Identification and characterization of two subpopulations of Encephalitozoon intestinalis

Microsporidia are obligate intracellular protozoa that have been shown to be pathogenic to most living creatures. The development of in vitro cell culture propagation methods has provided researchers with large numbers of spores and facilitated the study of these organisms. Here, we describe heterogeneity within cell culture-propagated Encephalitozoon intestinalis suspensions. Flow cytometer histograms depicting the log side scatter and forward-angle light scatter of spores from nine suspensions produced over 12 months consistently showed two populations differing in size. The suspensions were composed primarily of the smaller-spore subpopulation (76.4% +/- 5.1%). The presence of two subpopulations was confirmed by microscopic examination and image analysis (P < 0.001). Small subpopulation spores were noninfectious in rabbit kidney (RK13) cell culture infectivity assays, while the large spores were infectious when inocula included > or = 25 spores. The small spores stained brilliantly with fluorescein isothiocyanate-conjugated monoclonal antibody against Encephalitozoon genus spore wall antigen, while the large spores stained poorly. There was no difference in staining intensities using commercial (MicroSporFA) and experimental polyclonal antibodies. Vital-dye (DAPI [4',6'-diamidino-2-phenylindole], propidium iodide, or SYTOX Green) staining showed the spores of the small subpopulation to be permeable to all vital dyes tested, while spores of the large subpopulation were not permeable in the absence of ethanol pretreatment. PCR using primers directed to the 16S rRNA or beta-tubulin genes and subsequent sequence analysis confirmed both subpopulations as E. intestinalis. Our data suggest that existing cell culture propagation methods produce two types of spores differing in infectivity, and the presence of these noninfective spores in purified spore suspensions should be considered when designing disinfection and drug treatment studies.     

A Spore Counting Method and Cell Culture Model for Chlorine Disinfection Studies of Encephalitozoon syn. Septata intestinalis

The microsporidia have recently been recognized as a group of pathogens that have potential for waterborne transmission; however, little is known about the effects of routine disinfection on microsporidian spore viability. In this study, in vitro growth of Encephalitozoon syn. Septata intestinalis, a microsporidium found in the human gut, was used as a model to assess the effect of chlorine on the infectivity and viability of microsporidian spores. Spore inoculum concentrations were determined by using spectrophotometric measurements (percent transmittance at 625 nm) and by traditional hemacytometer counting. To determine quantitative dose-response data for spore infectivity, we optimized a rabbit kidney cell culture system in 24-well plates, which facilitated calculation of a 50% tissue culture infective dose (TCID₅₀) and a minimal infective dose (MID) for E. intestinalis. The TCID₅₀ is a quantitative measure of infectivity and growth and is the number of organisms that must be present to infect 50% of the cell culture wells tested. The MID is as a measure of a system's permissiveness to infection and a measure of spore infectivity. A standardized MID and a standardized TCID₅₀ have not been reported previously for any microsporidian species. Both types of doses are reported in this paper, and the values were used to evaluate the effects of chlorine disinfection on the in vitro growth of microsporidia. Spores were treated with chlorine at concentrations of 0, 1, 2, 5, and 10 mg/liter. The exposure times ranged from 0 to 80 min at 25°C and pH 7. MID data for E. intestinalis were compared before and after chlorine disinfection. A 3-log reduction (99.9% inhibition) in the E. intestinalis MID was observed at a chlorine concentration of 2 mg/liter after a minimum exposure time of 16 min. The log₁₀ reduction results based on percent transmittance-derived spore counts were equivalent to the results based on hemacytometer-derived spore counts. Our data suggest that chlorine treatment may be an effective water treatment for E. intestinalis and that spectrophotometric methods may be substituted for labor-intensive hemacytometer methods when spores are counted in laboratory-based chlorine disinfection studies.     

