Fatty acid composition of four microsporidian species compared to that of their host fishes

ABSTRACT. The fatty acid composition of four microsporidian species (Glugea atherinae, Spraguea lophii, Glugea americanus , and Pleistophora mirandellae) and their host fishes has been determined using gas chromatography. Twenty-four fatty acids were identified with differences in relative abundance of fatty acids among the four parasites. Certain even-saturated fatty acids were found in a very high proportion: palmitic acid (16:0) represented one-third of total fatty acids in Pleistophora mirandellae. The level of docosahexaenoic acid (22:6ω3) attained 26–28% in Glugea atherinae, Spraguea lophii , and Glugea americanus , but only 8–9% in P. mirandellae. With respect to fatty acid compositions of host organs, some significant differences were evident between marine and freshwater fishes. Palmitic acid was prevalent in the marine fishes, Atherinae boyeri and Lophius piscatorius , and oleic acid (18:1ω9) in the freshwater fish Leuciscus cephalus. The proportion of docosahexaenoic acid in marine fishes was two or three times as great as in freshwater fish Leuciscus. The high polyunsaturated fatty acid content in both parasites and host fishes may be related to the scavenging of these fatty acids by the parasites rather than a microsporidia-specific fatty acid biosynthesis pathway.     

Ribosomal DNA sequences of Glugea anomala, G. stephani, G. americanus and Spraguea lophii (Microsporidia): phylogenetic reconstruction

The microsporidian species Glugea anomala, G. stephani, G. americanus and Spraguea lophii were compared by using sequence data derived from their small subunit rDNA genes which were amplified by polymerase chain reaction and directly sequenced. These sequence data and published data of G. atherinae were analyzed and were used to infer a phylogenetic tree. The 5 microsporidian fish parasites appeared to be closely related. The higher sequence similarities demonstrated among G. anomala, G. stephani and G. atherinae suggest that these 3 parasites are in fact only 1 species of Glugea. Moreover, the higher sequence similarities between S. lophii and G. americanus support the transfer of the latter Glugea species into the genus Spraguea.     

Identification and Phylogenetic Relationships of Microsporidia by Riboprinting

ABSTRACT. The SSUrDNA and the ITS of different microsporidia from eight fishes, four insects and a shrimp were amplified and digested with restriction enzymes. The generated riboprints suggest a close evolutionary relationship between Glugea americanus and Spraguea lophii suggesting that Glugea americanus should be renamed Spraguea americanus and that the tissue infected and host origin should be considered of greater taxonomic importance for defining a genus than previously considered. Phylogenetic analysis of the riboprints demonstrates an unidentified microsporidium from a bumper fish ( Chloroscombrus chrysurus ) is related although not identical to Microgemma oviodea , a parasite from red band fish. We were also able to distinguish between Glugea anomala and Glugea atherinae and Glugea stephani but were not able to differenciate among the latter two. Insects isolates, Nosema costelytrae, N. bombycis, N. trichoplusiae, Nosema sp. and a shrimp isolate, Agmasoma penaei , are not related to the fish isolates.     

Development and use of flow cytometry for detection of airborne fungi

Traditional methods for the enumeration of airborne fungi are slow, tedious, and rather imprecise. In this study, the possibility of using flow cytometry (FCM) for the assessment of exposure to the fungus aerosol was evaluated. Epifluorescence microscopy direct counting was adopted as the standard for comparison. Setting up of the method was achieved with pure suspensions of Aspergillus fumigatus and Penicillium brevicompactum conidia at different concentrations, and then analyses were extended to field samples collected by an impinger device. Detection and quantification of airborne fungi by FCM was obtained combining light scatter and propidium iodide red fluorescence parameters. Since inorganic debris are unstainable with propidium iodide, the biotic component could be recognized, whereas the preanalysis of pure conidia suspensions of some species allowed us to select the area corresponding to the expected fungal population. A close agreement between FCM and epifluorescence microscopy counts was found. Moreover, data processing showed that FCM can be considered more precise and reliable at any of the tested concentrations.     

