scholarly journals Discrimination between Viable and Dead Encephalitozoon cuniculi (Microsporidian) Spores by Dual Staining with Sytox Green and Calcofluor White M2R

2000 ◽  
Vol 38 (10) ◽  
pp. 3811-3814 ◽  
Author(s):  
L. C. Green ◽  
P. J. LeBlanc ◽  
E. S. Didier
Keyword(s):  
Systemic Disease ◽  
Organ Transplant ◽  
Spore Wall ◽  
Staining Procedure ◽  
Excitation Wavelength ◽  
Encephalitozoon Cuniculi ◽  
Calcofluor White ◽  
Sytox Green ◽  
Intracellular Parasites ◽  
Dual Staining

Microsporidia are obligate intracellular parasites, recognized as causing chronic diarrhea and systemic disease in AIDS patients, organ transplant recipients, travelers, and malnourished children. Species of microsporidia that infect humans have been detected in drinking-water sources, and methods are needed to ascertain if these microsporidia are viable and capable of causing infections. In this study, Calcofluor White M2R and Sytox Green stains were used in combination to differentiate between live (freshly harvested) and dead (boiled)Encephalitozoon cuniculi spores. Calcofluor White M2R binds to chitin in the microsporidian spore wall. Dual-stained live spores appeared as turquoise-blue ovals, while dead spores appeared as white-yellow ovals at an excitation wavelength of 395 to 415 nm used for viewing the Calcofluor stain. Sytox Green, a nuclear stain, is excluded by live spores but penetrates compromised spore membranes. Dual-stained dead spores fluoresced bright yellow-green when viewed at an excitation wavelength of 470 to 490 nm, whereas live spores failed to stain with Sytox Green. After live and dead spores were mixed at various ratios, the number of viably stained spores detected in the dual-staining procedure correlated (P = 0.0025) with the expected numbers of viable spores. Spore mixtures were also assayed for infectivity in a focus-forming assay, and a correlation (P = 0.0002) was measured between the percentage of focus-forming microsporidia and the percentage of expected infectious spores in each mixture. By analysis of variance, no statistically significant differences were measured between the percentage of viably stained microsporidia and the percentage of infectious microsporidia (P = 0.964) in each mixture. These results suggest that Calcofluor White M2R and Sytox Green stains, when used together, may facilitate studies to identify viable microsporidia.

scholarly journals Identification and Characterization of Two Subpopulations of Encephalitozoon intestinalis

2003 ◽  
Vol 69 (8) ◽  
pp. 4966-4970 ◽  
Author(s):  
Rebecca M. Hoffman ◽  
Marilyn M. Marshall ◽  
David M. Polchert ◽  
B. Helen Jost
Keyword(s):  
Cell Culture ◽  
Polyclonal Antibodies ◽  
Fluorescein Isothiocyanate ◽  
Spore Wall ◽  
Rabbit Kidney ◽  
Sytox Green ◽  
Encephalitozoon Intestinalis ◽  
Propagation Methods ◽  
Small Subpopulation

ABSTRACT 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 ≥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 β-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.

scholarly journals The tribe Ehrlichieae and ehrlichial diseases.

1991 ◽  
Vol 4 (3) ◽  
pp. 286-308 ◽  
Author(s):  
Y Rikihisa
Keyword(s):  
Systemic Disease ◽  
The United States ◽  
Cross Reactivity ◽  
Etiologic Agent ◽  
16S Rrna Sequence ◽  
Intracellular Parasites ◽  
Potomac Horse Fever ◽  
Ehrlichia Risticii ◽  
16S Rrna Sequence Analysis ◽  
Human Ehrlichiosis

The tribe Ehrlichieae consists of gram-negative minute cocci that are obligate intracellular parasites classified in the family Rickettsiaceae. Although ehrlichial organisms have been observed in leukocytes for many years, only a few species have been cultured in quantities sufficient for biochemical and molecular analyses. Recents studies on 16S-rRNA sequence analysis and energy metabolism showed that the genus Ehrlichia is closely related to the genus Rickettsia. There is, however, no antigenic cross-reactivity between these genera. Ehrlichial organisms cause a disease called "ehrlichiosis," a noncontagious infectious disease known to be transmitted by a tick in several cases and by a fluke in one case. Ehrlichia spp. infect dogs, ruminants, horses, and humans. Recently, two new ehrlichial diseases, Potomac horse fever and human ehrlichiosis, were discovered in the United States. The etiologic agent of Potomac horse fever, Ehrlichia risticii, is closely related to the known human pathogen Ehrlichia sennetsu. The etiologic agent of human ehrlichiosis is related to Ehrlichia canis, a canine pathogen. In contrast to the genus Rickettsia, members of the tribe Ehrlichieae reside primarily in the cytoplasmic vacuoles of monocytes or granulocytes and cause hematologic abnormalities, lymphadenopathy, and other pathologic changes in the host. However, the actual mechanisms whereby Ehrlichia spp. infect leukocytes, multiply in them, and produce various forms of systemic disease have not been defined. Depending on the ehrlichial species involved, serologic or direct microscopic observation of stained blood smears is currently used to diagnose ehrlichial disease.

