scholarly journals Visualization of intestinal infections with astro- and sapovirus in mink (Neovison vison) kits by in situ hybridization

FEMS Microbes ◽  
2021 ◽  
Author(s):  
Julie Melsted Birch ◽  
Mikael Leijon ◽  
Søren Saxmose Nielsen ◽  
Tina Struve ◽  
Henrik Elvang Jensen
Keyword(s):  
In Situ Hybridization ◽  
Goblet Cells ◽  
Neovison Vison ◽  
Localization Pattern ◽  
Intestinal Infections ◽  
Specific Localization ◽  
The Impact ◽  
First Time ◽  
Mink Kits

Abstract Clarification of the infection microbiology remains a challenge in the pre-weaning diarrhea (PWD) syndrome in farmed mink (Neovison vison). Duodenal, jejunal and colon sections from 36 mink kits with PWD were systematically examined by chromogen in situ hybridization (CISH) targeting two incriminated viruses: Mink astrovirus and mink sapovirus. Using the RNAscope® 2.5 HD Duplex Assay astrovirus and sapovirus were visualized and simultaneously demonstrated in the gut tissue. Both viruses infect enterocytes in the small intestine with a specific localization pattern; astrovirus affects the two apical thirds of the villi, whereas sapovirus generally affects the basal parts of the villi. Furthermore, we demonstrated that astrovirus in mink does not target the goblet cells. This is the first time astro- and calicivirus have been visualized in mink kit gut tissue, and these findings might be important in clarification of the impact of these viruses in the PWD syndrome.

Fluorescence in situ hybridization for identification and visualization of microorganisms in infected heart valve tissue as addition to standard diagnostic tests improves diagnosis of endocarditis

2019 ◽  
Vol 29 (5) ◽  
pp. 678-684
Author(s):  
Simone Eichinger ◽  
Judith Kikhney ◽  
Annette Moter ◽  
Alexandra Wießner ◽  
Walter B Eichinger
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Diagnostic Tests ◽  
Diagnostic Testing ◽  
Bacterial Biofilm ◽  
Causative Organism ◽  
Histopathological Diagnosis ◽  
Duke Criteria ◽  
The Impact

Abstract OBJECTIVES In infective endocarditis (IE), identification of the causative organism and consecutive treatment are crucial for patient survival. Although the macroscopic aspect resembles infected tissue, standard diagnostic tests often fail to allow one to identify bacteria. Fluorescence in situ hybridization (FISH) is a molecular, culture-independent technique that allows one to identify and visualize microorganisms within tissue and to recognize their morphology, number and activity. We analysed the diagnostic benefit of FISH/polymerase chain reaction (PCR) by comparing its results to those of standard diagnostic tests. METHODS From September 2015 to April 2018, 128 patients underwent first-time or redo valve surgery to treat IE. Patients were designated according to the modified Duke criteria as definite (n = 61), possible (n = 34) or rejected (n = 33) IE. Tissue specimens obtained intraoperatively were analysed using FISH/PCR in addition to undergoing standard diagnostic testing and PCR alone. RESULTS We used blood cultures to detect microorganisms in 67/128 patients; valve cultures, in 34/128; PCR, in 67/128; histopathological diagnosis showed IE in 72/128 cases. We were able to detect microorganisms in 103/128 cases using FISH/PCR, with 55/61 in definite IE. Furthermore, we were able to identify 26 cases of bacterial biofilm using FISH/PCR, despite antibiotic treatment of 61 in the definite, 13 in the possible and 1 in the rejected group, including 8/33 patients in the rejected group with active bacteria. In all cases, the patient’s therapy was altered. CONCLUSIONS FISH/PCR was used to identify microorganisms in cases in which standard diagnostic tests failed to provide sufficient results for various reasons. Furthermore, FISH/PCR enabled us to identify bacterial biofilms and to differentiate between active versus degraded bacteria, thus indicating the impact of treatment. Therefore, we suggest FISH/PCR as an additional diagnostic tool in IE alongside standard diagnostic tests.

scholarly journals Investigation of an Acetate-Fed Denitrifying Microbial Community by Stable Isotope Probing, Full-Cycle rRNA Analysis, and Fluorescent In Situ Hybridization-Microautoradiography