Development and Use of Flow Cytometry for Detection of Airborne Fungi

Traditional methods for the enumeration of airborne fungi are slow, tedious, and rather imprecise. In this study, the possibility of using flow cytometry (FCM) for the assessment of exposure to the fungus aerosol was evaluated. Epifluorescence microscopy direct counting was adopted as the standard for comparison. Setting up of the method was achieved with pure suspensions of Aspergillus fumigatus and Penicillium brevicompactum conidia at different concentrations, and then analyses were extended to field samples collected by an impinger device. Detection and quantification of airborne fungi by FCM was obtained combining light scatter and propidium iodide red fluorescence parameters. Since inorganic debris are unstainable with propidium iodide, the biotic component could be recognized, whereas the preanalysis of pure conidia suspensions of some species allowed us to select the area corresponding to the expected fungal population. A close agreement between FCM and epifluorescence microscopy counts was found. Moreover, data processing showed that FCM can be considered more precise and reliable at any of the tested concentrations.     

Flow cytometric discrimination of mitotic cells: resolution of M, as well as G1, S, and G2 phase nuclei with mithramycin, propidium iodide, and ethidium bromide after fixation with formaldehyde

Cells in mitosis can be flow cytometrically discriminated from G1, S, and G2 cells by analysis of a nuclear suspension prepared with nonionic detergent, fixed with formaldehyde, and stained with mithramycin, propidium iodide, or ethidium bromide. With these DNA-fluorochromes, the fluorescence is quenched by formaldehyde less in mitotic nuclei than in interphase nuclei. Mitotic nuclei have a 20-40% increased mithramycin fluorescence and 30-60% decreased light scatter in comparison to those of G2 nuclei. There is a high correlation (r = 0.95; P less than 0.001) between microscope counts of mitotic figures in smear preparations of the initial cell suspension and the flow cytometrically estimated fraction of nuclei with increased mithramycin fluorescence. Flow sorting (FACS) demonstrates that the mitotic nuclei are confined to the peak of increased mithramycin fluorescence and decreased light scatter. The method has been applied to cultures of Yoshida ascites tumor cells, JB-1 reticulosarcoma cells, and PHA-stimulated human lymphocytes, incubated in the presence or absence of vinblastine for mitotic arrest. In a heteroploid mixture of fixed Yoshida (near-diploid) and JB-1 (hypotetraploid) nuclei, the mitotic fractions of the two cell lines could be estimated separately when analyzed with mithramycin fluorescence versus light scatter or with mithramycin fluorescence versus propidium iodide fluorescence.     

Lipids of three microsporidian species and multivariate analysis of the host-parasite relationship.

Sporal lipids of 3 microsporidia, Encephalitozoon cuniculi from mammals and Glugea atherinae and Spraguea lophii from fishes, were investigated. High phospholipid levels were found (54.8-64.5% of total lipids), which is in agreement with the presence of highly developed internal membranes in microsporidian spores. Sphingomyelin was not detected in G. atherinae. Triglycerides (less than 10% of total lipids), cholesterol, and free fatty acids were identified in all species. Analysis of fatty acids from the phospholipid fraction revealed the predominance of docosahexaenoic acid (30-40% of total phospholipid fatty acids) in G. atherinae and S. lophii and oleic acid (25.8% of total phospholipid fatty acids) in E. cuniculi. The 3 microsporidia possessed a significant amount of branched-chain fatty acids (iso and anteiso forms) not found in the hosts, supporting the existence of some parasite-specific metabolic steps for these fatty acids. On the basis of phospholipid fatty acid profiles, host-parasite relationships were investigated through correspondence factorial analysis. It shows 3 distinct clusters with the first corresponding to fishes, the second to fish parasites, and the third to E. cuniculi and its host cell. These data suggest that the mammal microsporidia developing within parasitophorous vacuoles are more dependent on host cells than the fish microsporidia that induce cystlike structures.     