Soybean and sunflower oils increase the infectivity of Colletotrichum gloeosporioides f. sp. aeschynomene to Northern Jointvetch

Soybean and sunflower oils increased the level of infection of northern jointvetch, Aeschynomene virginica, plants by Colletotrichum gloeosporioides f. sp. aeschynomene. Inoculation of seedlings with spore suspensions containing 10% (v:v) soybean oil or 10% sunflower oil resulted in more disease than when inoculated with suspensions of spores in water alone. The lengths of the dew periods required to establish equivalent levels of disease by spore suspensions containing 10% soybean or 10% sunflower oil were approximately 4–8 h less compared to aqueous suspensions. Incubation of spores in 10% soybean oil followed by removal and resuspension in water did not affect the infectivity of spores when compared to spores incubated in aqueous suspensions. Spore germination and appressoria formation were unaffected by either of the oils tested in in vitro assays; however, in in vivo assays, 10% soybean oil and 10% sunflower oil increased spore germination in comparison to spores that were suspended in water.     

Determination of the Biomasses of Small Bacteria at Low Concentrations in a Mixture of Species with Forward Light Scatter Measurements by Flow Cytometry

The forward light scatter intensity of bacteria analyzed by flow cytometry varied with their dry mass, in accordance with theory. A standard curve was formulated with Rayleigh-Gans theory to accommodate cell shape and alignment. It was calibrated with an extinction-culture isolate of the small marine organism Cycloclasticus oligotrophus, for which dry weight was determined by CHN analysis and ¹⁴C-acetate incorporation. Increased light scatter intensity due to formaldehyde accumulation in preserved cells was included in the standard curve. When differences in the refractive indices of culture media and interspecies differences in the effects of preservation were taken into account, there was agreement between cell mass obtained by flow cytometry for various bacterial species and cell mass computed from Coulter Counter volume and buoyant density. This agreement validated the standard curve and supported the assumption that cells were aligned in the flow stream. Several subpopulations were resolved in a mixture of three species analyzed according to forward light scatter and DNA-bound DAPI (4′,6-diamidino-2-phenylindole) fluorescence intensity. The total biomass of the mixture was 340 μg/liter. The lowest value for mean dry mass, 0.027 ± 0.008 pg/cell, was for the subpopulation of C. oligotrophus containing cells with a single chromosome. Calculations from measurements of dry mass, Coulter Counter volume, and buoyant density revealed that the dry weight of the isolate was 14 to 18% of its wet weight, compared to 30% for Escherichia coli. The method is suitable for cells with 0.005 to about 1.2 pg of dry weight at concentrations of as low as 10³ cells/ml and offers a unique capability for determining biomass distributions in mixed bacterial populations.     

Effect of Batch-Process Solar Disinfection on Survival of Cryptosporidium parvum Oocysts in Drinking Water

The results of batch-process solar disinfection (SODIS) of Cryptosporidium parvum oocysts in water are reported. Oocyst suspensions were exposed to simulated sunlight (830 W m⁻²) at 40°C. Viability assays (4′,6′-diamidino-2-phenylindole [DAPI]/propidium iodide and excystation) and infectivity tests (Swiss CD-1 suckling mice) were performed. SODIS exposures of 6 and 12 h reduced oocyst infectivity from 100% to 7.5% (standard deviation = 2.3) and 0% (standard deviation = 0.0), respectively.     

Survival of Clostridium botulinum Spores

Radiation survival curves of spores of Clostridium botulinum strain 33A exhibited an exponential reduction which accounted for most of the population, followed by a “tail” comprising a very small residual number [7 to 0.7 spore(s) per ml] which resisted death in the range between 3.0 and 9.0 Mrad dose levels. The “tail” was not caused by protective spore substances released into the suspensions during irradiation, by the presence of accumulated radiation “inactivated” spores, or by heat shock of pre-irradiated spores. The theoretical number of spore targets which must be inactivated by irradiation was estimated both by a graphical and by a computation method to be about 80, and the D value was calculated to be 0.295 and 0.396 Mrad, respectively, in buffer and in pork pea broth.     

Heat Resistance Correlated with DNA Content in Bacillus megaterium Spores

Two subpopulations of Bacillus megaterium spores (1.360 and 1.355 g/ml) were obtained by density gradient centrifugation. The heavier spores had a higher thermoresistance (e.g., D₈₀ = 186 versus 81 min) and a higher DNA content (1.25 × 10⁻¹⁴ versus 0.65 × 10⁻¹⁴ g per spore, apparently corresponding to digenomic versus monogenomic spores). No appreciable differences were found in the mineral and dipicolinic acid contents or in the inactivation kinetics of the two subpopulations. The implications of the findings are discussed with regard to mechanisms of heat resistance and of inactivation.     