2005 ◽  
Vol 71 (12) ◽  
pp. 8683-8691 ◽  
Author(s):  
Maneesha P. Ginige ◽  
Jürg Keller ◽  
Linda L. Blackall
Keyword(s):  
In Situ Hybridization ◽  
Stable Isotope ◽  
Activated Sludge ◽  
Fluorescent In Situ Hybridization ◽  
Batch Reactor ◽  
External Source ◽  
Stable Isotope Probing ◽  
Rrna Gene ◽  
The Impact

ABSTRACT The acetate-utilizing microbial consortium in a full-scale activated sludge process was investigated without prior enrichment using stable isotope probing (SIP). [13C]acetate was used in SIP to label the DNA of the denitrifiers. The [13C]DNA fraction that was extracted was subjected to a full-cycle rRNA analysis. The dominant 16S rRNA gene phylotypes in the 13C library were closely related to the bacterial families Comamonadaceae and Rhodocyclaceae in the class Betaproteobacteria. Seven oligonucleotide probes for use in fluorescent in situ hybridization (FISH) were designed to specifically target these clones. Application of these probes to the sludge of a continuously fed denitrifying sequencing batch reactor (CFDSBR) operated for 16 days revealed that there was a significant positive correlation between the CFDSBR denitrification rate and the relative abundance of all probe-targeted bacteria in the CFDSBR community. FISH-microautoradiography demonstrated that the DEN581 and DEN124 probe-targeted cells that dominated the CFDSBR were capable of taking up [14C]acetate under anoxic conditions. Initially, DEN444 and DEN1454 probe-targeted bacteria also dominated the CFDSBR biomass, but eventually DEN581 and DEN124 probe-targeted bacteria were the dominant bacterial groups. All probe-targeted bacteria assessed in this study were denitrifiers capable of utilizing acetate as a source of carbon. The rapid increase in the number of organisms positively correlated with the immediate increase in denitrification rates observed by plant operators when acetate is used as an external source of carbon to enhance denitrification. We suggest that the impact of bacteria on activated sludge subjected to intermittent acetate supplementation should be assessed prior to the widespread use of acetate in the wastewater industry to enhance denitrification.

Localization of the sites of synthesis and action of prolactin by immunocytochemistry and in-situ hybridization within the human utero-placental unit

1991 ◽  
Vol 7 (3) ◽  
pp. 241-247 ◽  
Author(s):  
W.-X. Wu ◽  
J. Brooks ◽  
M. R. Millar ◽  
W. L. Ledger ◽  
P. T. K. Saunders ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Culture Medium ◽  
Cdna Probe ◽  
Specific Hybridization ◽  
Decidual Cells ◽  
Its Gene ◽  
First Time ◽  
Silver Grains ◽  
Prolactin Mrna

ABSTRACT While the fetal pituitary synthesizes and releases prolactin, it is also produced within the utero-placental unit during pregnancy in women and has been localized in the amnion, chorion and decidua. However, it is not clear whether prolactin is synthesized within all these non-fetal pituitary tissues. We have investigated prolactin production and its gene expression using tissue culture, immunocytochemistry and in-situ hybridization techniques. Prolactin was immunolocalized not only in the decidua but also in amnion and trophoblast cells. In contrast, the in-situ hybridization results showed that silver grains, formed by specific hybridization of a prolactin cDNA probe to prolactin mRNA, were confined to decidual cells of early and term pregnancy. The results from tissue cultures correlated well with those of in-situ hybridization, that is that only the decidua made detectable prolactin, while it was undetectable in the culture medium from trophoblast tissue, irrespective of the stage of pregnancy. This study, for the first time, establishes that only decidualized cells are involved in biosynthesis of prolactin; other prolactin-containing cells in the amnion and trophoblast appear to sequester prolactin, possibly via receptors, suggesting that prolactin may play an important paracrine role within the amnion and syncitio- and cytotrophoblast of the utero-placental unit.

scholarly journals Fluorescence In Situ Hybridization (FISH) on Human Chromosomes Using Photoprobe Biotin-labeled Probes

2003 ◽  
Vol 51 (4) ◽  
pp. 549-551 ◽  
Author(s):  
Anja Weise ◽  
Peter Harbarth ◽  
Uwe Claussen ◽  
Thomas Liehr
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Yeast Artificial Chromosome ◽  
Artificial Chromosome ◽  
Dna Probes ◽  
Human Chromosomes ◽  
First Time ◽  
Established Technique ◽  
Whole Chromosome Painting

