as a pathogen of coccidiosis in chicken, and Cryptosporidium causes cryptospiridiosis in cattle and other farm animals. Toxoplasma gondii can cause infection that results in abortion, central nervous disorders and death of stressed and immunocompromised farm animals. Common to all Apicomplexan parasites is an unconventional motility system that is thought to be actively involved in host-cell invasion. However, the structural and biochemical characterization of Apicomplexan parasites has proven more difficult than previously thought because of genetic and behavioral differences of the parasites’ cytoskeletal proteins that differ from those in well studied mammalian systems.

Hepatitis C virus (HCV) is an RNA virus of the family Flaviviridae. In the decade since its identification,1 it has emerged as a leading cause of chronic hepatitis and cirrhosis. Easily transmissible by parenteral and sexual routes, fastidious in culture, highly limited in host range, and difficult to detect by most microscopic and immunohistochemical methods, HCV qualifies as an extreme pathogen. Another form of extremity exhibited by HCV is the bewildering array of renal disorders that occur in the setting of HCV-induced hepatitis.

Several different mechanisms may underlie the development of renal disease in patients infected with HCV. Immune complexes of anti-HCV antibodies and viral components, frequently bound to anti-immunoglobulin rheumatoid factors, can form in the circulation and be deposited in the kidney. HCV has also been shown to induce immune responses to a variety of autoantigens, some of which are expressed in the kidney. Hepatic injury caused by HCV may produce downstream effects on other organs, including the kidney.

Biofilms are collections of microorganisms at interfaces. Because of the high proportion of interfacial regions in the natural environment, biofilms are likely to be the repository for most of the world's microbial biomass. The close proximity of organisms within the biofilm presents the opportunity for intra- and interspecies signaling on a physical level through direct contact of organisms with one another, and on a metabolic level through production/excretion of intermediary molecules of metabolism and of specific cell signaling molecules. One of the best studied biofilms is dental plaque, which has been investigated from a microscopy standpoint since the time of van Loewenhouk and from a classical bacteriological standpoint since the relatively recent (20th century) development of anaerobic culture techniques. Through the even more recent (the last decade) application of molecular methods, it has been shown that perhaps half of all bacteria present in plaque are culturable and, at present, this consists of some 500 species.

One-third of the worldwide sodium chloride (salt) production (about 200 million tons per year) is manufactured in solar saltworks. A solar saltworks is a series of one or more interconnected, shallow concentrating ponds (40 to 70 cm deep) through which seawater flows, evaporates by wind and sun, and deposits sodium chloride on the floors of crystallizer ponds (30 cm to 50 cm deep). In the downstream flow, salts of low solubility (in respect to sodium chloride) precipitate, with calcium carbonate first dropping out near three times, and calcium sulfate (gypsum) first appearing near four times seawater salinity. Shortly before reaching saturation with sodium chloride (about S.G. 1.214), the brine flows into crystallizer ponds (crystallizers), where evaporation continues until 5 to 20 cm of sodium chloride is deposited on the floors. The supernatant liquid, rich in compounds of calcium, magnesium, chloride, and sulfate, is removed, and the salt is harvested, washed, stockpiled for a time to decrease contaminants, and marketed.

A seldom studied and poorly understood feature of cyanobacterial growth and development is the formation of ordered macroscopic structures from microscopic cells, trichomes, and filaments. The forms range from nostoc balls and layered mats of comparatively simple structure to more complex erect tree-like (dendroid) macro-structures. Using macrophotography, time lapse micro-cinematography, light and scanning electron microscopy of Nostoc species in pure culture, it has been possible to show how motility, adhesion and aggregation of photo-induced motile trichomes (hormogonia) result in the macro-morphogenesis of dendroid forms.

Nostocacean development proceeds by the differentiation of cells in a cyclic sequence of stages including: long chains of cells in heterocystous filaments, motile hormogonia, and large rounded cells termed the aseriate phase. When grown from localized inocula on solid media, sequential development recycles across the agar surface of the culture. Different strains and species vary in the form and duration of each stage and the photo-response of hormogonia.