Dual-parameter scatter-flow immunofluorescence analysis of Bacillus spores

Using a commercial flow cytometer (Cyto-fluorograf), narrow-forward-angle (NFA) light-scatter signals were detected for spore preparations of Bacillus anthracis Vollum, B. anthracis Sterne, B. cereus NCTC 8035, and B. subtilis var niger. In the flow immunofluorescence (FIF) analysis of spores stained with fluorescein-conjugated hyperimmune antibody to B. anthracis Vollum spores, fluorescence histograms could be acquired by selecting on NFA scatter. Fluorescence data selected on ninety degree scatter were rather noisier. Fluorescence analysis by dual parameter NFA scatter-FIF techniques was shown to have several advantages over the subtraction FIF method reported earlier. The implication from FIF analysis of spore suspensions and corresponding cell-free supernatants that the peak in the fluorescence histogram was caused by signals from fluorescing spores, was confirmed by use of the cell sorter and subsequent microscopy of the sorted samples. Although a proportion of spore aggregates was present in samples sorted from the right-hand tail of the fluorescence histogram, it was demonstrated that the majority of the observed distribution of fluorescence was not due to the formation of aggregates but was rather an expression of variation in the degree of staining of individual spores.     

Cyclic Guanosine 3′,5′-Monophosphate in the Dimorphic Fungus Mucor racemosus

The dimorphic fungus Mucor racemosus was found to contain the cyclic nucleotide guanosine 3′,5′-monophosphate (cGMP). Approximately equivalent amounts of the compound were found in ungerminated spores, yeastlike cells, and mycelia. Germinating spores contained severalfold higher amounts of cGMP than the other cell forms. cGMP levels did not change significantly during the morphogenetic conversion of yeast to mycelia. Added exogenous cGMP or the dibutyryl derivative did not influence cell morphology in any way and did not alter the effect that cyclic adenosine 3′,5′-monophosphate has upon cell morphology.     

Uptake of and Resistance to the Antibiotic Berberine by Individual Dormant,Germinating and Outgrowing Bacillus Spores as Monitored by Laser Tweezers Raman Spectroscopy

Berberine, an alkaloid originally extracted from the plant Coptis chinensis and other herb plants, has been used as a pharmacological substance for many years. The therapeutic effect of berberine has been attributed to its interaction with nucleic acids and blocking cell division. However, levels of berberine entering individual microbial cells minimal for growth inhibition and its effects on bacterial spores have not been determined. In this work the kinetics and levels of berberine accumulation by individual dormant and germinated spores were measured by laser tweezers Raman spectroscopy and differential interference and fluorescence microscopy, and effects of berberine on spore germination and outgrowth and spore and growing cell viability were determined. The major conclusions from this work are that: (1) colony formation from B. subtilis spores was blocked ~ 99% by 25 μg/mL berberine plus 20 μg/mL INF55 (a multidrug resistance pump inhibitor); (2) 200 μg/mL berberine had no effect on B. subtilis spore germination with L-valine, but spore outgrowth was completely blocked; (3) berberine levels accumulated in single spores germinating with ≥ 25 μg/mL berberine were > 10 mg/mL; (4) fluorescence microscopy showed that germinated spores accumulated high-levels of berberine primarily in the spore core, while dormant spores accumulated very low berberine levels primarily in spore coats; and (5) during germination, uptake of berberine began at the time of commitment (T₁) and reached a maximum after the completion of CaDPA release (T_release) and spore cortex lysis (T_lysis).     

Spore Yield and Microcycle Conidiation of Colletotrichum gloeosporioides in Liquid Culture