scholarly journals Methods for Assessment of Viability and Germination of Plasmodiophora brassicae Resting Spores

2022 ◽  
Vol 12 ◽  
Author(s):  
Yao Wang ◽  
Birger Koopmann ◽  
Andreas von Tiedemann
Keyword(s):  
Evans Blue ◽  
Plasmodiophora Brassicae ◽  
Germination Rate ◽  
Inoculum Potential ◽  
Differential Interference Contrast Microscopy ◽  
Calcofluor White ◽  
Clubroot Disease ◽  
Resting Spores ◽  
Dual Staining

Clubroot caused by the obligate biotrophic parasite Plasmodiophora brassicae is a destructive soil borne disease of cruciferous crops. Resting spores of P. brassicae can survive in the soil for a long period without hosts or external stimulants. The viability and germination rate of resting spores are crucial factors of the inoculum potential in the field. The accurate assessment of viability and germination rate is the foundation to evaluate the effect of control methods. In this study, we evaluated several methods for the assessment of viability and germination rate of P. brassicae resting spores. Dual staining with calcofluor white-propidium iodide (CFW-PI) or single stain with Evans blue showed reliable accuracy in estimating viability. CFW-PI was capable of reliably determining the viability within 10 min, while Evans blue required overnight incubation to obtain accurate results. Due to DNA degradation of heat treatments, acetone was selected to evaluate the efficiency of propidium monoazide (PMA)–quantitative PCR (qPCR) used for the quantification of DNA from viable cells. The staining with 4,6-Diamidine-2-phenylindole dihydrochloride (DAPI) and the use of differential interference contrast microscopy were suitable for the determination of resting spore germination rates. The latter method also allowed recording individual germination states of spores. Alternatively, dual staining with CFW-Nile red was successfully used to assess the germination rate of resting spores with a lethal pre-treatment. This study evaluates and confirms the suitability of various microscopic and molecular genetic methods for the determination of viability and germination of P. brassicae resting spores. Such methods are required to study factors in the soil regulating survival, dormancy and germination of P. brassicae resting spores causing clubroot disease in Brassicaceae hosts and therefore are fundamental to develop novel strategies of control.

scholarly journals Branching Network of Proteinaceous Filaments within the Parasitophorous Vacuole ofEncephalitozoon cuniculiandEncephalitozoon hellem

2011 ◽  
Vol 79 (3) ◽  
pp. 1374-1385 ◽  
Author(s):  
Kaya Ghosh ◽  
Eddie Nieves ◽  
Patrick Keeling ◽  
Jean-Francois Pombert ◽  
Philipp P. Henrich ◽  
...  
Keyword(s):  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Spore Wall ◽  
Human Pathogens ◽  
Polar Tube ◽  
Molecular Approaches ◽  
Resting Spore ◽  
Intracellular Parasites ◽  
Significant Pathology ◽  
Microsporidian Spore

ABSTRACTThe microsporidia are a diverse phylum of obligate intracellular parasites that infect all major animal groups and have been recognized as emerging human pathogens for which few chemotherapeutic options currently exist. These organisms infect every tissue and organ system, causing significant pathology, especially in immune-compromised populations. The microsporidian spore employs a unique infection strategy in which its contents are delivered into a host cell via the polar tube, an organelle that lies coiled within the resting spore but erupts with a force sufficient to pierce the plasma membrane of its host cell. Using biochemical and molecular approaches, we have previously identified components of the polar tube and spore wall of the Encephalitozoonidae. In this study, we employed a shotgun proteomic strategy to identify novel structural components of these organelles inEncephalitozoon cuniculi. As a result, a new component of theE. cuniculideveloping spore wall was identified. Surprisingly, using the same approach, a heretofore undescribed filamentous network within the lumen of the parasitophorous vacuole was discovered. This network was also present in the parasitophorous vacuole ofEncephalitozoon hellem. Thus, in addition to further elucidating the molecular composition of seminal organelles and revealing novel diagnostic and therapeutic targets, proteomic analysis-driven approaches exploring the spore may also uncover unknown facets of microsporidian biology.