Automobile air conditioning systems (ACS) might be considered an extreme environment for many microorganisms. Organisms surviving and proliferating in these systems may be presented with temperature changes ranging from subzero to over 140°F, water activity from saturation to dryness and a nutrient complexity including varying levels of hydrocarbons. Microbial communities develop in ACS and sometimes proliferate to the extent of massive colonization and production of objectionable odors.

In a few instances microorganisms emanating from ACS have been associated with hypersensitivity pneumonitis and other allergic reactions. Previously we demonstrated that foam insulation and glues, in particular, on ACS insulations were colonized by fungi such as Aspergillus, Aureobasidium, Cladosporium, and Penicillium. Such fungi often are implicated in colonization of similar substrates in buildings categorized with the sick building syndrome.

A total of 42 ACS from the United States, Europe and Central America have been examined by cabin air sampling culture swab, direct microscopy or a combination of these techniques.

The small (0.5-0.75 um) Clostridial endospores are highly resistant to solvents, acids, bases, radiation, heat, cold, pressure, vacuum and most sporicides. C.difficile causes antibiotic induced pseudomembranous colitis, and is responsible for almost 20% of nosocomial diarrheas in todays hospitals. These endospores have been found on hospital floors, sheets and bedding, as well as on the hands and white coats of hospital personnel.

Our previous studies revealed that these endospores attach to agar by using a two phase attachment process. To examine if this mechanism for spore attachment is also responsible for C.difficile pathogenesis in the human colon, spore attachment and invasion of monolayers of human colon carcinoma cells (Caco-2 cells) using two strains of C.difficile,(ATCC 43594 an isolate from a patient with pseudomembranous colitis [FIG.1A] and ATCC 9689 a clinical microbology laboratory quality control strain) was studied. Cell monolayers grown on 6mm coverslips were inoculated with 10 ul of spore suspension (0.5 McFarland turbidity in sterile water),

Rickettsias are small, gram-negative Alpha Proteobacteria that are, with one exception, obligate intracellular parasites. They are found in a wide range of arthropods. Some of these bacteria are arthropod-vectored disease agents of vertebrates. For example, rickettsias are the causative agents of human typhus and Rocky Mountain spotted fever. Others, such as Wolbachia, are important manipulators of their arthropod host's reproductive capabilities and are not currently known to be disease agents of vertebrates.

Research on Wolbachia and related rickettsias has increased dramatically in recent years. Most of the attention has been focused on insects, although there are occasional reports of rickettsiae being found in isopods and mites. Most recently, the use of molecular techniques has established that Wolbachia is widespread in spider and predatory mites. However, Wolbachia or other rickettsias have not been demonstrated ultrastructurally in the spider mites. This report documents the presence of a rickettsia in the ovary of the adult female twospotted spider mite, Tetranchus urticae Koch, an economically important pest of ornamental and crop plants.

In the yeast Saccharomyces cerevisiae, [PST] acts as an epigenetic modifier of translation termination efficiency. [PSI+] can be passed through generations of yeast cells via changes in protein conformation rather than changes in DNA or RNA, and has thus been referred to as a yeast prion. The [PSI+] determinant is the Sup35 protein. Sup35 can exist in two states - soluble and insoluble. Soluble Sup35 functions in translation termination, but when insoluble, stop codons are read through, resulting in incorrect protein products.

Sup35 is composed of three distinct domains, N, M, and C. The N region is rich in glutamine and asparagine and is required for the [PST] phenotype to exist. M is a highly charged domain, and no specific function has been assigned to it. C is essential in yeast, as it is responsible for translation termination. The insoluble form of Sup35 has characteristics reminiscent of other prion proteins - in vitro it binds to the dye Congo Red and it exhibits apple green birefringence in polarized light.