The effect of V8 juice concentration (5 to 40%, vol/vol), spore inoculum density (10⁵ and 10⁷ spores per ml), and liquid batch or fed-batch culture condition on mycelium and spore production by Colletotrichum gloeosporioides was evaluated. The amount of mycelium produced, the time required for initiation of sporulation following attainment of maximum mycelium, and the time for attainment of maximum spore concentration increased with increasing V8 juice concentration in batch culture. Cultures containing V8 juice at >10% achieved a similar spore density (apparent spore-carrying capacity) of about 0.8 mg of spores per ml (1 × 10⁷ to 2 × 10⁷ spores per ml) independent of inoculum density and V8 juice concentration. The relative spore yield decreased from a high of 64% of the total biomass for the low-inoculum 5% V8 culture, through 13% for the analogous 40% V8 culture, to a low of 2% for the high-inoculum 27% V8 culture. Fed-batch cultures were used to establish conditions of high spore density and low substrate availability but high substrate flux. The rate of addition of V8 juice was adjusted to approximate the rate of substrate utilization by the (increasing) biomass. The final spore concentration was about four times higher (3.0 mg of spores per ml) than the apparent spore-carrying capacity in batch culture. This high spore yield was obtained at the expense of greatly reduced mycelium, resulting in a high relative spore yield (62% of the total biomass). Microcycle conidiation occurred in the fed-batch but not batch systems. These data indicate that substrate-limited, fed-batch culture can be used to increase the amount and efficiency of spore production by C. gloeosporioides by maintaining microcycle conidiation conditions favoring allocation of nutrients to spore rather than mycelium production.     

The fission yeast spore is coated by a proteinaceous surface layer comprising mainly Isp3

The spore is a dormant cell that is resistant to various environmental stresses. As compared with the vegetative cell wall, the spore wall has a more extensive structure that confers resistance on spores. In the fission yeast Schizosaccharomyces pombe, the polysaccharides glucan and chitosan are major components of the spore wall; however, the structure of the spore surface remains unknown. We identify the spore coat protein Isp3/Meu4. The isp3 disruptant is viable and executes meiotic nuclear divisions as efficiently as the wild type, but isp3∆ spores show decreased tolerance to heat, digestive enzymes, and ethanol. Electron microscopy shows that an electron-dense layer is formed at the outermost region of the wild-type spore wall. This layer is not observed in isp3∆ spores. Furthermore, Isp3 is abundantly detected in this layer by immunoelectron microscopy. Thus Isp3 constitutes the spore coat, thereby conferring resistance to various environmental stresses.     

Genetic interactions of smc, ftsK, and parB genes in Streptomyces coelicolor and their developmental genome segregation phenotypes

The mechanisms by which chromosomes condense and segregate during developmentally regulated cell division are of interest for Streptomyces coelicolor, a sporulating, filamentous bacterium with a large, linear genome. These processes coordinately occur as many septa synchronously form in syncytial aerial hyphae such that prespore compartments accurately receive chromosome copies. Our genetic approach analyzed mutants for ftsK, smc, and parB. DNA motor protein FtsK/SpoIIIE coordinates chromosome segregation with septum closure in rod-shaped bacteria. SMC (structural maintenance of chromosomes) participates in condensation and organization of the nucleoid. ParB/Spo0J partitions the origin of replication using a nucleoprotein complex, assembled at a centromere-like sequence. Consistent with previous work, we show that an ftsK-null mutant produces anucleate spores at the same frequency as the wild-type strain (0.8%). We report that the smc and ftsK deletion-insertion mutants (ftsK′ truncation allele) have developmental segregation defects (7% and 15% anucleate spores, respectively). By use of these latter mutants, viable double and triple mutants were isolated in all combinations with a previously described parB-null mutant (12% anucleate spores). parB and smc were in separate segregation pathways; the loss of both exacerbates the segregation defect (24% anucleate spores). For a triple mutant, deletion of the region encoding the FtsK motor domain and one transmembrane segment partially alleviates the segregation defect of the smc parB mutant (10% anucleate spores). Considerable redundancy must exist in this filamentous organism because segregation of some genomic material occurs 90% of the time during development in the absence of three functions with only a fourfold loss of spore viability. Furthermore, we report that scpA and scpAB mutants (encoding SMC-associated proteins) have spore nucleoid organization defects. Finally, FtsK-enhanced green fluorescent protein (EGFP) localized as bands or foci between incipient nucleoids, while SMC-EGFP foci were not uniformly positioned along aerial hyphae, nor were they associated with every condensing nucleoid.     

Microsporidian Species Known To Infect Humans Are Present in Aquatic Birds: Implications for Transmission via Water?