Immunocytochemical Identification of Spore Proteins in Two Microsporidia, with Emphasis on Extrusion Apparatus

Microsporidia can form small spores with a unique invasive apparatus featuring a long polar tube whose extrusion allows entry of infectious sporoplasm into a host cell. The reactivity of mouse polyclonal antibodies raised against sporal proteins from two microsporidian species belonging to different genera ( Glugea atherinae and Encephalitozoon cuniculi ) was studied by western blotting and indirect immunofluorescence. Whole protein antisera provided a few cross-reactions relatable to some proteins of the spore envelope or polar tube. Ultrastructural immunocytochemistry with murine antibodies against protein bands separated by sodium dodecylsulphate polyacrylamide gel electrophoresis allowed the assignment of several proteins to the polar tube (34, 75 and 170 kDa in Glugea , 35, 55 and 150 kDa in Encephalitozoon ). Antigenic similarities were detected for the Glugea 34 kDa and Encephalitozoon 35 kDa polar tube proteins. Species-specific proteins were shown to be located in either the lamellar polaroplast of Glugea or the spore envelope of Encephalitozoon.     

Redescription of Microsporidium takedai (Awakura, 1974) as Kabatana takedai (Awakura, 1974) comb. n

Ultrastructural study of the microsporidian Microsporidium takedai from the muscles of masu salmon Oncorhynchus masou proved that this species can be assigned to the genus Kabatana Lom, Dyková and Tonguthai, 2000. The parasites develop within disintegrated sarcoplasm without any delimiting boundary or cyst. Cylindrical multinucleate meronts proliferate by serial constrictions into uninucleate stages which repeat the process. Eventually, the uninucleate stages transform into uninucleate sporonts, which divide once to produce sporoblasts, thus functioning as sporoblast mother cells. Spores, with a subterminally located anchoring disc and 3 to 4 turns of the polar tube coil, average 3.3 by 1.9 microm in size. The exospore is divided into small fields; the endospore frequently makes small invaginations into the spore inside. Phylogenetic analysis using SSU rDNA sequence consistently placed Kabatana takedai in a group consisting of Microgemma sp., Spraguea lophii and Glugea americanus. The K. takedai could easily be separated from the other species in the same group by 2 inserts in the SSU rDNA sequence.     

Microsporidians in penaeid shrimp along the west coast of Madagascar

Three species of penaeid shrimp, Fenneropenaeus indicus, Penaeus monodon and P. semisulcatus, found in trawler catches off the west coast of Madagascar were infected with microsporidian parasites. The infections were evident as muscular lesions with a cottony appearance when abundant. Spore size (2.6 x 1.6 microm) and morphology (ovoid) for the parasites infecting both F. indicus and P. semisulcatus were not significantly different, suggesting that they might be the same microsporidian species. Spore size (1.4 x 1.1 microm) and morphology (sub-globose to ovoid) in P. monodon infections were significantly different from those in the other 2 shrimp species, suggesting that it was a different parasite. The presence of microsporidians in this biogeographical zone means that there is a potential risk of infections of cultured shrimp in farms situated in the vicinity. This must be assessed by increasing current knowledge of the parasites.     

Fringe-scan flow cytometry

We describe the development of a scanning flow cytometer capable of measuring the distribution of fluorescent dye along objects with a spatial resolution of 0.7 micron. The heart of this instrument, called a fringe-scan flow cytometer, is an interference field (i.e., a series of intense planes of illumination) produced by the intersection of two laser beams. Fluorescence profiles (i.e., records showing the intensity of fluorescence measured at 20 ns intervals) are recorded during the passage of objects through the fringe field. The shape of the fringe field is determined by recording light scatter profiles as 0.25 micron diameter microspheres traverse the field. The distribution of the fluorescent dye along each object passing through the fringe field is estimated from the recorded fluorescence profile using Fourier deconvolution. We show that the distribution of fluorescent dye along microsphere doublets and along propidium iodide stained human chromosomes can be determined accurately using fringe-scan flow cytometry. The accuracy of fringe-scan shape analysis was determined by comparing fluorescence profiles estimated from fringe-scan profiles for microspheres and chromosomes with fluorescence profiles for the same objects measured using slit-scan flow cytometry.     