Economically important insects include pests of plants, animals and stored products as well as insects produced commercially (honey bees, silkworms, insects for fish bait and food for birds and zoo animals). Other insects are produced in large numbers for experimental purposes, biological control of insect pests and weeds and the production of sterile insects for population suppression. Insect viruses may affect morphology, physiology and behavior, often reducing longevity and reproductive potential. Thus insect viruses can be used as biological control agents of pest insects. Insect viruses may also interfere with the production or function of biological control agents (e.g., parasitoids and predators) and insects used for research purposes. Therefore, it is advantageous to screen commercial and research colonies and imported insects for viruses.

Electron microscopy of negatively stained specimens (NS EM) offers a relatively rapid and inexpensive means of screening populations of insects for the presence of viruses or viruslike particles and to monitor the progress of virus control programs.

Previously, we uncovered evidence of an infectious agent, Chlamydia pneumoniae, associated with sporadic Alzheimer's disease (AD). We demonstrated that C. pneumoniae was present in 90% of brain materials examined from AD patients, compared with 5% in age-matched, non-AD patients. We further showed that microglia and astroglia in the CNS are host cells for the bacterium. RT-PCR analyses indicated that the bacterium is metabolically-active in the brain and could be cultured in a human monocyte cell line (THP-1). Importantly, we showed that the organism is concentrated in areas of AD-neuropathology also containing tau and beta-amyloid. The present study was designed to investigate the effects of C. pneumoniae infection on beta amyloid production in human monocytes (THP-1), astroglial (CCF-STTG1), epithelial (Hep 2), and endothelial (HBMEC) cell lines. We investigated the host pathogen relationship with C. pneumoniae and whether entities characteristic of AD neuropathology such as betaamyloid are associated directly with this infectious agent.

The purpose of this work was to examine, by electron microscopy, the mechanism of bacterial binding to oral epithelium in patients with stomatitis using ruthenium red in combination with osmium tetroxide.

Bacteriological semi-quantitative estimation of smear from the palatal mucosa was performed in patients with stomatitis and only patients with viridans Streptococci were further included in the present investigation. Biopsies were taken from the inflamed keratinizing mucosa under regional block anesthesia in agreement with Vancouver guidelines 1978. Control samples were fixed with 1.2% glutaraldehyde (buffered at pH 6.5 with 0.1 M sodium cacodylate) for 2 h at 4° C. Postfixation was performed with 1% osmium tetroxide (buffered at pH 6.5 with 0.1 M sodium cacodylate) for 2 h at 4° C. The other samples were fixed and postfixed by the fixatives mentioned before with addition of 1.6 % ruthenium red in each case.

The eastern coast of north central Florida is home to a habitat unique in the southeastern United States. Some areas of the north to north-central Florida beaches consist of outcroppings of coquina, a beach rock formed by shell fragments and sand held together by a calcareous cement. The major constituents of coquina are shell fragments of the surf clam Donax variabilis, while fragments of Area sp. and Dinocardium sp. are also generally present. These same shell fragments are also found loose comprising most of the beach “sand” in this area. The coquina serves as a substrate for a variety of attatched seaweeds and fouling organisms. Microalgae and cyanophytes of interest to this study can be classified as either epilithic or endolithic. Epilithic microorganisms inhabit the surface, using the rock primarily as a site of attatchment. The endolithic microorganisms are those algae and cyanophytes that actively bore into the coquina rock's shell fragments.

Cryo techniques of specimen preparation have become the standard for cytological studies of biological specimens. From the pioneering work of Feder and Sidman, and Zalokar, the now ubiquitous application of freeze substitution for transmission electron microscope studies of fungi was a direct result of work reported in 1979. Since then, cryopreparative methods have also become the standard for SEM studies and may, for certain purposes, replace conventional methods of fixation for light microscopy as well. Undoubtedly, there are instances where non-cryo methods might be preferred, or where comparisons of results using cryo vs. chemical fixations can provide unique information. On the whole, however, the advantages offered to mycologists and plant pathologists by cryo fixation over any chemical methods are many, and include (a) the opportunity to preserve specimens in a non-aqueous environment, (b) preservation of labile structures such as certain organelles, extracellular matrices, or various cellular content such as ions or that of vacuoles, (c) preservation of cells in a more life-like state,