Fluorescence in situ hybridization (FISH) on human chromosomes in meta-and interphase is a well-established technique in clinical and tumor cytogenetics and for studies of evolution and interphase architecture. Many different protocols for labeling the DNA probes used for FISH have been published. Here we describe for the first time the successful use of Photoprobe biotin-labeled DNA probes in FISH experiments. Yeast artificial chromosome (YAC) and whole chromosome painting (wcp) probes were tested.

scholarly journals Catalyzed Reported Deposition-Fluorescence In Situ Hybridization Protocol To Evaluate Phagotrophy in Mixotrophic Protists

2005 ◽  
Vol 71 (11) ◽  
pp. 7321-7326 ◽  
Author(s):  
Juan M. Medina-Sánchez ◽  
Marisol Felip ◽  
Emilio O. Casamayor
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Signal Amplification ◽  
Cell Attachment ◽  
Cell Loss ◽  
Alexa Fluor ◽  
Hybridization Protocol ◽  
Card Fish ◽  
First Time

ABSTRACT We describe a catalyzed reported deposition-fluorescence in situ hybridization (CARD-FISH) protocol particularly suited to assess the phagotrophy of mixotrophic protists on prokaryotes, since it maintains cell and plastid integrity, avoids cell loss and egestion of prey, and allows visualization of labeled prey against plastid autofluorescence. This protocol, which includes steps such as Lugol's-formaldehyde-thiosulfate fixation, agarose cell attachment, cell wall permeabilization with lysozyme plus achromopeptidase, and signal amplification with Alexa-Fluor 488, allowed us to detect almost 100% of planktonic prokaryotes (Bacteria and Archaea) and, for the first time, to show archaeal cells ingested by mixotrophic protists.

scholarly journals EnVision+, a New Dextran Polymer-based Signal Enhancement Technique for In Situ Hybridization (ISH)

2001 ◽  
Vol 49 (9) ◽  
pp. 1067-1071 ◽  
Author(s):  
Klaus Hermann Wiedorn ◽  
Torsten Goldmann ◽  
Christof Henne ◽  
Heike Kühl ◽  
Ekkehard Vollmer
Keyword(s):  
In Situ Hybridization ◽  
Signal Amplification ◽  
Detection System ◽  
Lower Sensitivity ◽  
The Novel ◽  
Visualization System ◽  
Integrated Optical ◽  
Assay Time ◽  
First Time

Seventy paraffin-embedded cervical biopsy specimens and condylomata were tested for the presence of human papillomavirus (HPV) by conventional in situ hybridization (ISH) and ISH with subsequent signal amplification. Signal amplification was performed either by a commercial biotinyl-tyramide-based detection system [GenPoint (GP)] or by the novel two-layer dextran polymer visualization system EnVision+ (EV), in which both EV–horseradish peroxidase (EV–HRP) and EV–alkaline phosphatase (EV–AP) were applied. We could demonstrate for the first time, that EV in combination with preceding ISH results in a considerable increase in signal intensity and sensitivity without loss of specificity compared to conventional ISH. Compared to GP, EV revealed a somewhat lower sensitivity, as measured by determination of the integrated optical density (IOD) of the positively stained cells. However, EV is easier to perform, requires a shorter assay time, and does not raise the background problems that may be encountered with biotinyl–tyramide-based amplification systems. (J Histochem Cytochem 49:1067–1071, 2001)

Effects of selected pharmaceuticals on riverine biofilm communities

2005 ◽  
Vol 51 (8) ◽  
pp. 655-669 ◽  
Author(s):  
John R Lawrence ◽  
George D.W Swerhone ◽  
Leonard I Wassenaar ◽  
Thomas R Neu
Keyword(s):  
In Situ Hybridization ◽  
Algal Biomass ◽  
Lectin Binding ◽  
Stable Carbon Isotope ◽  
Bacterial Biomass ◽  
Relative Increase ◽  
Carbon Utilization ◽  
The Impact ◽  
Isotope Analyses