Flow cytometric, phase-resolved fluorescence measurement of propidium iodide uptake in macrophages containing phagocytized fluorescent microspheres

BACKGROUND: Spectral interference (overlap) from phagocytosed green-yellow (GY) microspheres in the flow cytometric, red fluorescence emission measurement channel causes errors in quantifying damaged/dead alveolar macrophages by uptake of propidium iodide. METHODS: Particle burdens of uniform GY fluorescent microspheres phagocytosed by rat alveolar macrophages and the discrimination of damaged/dead cells as indexed by propidium iodide uptake were assessed with conventional and phase-sensitive flow cytometry. RESULTS: The fluorescence spectral emission from phagocytosed microspheres partly overlapped the propidium iodide red fluorescence emission and interfered with the measurement of damaged/dead cells when using conventional flow cytometry without subtractive compensation. This caused errors when estimating the percentage of nonviable, propidium iodide-positive, phagocytic macrophages. The interference was eliminated by employing phase-sensitive detection in the red fluorescence measurement channel based on differences in fluorescence lifetimes between the fluorescent microspheres and propidium iodide. Intrinsic cellular autofluorescence, whose fluorescence lifetime is approximately the same as that of the phagocytosed microspheres, also was eliminated in the phase-sensitive detection process. Because there was no detectable spectral interference of propidium iodide in the green fluorescence (phagocytosis) measurement channel, conventional fluorescence detection was employed. CONCLUSIONS: Phase-resolved, red fluorescence emission measurement eliminates spectral overlap errors caused by autofluorescent phagocytes that contain fluorescent microspheres in the analyses of propidium iodide uptake.Cytometry 39:45-55, 2000. Published 2000 Wiley-Liss, Inc.     

Flow cytometric application of the Sabin and Feldman dye test in the diagnosis of toxoplasmosis.

Although time-consuming and requiring live parasites, the Sabin and Feldman dye test (DT) is still considered the 'gold standard' among the serological tests for toxoplasmosis diagnosis. The present study was initiated to compare detection of dead parasites using optical microscopy with flow cytometry and a fluorescent nonvital dye, propidium iodide. After incubation with sera (N = 150) and a complement source, tachyzoites were washed, then stained using a fluorescein-conjugated Toxoplasma-specific antiserum. Dead tachyzoites were detected by flow cytometry after addition of propidium iodide. Intra- and inter-assay reproducibilities of percentages of dead parasites varied between 7 and 14%, and 8 and 21%, respectively. When comparing flow cytometry with the classical DT, no discrepancy was noted for positive (N = 118) and negative sera (N = 32). Correlation was good (r = 0.85) for positive sera. In conclusion, when easily available, flow cytometry is a very sensitive, specific and time-sparing method to detect specific antibodies to Toxoplasma gondii.     

A flow-cytometric method for the separation and quantitation of normal and apoptotic thymocytes.

Using flow cytometry, we describe a method for separating and quantifying normal and apoptotic thymocytes. Apoptosis was induced in isolated thymocytes from immature rats by treatment with the glucocorticoid dexamethasone or the antitumor agent etoposide. Subsequent incubation with the vital bisbenzimidazole dye Hoechst 33342 and the DNA intercalating agent propidium iodide enabled three distinct populations of cells to be identified and sorted by flow cytometry. Dead cells fluoresced red due to propidium iodide whereas normal and apoptotic cells fluoresced blue due to Hoechst 33342. Apoptotic cells were distinguished from normal thymocytes both by their higher intensity of blue fluorescence and by their smaller size as determined by a reduction in forward light scatter. The larger cells, with low blue fluorescence, showed normal thymocyte morphology by electron microscopy and the absence of any DNA fragmentation as measured by agarose gel electrophoresis. In contrast, the smaller cells showed both the morphological characteristics of apoptosis and extensive internucleosomal fragmentation of DNA to multiples of approximately 180 bp. Using this method, a time-dependent induction of apoptosis by dexamethasone, which was inhibited by cycloheximide, actinomycin D, and aurin tricarboxylate, was observed. The method should facilitate mechanistic studies on the induction of apoptosis in thymocytes.     