Allomyces macrogynus is a zoosporic fungus (Chytridiomycota) that exhibits a true alternation of generations between the diploid and haploid thalli. During the diploid stage in the life cycle, sporothalli are produced which are capable of forming asexual zoosporangia containing a multinucleate protoplast bound by a single plasma membrane and cell wall. Though able to remain in a quiescent, coenocytic state for an extended period of time, under certain environmental conditions the cytoplasm of mature zoosporangia is cleaved and gives rise to numerous uninucleate, uniflagellate zoospores within 40 to 50 min. Previous studies of cleavage membrane development m Allomyces zoosporangia have been conducted using light microscopy and transmission electron microscopy (TEM). Despite these investigations, our understanding of cytokinesis in zoosporangia of Allomyces and other zoosporic organisms remains incomplete. This is due, in part, to the difficulty of detecting cleavage elements using standard light microscope optics (i.e., phase contrast, differential interference contrast) and because ultrastructural analysis often provides limited temporal and spatial detail and may suffer from artifacts of sample preparation.

Low temperature, low voltage field emission scanning electron microscopy (cryoSEM) allows brilliant imaging of superbly preserved biological material without suffering the specimen destruction inherent in conventional SEM techniques. Confocal laser scanning microscopy (CLSM) allows observation of fluorescence-labeling within host tissues, utilizing the optical sectioning capability of the CLSM to construct composite stereo images of specimens in three dimensions. These technologies were invaluable in elucidating the septoria tritici blotch infection process on wheat, caused by Mycosphaerella graminicola (anamorph Septoria tritici).

Seedlings inoculated with M. graminicola were incubated for the first 48 h in a dew chamber with constant misting, 20°C days and 16°C nights, then transferred to a growth chamber under the same temperature conditions at 90% RH until development of mature pycnidia, visible macroscopically at 11 days post-inoculation. Samples for cryoSEM were attached to specimen blocks with Histo Prep (Fischer Scientific, Orangeburg, NY) and frozen by plunging blocks into liquid nitrogen slush.

Specific gene products of both pathogens and hosts have been implicated as decisive elements during plant pathogenesis. While expression of some of these genes is constitutive, that of others is likely ephemeral and activated only during a particular stage of the interaction. Because the relative timing of expression may be critical, transcription and translation have often been addressed by extracting mRNA and proteins from infected plant tissue. This approach, however, cannot readily detect proteins of low abundance in bulk samples nor offer much useful information on cell-cell interaction. Only a cytological analysis that employs microscopy can resolve the temporal and spatial details of gene expression. Typically, such protein localization studies have required specific antibodies, but these large probe molecules do not diffuse into living or conventionally fixed cells of either fungal pathogens or plant hosts. For TEM analysis, these permeability-imposed limitations have been reduced by thin sectioning to render accessible antibody binding sites.

The high-pressure freezing instrument exposes a sample to a pressure of 2100 bar, which lowers the freezing point and, as a result, reduces the rate of ice nucleation and ice-crystal growth. The reduced critical cooling rate allows adequate freezing of samples up to 0,3 mm in thickness without using cryoprotectants. Before pressure application, the sample is sandwiched between specimen holders. To optimize heat conductivity and to avoid damage by the high pressure impact, the free space inside the specimen holders and within the sample has to be filled with liquid. This means that plant leaves need to be infiltrated to remove gas from the intercellular space. We have used water, 3-8% methanol in water, 1-hexadecene or heptane as infiltration medium. Subsequently, samples were freeze substituted in unhydrous acetone with 2% Os04 for 24 h at -90°C. Samples were slowly warmed up to 4°C and embedded in Unicryl, or warmed up to room temperature and embedded in epoxide resin.