Human microsporidiosis, a serious disease of immunocompetent and immunosuppressed people, can be due to zoonotic and environmental transmission of microsporidian spores. A survey utilizing conventional and molecular techniques for examining feces from 570 free-ranging, captive, and livestock birds demonstrated that 21 animals shed microsporidian spores of species known to infect humans, including Encephalitozoon hellem (20 birds; 3.5%) and Encephalitozoon intestinalis (1 bird; 0.2%). Of 11 avian species that shed E. hellem and E. intestinalis, 8 were aquatic birds (i.e., common waterfowl). The prevalence of microsporidian infections in waterfowl (8.6%) was significantly higher than the prevalence of microsporidian infections in other birds (1.1%) (P < 0.03); waterfowl fecal droppings contained significantly more spores (mean, 3.6 × 10⁵ spores/g) than nonaquatic bird droppings contained (mean, 4.4 × 10⁴ spores/g) (P < 0.003); and the presence of microsporidian spores of species known to infect humans in fecal samples was statistically associated with the aquatic status of the avian host (P < 0.001). We demonstrated that a single visit of a waterfowl flock can introduce into the surface water approximately 9.1 × 10⁸ microsporidian spores of species known to infect humans. Our findings demonstrate that waterborne microsporidian spores of species that infect people can originate from common waterfowl, which usually occur in large numbers and have unlimited access to surface waters, including waters used for production of drinking water.     

Development and optimization of a quantitative cell culture infectivity assay for the microsporidium Encephalitozoon intestinalis and application to ultraviolet light inactivation

Microsporidia are unique parasites recognized as a major cause of intestinal illness among immunocompromised patients and occasionally in otherwise healthy hosts. These organisms have been detected in water and are likely transmitted by the fecal-oral route. The most common human pathogenic microsporidia for which cell culture methods have been established is Encephalitozoon intestinalis. This study describes the development of a quantitative cell culture infectivity assay for E. intestinalis and its application to assess inactivation by ultraviolet (UV) light irradiation. The method described here employs calcofluor white, a fluorescent brightener that targets the chitin spore wall, to visualize groups of developing spores in order to confirm infectivity. Serial dilutions of the spore suspension were seeded into tissue culture well slides containing RK-13 cells. Slides were then rinsed, fixed in methanol and stained with calcofluor white and examined microscopically. Large masses of developing spores were easily visible on infected cell monolayers. Positive and negative wells at each dilution step were used to quantify the number of infectious spores in the original suspension using a most-probable-number (MPN) statistical analysis. This assay was used to evaluate the disinfecting potential of ultraviolet light on E. intestinalis spores in water. The ultraviolet dose required for a 3-log(10) or 99.9% reduction in the number of infective spores was determined to be 8.43 mW s/cm(2).     

Cellular damage during drying and storage of Trichoderma harzianum spores

Factors that cause cellular damage during the drying and storage of Trichoderma harzianum conidia were independently studied to determine their effects on spore viability. Specifically, thermal stress and dehydration levels (water activity, a_w = 0.1–0.7) were assessed for their effect on spore survival. In addition, environmental conditions, such as water activity and temperature, were evaluated during storage of the spores. T. harzianum spores produced in liquid culture are highly sensitive to thermal stress, but dehydration does not seem to be a factor that influences spore death during desiccation. An inverse correlation between spore survival and the specific concentration of malondialdehyde (MDA) was observed during storage, especially when the conidia moisture levels were lower than the monolayer moisture levels. We prepared spore suspensions without additives and spray-dried the samples. Our data showed that reduced sample viability was mainly caused by the temperature of the drying process, an effect that appears to be independent of water activity.     

Sporulation Temperature Reveals a Requirement for CotE in the Assembly of both the Coat and Exosporium Layers of Bacillus cereus Spores

The Bacillus cereus spore surface layers consist of a coat surrounded by an exosporium. We investigated the interplay between the sporulation temperature and the CotE morphogenetic protein in the assembly of the surface layers of B. cereus ATCC 14579 spores and on the resulting spore properties. The cotE deletion affects the coat and exosporium composition of the spores formed both at the suboptimal temperature of 20°C and at the optimal growth temperature of 37°C. Transmission electron microscopy revealed that ΔcotE spores had a fragmented and detached exosporium when formed at 37°C. However, when produced at 20°C, ΔcotE spores showed defects in both coat and exosporium attachment and were susceptible to lysozyme and mutanolysin. Thus, CotE has a role in the assembly of both the coat and exosporium, which is more important during sporulation at 20°C. CotE was more represented in extracts from spores formed at 20°C than at 37°C, suggesting that increased synthesis of the protein is required to maintain proper assembly of spore surface layers at the former temperature. ΔcotE spores formed at either sporulation temperature were impaired in inosine-triggered germination and resistance to UV-C and H₂O₂ and were less hydrophobic than wild-type (WT) spores but had a higher resistance to wet heat. While underscoring the role of CotE in the assembly of B. cereus spore surface layers, our study also suggests a contribution of the protein to functional properties of additional spore structures. Moreover, it also suggests a complex relationship between the function of a spore morphogenetic protein and environmental factors such as the temperature during spore formation.     