Palmitate induces structural alterations in nuclei of cardiomyocytes

The objective of this study was to examine the hypothesis that the alterations of cardiac nuclei, that has been noted in some cardiomyopathies, can be produced by palmitate, a saturated fatty acid present in high circulating concentrations in patients with conditions associated with a high probability of developing cardiomyopathy. Cardiomyocytes isolated from embryonic chick ventricle were maintained in culture for 72 h and then treated with palmitate, 100 microM for 24 h. Cells were stained with acridine orange or Giemsa and examined microscopically. Cell size and nuclear size were examined by forward light scatter during flow cytometry. Cells were permeabilized and their nuclei were stained with propidium iodide and examined by flow cytometry on populations of 10,000 cells. Cardiomyocytes treated with palmitate displayed changes in nuclear appearance as nuclei were larger, relative to cell size, with more intense acridine orange staining in a peripheral location. Nucleoli were often disrupted. Palmitate produced a significant (P < 0.001) and 17% increase in nuclear size compared to untreated cells using flow cytometry analysing forward light scatter to estimate nuclear and whole cells size. There were no significant changes in the size of the whole cell and ratio of nucleus to whole cell was significantly (P < 0.01) increased compared to control cells. Fluorescent activating cell sorting analysis of propidium iodide stained nuclei demonstrated that the nuclear enlargement was not due to cell mitosis as the proportion of nuclei in Go/G1, S or M was not changed by palmitate. In summary, these studies identify that palmitate can induce structural abnormalities of cardiomyocytes nuclei by producing increased nuclear size and nucleolar destruction.     

Flow cytometric discrimination of mitotic nuclei by right-angle light scatter

Flow cytometry has been used to demonstrate alterations in protein, RNA, and DNA content of cells as they traverse the cell cycle. Employing fluorescein isothiocyanate (FITC) to stain protein and propidium iodide (PI) to stain nucleic acids, multiple regions within the G1 and G2 phases of the cell cycle, in addition to the M phase, can be distinguished. In this study, cytograms of the 90 degree light scatter signal vs. PI fluorescence were remarkably similar to those of FITC fluorescence vs. PI fluorescence, suggesting a relationship between 90 degree light scatter and protein content. M-phase nuclei can be distinguished from G2-phase nuclei on cytograms of 90 degree light scatter vs. PI fluorescence. However, the percentage of mitotic nuclei obtained by this technique is less than that found by light microscopic analysis. Flow cytometric parameters of nuclei prepared by nonionic detergent (NP40) lysis in Dulbecco's PBS, Vindelov's buffer, or Pollack's hypotonic EDTA/Tris buffer were compared. The best resolution of mitotic nuclei was obtained in Pollack's buffer. However, the stainability of the M-phase nuclei is reduced, and the nuclei are located in the late S/G2 region of the single-parameter histogram.     

微孢子虫一新种的描述及分子鉴定

随着集约化、高密度养殖业的发展, 传播性寄生虫病给水产业造成了严重的损失, 微孢子虫是养殖鱼类的主要病原生物之一, 迄今, 已报道的鱼类寄生微孢子虫多达100 余种。       

Flow cytometric investigation of heterogeneous copper-sensitivity in asynchronously grown Saccharomyces cerevisiae