scholarly journals Evaluation of a Dual-Staining Method for Acid-Fast Bacilli

1975 ◽  
Vol 2 (2) ◽  
pp. 149-150
Author(s):  
N. M. Burdash ◽  
M. E. West ◽  
E. R. Bannister ◽  
C. Dyar ◽  
R. C. Duncan
Keyword(s):  
Staining Method ◽  
Staining Procedure ◽  
Poor Correlation ◽  
Acid Fast Bacilli ◽  
Staining Techniques ◽  
Dual Staining

A dual-staining procedure for acid-fast bacilli was found to have poor correlation with the Ziehl-Neelsen and auramine-rhodamine staining techniques.

scholarly journals Mycobacterial disease, immunosuppression, and acquired immunodeficiency syndrome.

1989 ◽  
Vol 2 (4) ◽  
pp. 360-377 ◽  
Author(s):  
F M Collins
Keyword(s):  
Human Immunodeficiency Virus ◽  
Acquired Immunodeficiency Syndrome ◽  
Systemic Disease ◽  
Human Pathogens ◽  
Mesenteric Lymph Nodes ◽  
Aids Epidemic ◽  
Intracellular Parasites ◽  
Immunodeficiency Virus ◽  
Acquired Immunodeficiency ◽  
Immunodeficiency Syndrome

The mycobacteria are an important group of acid-fast pathogens ranging from obligate intracellular parasites such as Mycobacterium leprae to environmental species such as M. gordonae and M. fortuitum. The latter may behave as opportunistic human pathogens if the host defenses have been depleted in some manner. The number and severity of such infections have increased markedly with the emergence of the acquired immunodeficiency syndrome (AIDS) epidemic. These nontuberculous mycobacteria tend to be less virulent for humans than M. tuberculosis, usually giving rise to self-limiting infections involving the cervical and mesenteric lymph nodes of young children. However, the more virulent serovars of M. avium complex can colonize the bronchial and intestinal mucosal surfaces of healthy individuals, becoming virtual members of the commensal gut microflora and thus giving rise to low levels of skin hypersensitivity to tuberculins prepared from M. avium and M. intracellulare. Systemic disease develops when the normal T-cell-mediated defenses become depleted as a result of old age, cancer chemotherapy, or infection with human immunodeficiency virus. As many as 50% of human immunodeficiency virus antibody-positive individuals develop mycobacterial infections at some time during their disease. Most isolates of M. avium complex from AIDS patients fall into serotypes 4 and 8. The presence of these drug-resistant mycobacteria in the lungs of the AIDS patient makes their effective clinical treatment virtually impossible. More effective chemotherapeutic, prophylactic, and immunotherapeutic reagents are urgently needed to treat this rapidly increasing patient population.

scholarly journals Effect of Three Drugs against Encephalitozoon cuniculi Infection in Immunosuppressed Mice

2013 ◽  
Vol 57 (7) ◽  
pp. 3067-3071 ◽  
Author(s):  
Maria Anete Lallo ◽  
Lidiana F. Vidoto da Costa ◽  
João Manoel de Castro
Keyword(s):  
T Cells ◽  
The Elderly ◽  
Encephalitozoon Cuniculi ◽  
Content Type ◽  
Albendazole Sulfoxide ◽  
Intracellular Parasites ◽  
Immunosuppressed Patients ◽  
Short Time

ABSTRACTMicrosporidia comprise a large group of obligate intracellular parasites. The microsporidianEncephalitozoon cuniculicauses disseminated infection in immunosuppressed patients with HIV, cancer, or transplants and in the elderly.In vivoandin vitrostudies on the effectiveness of drugs are controversial. Currently, there is no effective treatment. We tested albendazole, albendazole sulfoxide, metronidazole, and cyclosporine in mice immunosuppressed with cyclophosphamide and inoculated by the intraperitoneal route with 107E. cuniculispores. One week after experimental inoculation, the mice were treated with albendazole, albendazole sulfoxide, metronidazole, and cyclosporine. Histological and morphometric analyses were performed to compare the treated groups. The state of immunosuppression was evaluated by phenotyping CD4+and CD8+T cells by flow cytometry. Nontreated mice showed acute disseminated and fatal encephalitozoonosis. The treatment with benzimidazoles significantly reduced infection until 30 days posttreatment (p.t.), but at 60 days p.t., the infection had recurred. Metronidazole decreased infection by a short time, and cyclosporine was not effective. All animals were immunosuppressed by all the experiments, as demonstrated by the low number of CD4+and CD8+T cells. We conclude that no drug was effective againstE. cuniculi, but the benzimidazoles controlled the infection transiently.