Aphidicolin Inhibition of DNA Synthesis and Germination in Spores of Anemia phyllitidis L. Sw

Aphidicolin inhibits DNA synthesis and nuclear division in spores of Anemia phyllitidis. In spite of blocked DNA replication, spores germinate under continuous dark conditions, if induced by addition of 5 × 10⁻⁵ grams per milliliter gibberellic acid. Differentiation of aphidicolin-treated prothallia indicate the existence of a prepattern in the dry spore which is realized independent of cell division during early events of spore germination.     

Thermal Inactivation of Nonproteolytic Clostridium botulinum Type E Spores in Model Fish Media and in Vacuum-Packaged Hot-Smoked Fish Products

Thermal inactivation of nonproteolytic Clostridium botulinum type E spores was investigated in rainbow trout and whitefish media at 75 to 93°C. Lysozyme was applied in the recovery of spores, yielding biphasic thermal destruction curves. Approximately 0.1% of the spores were permeable to lysozyme, showing an increased measured heat resistance. Decimal reduction times for the heat-resistant spore fraction in rainbow trout medium were 255, 98, and 4.2 min at 75, 85, and 93°C, respectively, and those in whitefish medium were 55 and 7.1 min at 81 and 90°C, respectively. The z values were 10.4°C in trout medium and 10.1°C in whitefish medium. Commercial hot-smoking processes employed in five Finnish fish-smoking companies provided reduction in the numbers of spores of nonproteolytic C. botulinum of less than 10³. An inoculated-pack study revealed that a time-temperature combination of 42 min at 85°C (fish surface temperature) with >70% relative humidity (RH) prevented growth from 10⁶ spores in vacuum-packaged hot-smoked rainbow trout fillets and whole whitefish stored for 5 weeks at 8°C. In Finland it is recommended that hot-smoked fish be stored at or below 3°C, further extending product safety. However, heating whitefish for 44 min at 85°C with 10% RH resulted in growth and toxicity in 5 weeks at 8°C. Moist heat thus enhanced spore thermal inactivation and is essential to an effective process. The sensory qualities of safely processed and more lightly processed whitefish were similar, while differences between the sensory qualities of safely processed and lightly processes rainbow trout were observed.     

Production of Vesicular-Arbuscular Mycorrhizal Fungus Inoculum in Aeroponic Culture

Bahia grass (Paspalum notatum) and industrial sweet potato (Ipomoea batatas) colonized by Glomus deserticola, G. etunicatum, and G. intraradices were grown in aeroponic cultures. After 12 to 14 weeks, all roots were colonized by the inoculated vesicular-arbuscular mycorrhizal fungi. Abundant vesicles and arbuscules formed in the roots, and profuse sporulation was detected intra-and extraradically. Within each fungal species, industrial sweet potato contained significantly more roots and spores per plant than bahia grass did, although the percent root colonization was similar for both hosts. Mean percent root colonization and sporulation per centimeter of colonized root generally increased with time, although with some treatments colonization declined by week 14. Spore production ranged from 4 spores per cm of colonized root for G. etunicatum to 51 spores per cm for G. intraradices. Infectivity trials with root inocula resulted in a mean of 38, 45, and 28% of bahia grass roots colonized by G. deserticola, G. etunicatum, and G. intraradices, respectively. The germination rate of G. etunicatum spores produced in soil was significantly higher than that produced in aeroponic cultures (64% versus 46%) after a 2-week incubation at 28°C. However, infectivity studies comparing G. etunicatum spores from soil and aeroponic culture indicated no biological differences between the spore sources. Aeroponically produced G. deserticola and G. etunicatum inocula retained their infectivity after cold storage (4°C) in either sterile water or moist vermiculite for at least 4 and 9 months, respectively.