The variable stress-sensitivity of individual cells within pure cultures is widely noted but generally unexplained. Here, factors determining the heterogeneous susceptibility to copper toxicity in Saccharomyces cerevisiae were examined with a rapid non-perturbing approach based on flow cytometry. By determination of the DNA content (with propidium iodide) in cell fractions gated by forward angle light scatter (an indicator of the cell volume), it was shown that forward angle light scatter measurements gave an approximation of the cell cycle stage. Thus, our observation that cells in different forward angle light scatter fractions displayed differing Cu-sensitivities indicated that heterogeneous Cu-sensitivity is a function of the cell cycle stage. Furthermore, cells sorted by their Cu-sensitivity and-resistance and subsequently analyzed for DNA content were found predominantly to occupy G1/S and G2/M cell cycle stages, respectively. The oxidant-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate was used to show that the Cu-sensitivity of G2/M phase S. cerevisiae was correlated with greater levels of pre-existing reactive oxygen species in these cells. The results indicate that differential Cu-sensitivity in a S. cerevisiae culture is linked to the cell cycle stage and this link may be determined partly by cell cycle-dependent fluctuations in basal reactive oxygen species generation.     

Flow cytometric quantification of electroporation-mediated uptake of macromolecules into plant protoplasts

Summary Flow cytometry was used to provide a rapid and accurate assessment of electroporation-induced uptake of macromolecules into plant protoplasts. Rice protoplasts were electroporated in the presence of fluorescein isothiocyanate-conjugated dextran (FITC-dextran). After washing, the protoplasts were resuspended in a solution containing propidium iodide which intercalates with DNA, but which is excluded by an intact plasma membrane. Electroporation in the presence of FITC-dextran gave rise to populations of protoplasts that fluoresced green or yellow due to the presence of non-conjugated FITC. Non-viable protoplasts fluoresced red because of their inability to exclude propidium iodide molecules. Flow cytometry was used to resolve and quantify these protoplast populations and thus identify optimal conditions for macromolecule uptake. A direct relationship was observed between FITC-dextran uptake and transient gene expression following plasmid uptake. Thus, simultaneous electroporation of protoplasts with foreign DNA and FITC-dextran followed by fluorescence activated cell sorting may permit partial selection of transformed cells and so reduce the need for a selectable marker.Abbreviations ADC analogue to digital converter - CAT chloramphenicol acetyl transferase (enzyme) - cat chloramphenicol acetyl transferase (gene) - CPW solution cell and protoplast wash solution - DC direct current - EF electrofusion - FALS forward angle light scatter - FITC fluorescein isothiocyanate - FITC-dextran fluorescein isothiocyanate conjugated dextran - PI propidium iodide - PMT photomultipliertube - TLC thin layer chromatography     

Identification of nuclei from apoptotic, necrotic, and viable lymphoid cells by using multiparameter flow cytometry.

BACKGROUND: Methods widely used to detect apoptosis do not allow us to easily distinguish between nuclei from viable or necrotic cells. Even if apoptosis and necrosis seem to occur as alternatives at the single cell level, they could be present simultaneously in a cell population much more frequently than expected. For this reason, attention was focused on attempting to recognize, by multiparameter flow cytometry, the characteristics of viable cells and of apoptotic or necrotic dead cells. METHODS: Apoptosis and necrosis were induced in vitro in murine thymocytes and lymphocytes from adult peripheral blood by using dexamethasone or prostaglandin E2 treatment and heat shock at 60 degrees C or hydrogen peroxide, respectively. Traditional methods, such as DNA gel electrophoresis and propidium iodide staining followed by single-fluorescence analysis or annexin-V-fluorescein isothiocyanate plus propidium iodide staining by using flow cytometry, were compared with a new method. This method consisted of combined light-scatter and red fluorescence analysis by flow cytometry after isolation of nuclei by hypotonic solution as well as high-dose detergent treatment and DNA staining with propidium iodide. RESULTS: Results showed that, although traditional methods such as DNA-gel electrophoresis and single-parameter fluorescence flow cytometry analysis were unable, as expected, to discriminate among viability, apoptosis, and necrosis, our new method has enabled us to easily identify nuclei from viable, apoptotic, and necrotic cells. Results obtained by using our method were comparable to those obtained by using two-color analysis of cells after propidium iodide/annexin V staining. CONCLUSIONS: A highly reproducible, inexpensive, rapid, and easily accessible method of analysis has been developed for simultaneously detecting apoptosis and necro sis.