scholarly journals Histochemical study of Encephalitozoon cuniculi spores in the kidneys of naturally infected New Zealand rabbits

2017 ◽  
Vol 29 (3) ◽  
pp. 269-277 ◽  
Author(s):  
Luis E. Rodríguez-Tovar ◽  
Alejandra Villarreal-Marroquín ◽  
Alicia M. Nevárez-Garza ◽  
Uziel Castillo-Velázquez ◽  
Heidi G. Rodríguez-Ramírez ◽  
...  
Keyword(s):  
Light Microscopy ◽  
Background Noise ◽  
Histochemical Study ◽  
Periodic Acid ◽  
Encephalitozoon Cuniculi ◽  
Calcofluor White ◽  
Periodic Acid Schiff ◽  
Hematoxylin And Eosin ◽  
Schiff Reaction ◽  
Host Tissues

Encephalitozoon cuniculi is an important microsporidian pathogen that is considered an emergent, zoonotic, and opportunistic. It infects both domestic and laboratory rabbits, generating severe chronic interstitial and granulomatous nephritis with fibrosis and granulomatous encephalitis. Encephalitozoonosis is diagnosed in paraffin-embedded sections by examining the spores in the host tissues. The spores are difficult to observe when the samples are stained with hematoxylin and eosin (H&E), particularly when there is an inflammatory reaction and tissue damage. The spores are easily mistaken for other microorganisms, such as fungi (yeasts), protozoa, and bacteria. In our study, we used kidney samples from E. cuniculi–positive rabbits and employed 14 recommended histologic stains for detecting microsporidia spores: alcian blue, calcofluor white, Giemsa, Gram, Grocott, H&E, Luna, Luxol fast blue, Masson trichrome, modified trichrome stain (MTS), periodic acid–Schiff reaction (PAS), Van Gieson, Warthin–Starry (WS), and Ziehl–Neelsen (ZN).We concluded that MTS and Gram stain, detected by light microscopy, and calcofluor white stain, detected by ultraviolet light microscopy, are the best stains for detecting spores of E. cuniculi in paraffin-embedded tissues from infected rabbits. These stains were superior to WS, ZN, Giemsa, and PAS for identifying spores without background “noise” or monochromatic interference. Also, they allow individual spores to be discerned in paraffin-embedded tissues. MTS allows observation of the polar tube, polaroplast, and posterior vacuole, the most distinctive parts of the spore.

scholarly journals Interleukin-12-Producing CD103+CD11b−CD8+Dendritic Cells Are Responsible for Eliciting Gut Intraepithelial Lymphocyte Response against Encephalitozoon cuniculi

2015 ◽  
Vol 83 (12) ◽  
pp. 4719-4730 ◽  
Author(s):  
Magali M. Moretto ◽  
Danielle I. Harrow ◽  
Teresa S. Hawley ◽  
Imtiaz A. Khan
Keyword(s):  
Dendritic Cells ◽  
Natural Infection ◽  
Organ Transplant ◽  
Intraepithelial Lymphocyte ◽  
Interleukin 12 ◽  
Encephalitozoon Cuniculi ◽  
Mesenteric Lymph Nodes ◽  
Content Type ◽  
Specific Subset

Microsporidia, which belong to the kingdomFungi, are important opportunistic pathogens in HIV-infected populations and organ transplant recipients that are often associated with a broad range of symptoms, such as diarrhea, nephritis, and encephalitis. Natural infection occurs via the oral route, and as a consequence, gut immunity plays an important role in restricting the dissemination of these pathogens. Studies from our laboratory have reported that the pathogens induce a rapid intraepithelial lymphocyte (IEL) response important for host protection. Although mucosal dendritic cells (DC) are likely involved in triggering an antigen-specific IEL response, the specific subset(s) responsible has yet to be identified. Toward this goal, we demonstrate a very important role for mucosal CD11b−CD8+DC in the initiation of an antigen-specific IELin vivo. Effectively, afterEncephalitozoon cuniculiinfection, CD11b−CD8+DC were activated in the lamina propria (LP) and acquired the ability to process retinoic acid (RA). However, this subset did not produce interleukin 12 (IL-12) but upregulated CD103, which is essential for migration to the mesenteric lymph nodes (MLN). Interestingly, CD103+CD11b−CD8+DC in the MLN, in addition to processing RA, also secreted IL-12 and were responsible for gut imprinting specificity on mucosal CD8 T cells. To the best of our knowledge, this is the first report describing the importance of MLN CD103+CD11b−CD8+DC isolated from infected animals in the generation of an IEL response against a live pathogen.

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