Although pharmaceutical and therapeutic products are widely found in the natural environment, there is limited understanding of their ecological effects. Here we used rotating annular bioreactors to assess the impact of 10 µg·L–1of the selected pharmaceuticals ibuprofen, carbamazepine, furosemide, and caffeine on riverine biofilms. After 8 weeks of development, community structure was assessed using in situ microscopic analyses, fluor-conjugated lectin binding, standard plate counts, fluorescent in situ hybridization, carbon utilization spectra, and stable carbon isotope analyses. The biofilm communities varied markedly in architecture although only caffeine treated biofilms were significantly thicker. Cyanobacteria were suppressed by all 4 compounds, whereas the nitrogen containing caffeine, furosemide, and carbamazepine increased algal biomass. Ibuprofen and carbamazepine reduced bacterial biomass, while caffeine and furosemide increased it. Exopolymer content and composition of the biofilms was also influenced. Significant positive and negative effects were observed in carbon utilization spectra. In situ hybridization analyses indicated all treatments significantly decreased the gamma-proteobacterial populations and increased beta-proteobacteria. Ibuprofen in particular increased the alpha-proteobacteria, beta-proteobacteria, cytophaga-flavobacteria, and SRB385 probe positive populations. Caffeine and carbamazepine additions resulted in significant increases in the high GC354c and low GC69a probe positive cells. Live–dead analyses of the biofilms indicated that all treatments influenced the ratio of live-to-dead cells with controls having a ratio of 2.4, carbamazepine and ibuprofen being 3.2 and 3.5, respectively, and furosemide and caffeine being 1.9 and 1.7, respectively. Stable isotope analyses of the biofilms indicated δ13C values shifted to more negative values relative to control biofilms. This shift may be consistent with proportional loss of cyanobacteria and relative increase in algal biomass rather than incorporation of pharmaceutical carbon into microbial biofilm. Thus, at 10 ug·L–1levels pharmaceuticals exhibit both nutrient-like and toxic effects on riverine microbial communities.Key words: carbamazepine, ibuprofen, furosemide, caffeine, laser microscopy, digital imaging, in situ hybridization, stable isotopes.

scholarly journals Method to obtain homozygous introgressing segments in Drosophila to identify the presence of hybrid sterility genes of the reproductive isolation

2020 ◽  
Author(s):  
Francisco García-Franco ◽  
Lilian Barandica-Cañon ◽  
Ezel Galindo-Pérez ◽  
Martha Martínez García ◽  
Blanca Chávez-Sandoval
Keyword(s):  
In Situ Hybridization ◽  
Physical Map ◽  
Hybrid Sterility ◽  
Bioinformatic Analysis ◽  
Chromosome 4 ◽  
Genomic Map ◽  
Hybrid Lines ◽  
First Time ◽  
Polythene Chromosomes

Abstract Here, we present for the first time, a method to generate homozygous segmental introgressions, by means of crosses between a pair of synmorphic species. The introgressions were monitored by the cytogenetic method of polygenic chromosome asynapses. Later the introgressions were evaluated in their capacity to produce sterility in segmental males. Also, the smallest segment with the capacity to produce sterility in segmental males was mapped by in situ hybridization of polythene chromosomes, using 8 sequences of BACs clones as probes. Finally, a bioinformatic analysis was carried out to identify the presence of particular genes. From 2 parental strains, D. buzzatii and D. koepferae, 6 simple segmental hybrid lines were generated, whose introgressing segments are distributed along chromosome 4 of these species. From the 6 simple segmental lines and by means of a new crossing strategy, the 6 respective homozygous segmental hybrid offspring were obtained, each of them carrying a specific homozygous introgression. None of the 6 heterozygous introgressions was capable of producing sterility in segmental males, while 4 of the same homozygous introgressions produced total sterility in segmental males, including in this group the two smallest introgressive segments, one of 5.03 % and the other 7.87% with respect to the total length of chromosome 4, which are located in the region F2 to F4 of the standard cytological map based on polythene chromosomes of the Drosophila Repleta group. In situ hybridization, using 8 clones from contig 1065 located along the F2 to F4 region of the physical map of D. buzzattii constructed in BACs, confirmed the precise location of the 6 clones in the chromosomal region F2 to F4 of chromosome 4 of the polygenic chromosomes of both D. buzzatii and D. mojavensis. The bioinformatic analysis of the F2 to F4 region, using the complete genetic sequence of the contig 1065 of D. buzzatti shows the presence of two predicted genes in the genomic map of D. buzzatii (g.1313.t1 and g.1314.t1), and the orthologous association of these 2 genes both with the D. moj_GI22766 gene of D. mojavensis and with the Trivet gene of D. melanogaster.

scholarly journals Shifting into high gear: how interstitial cells of Cajal change the motility pattern of the developing intestine

2020 ◽  
Vol 319 (4) ◽  
pp. G519-G528
Author(s):  
Nicolas R. Chevalier ◽  
Yanis Ammouche ◽  
Anthony Gomis ◽  
Clémence Teyssaire ◽  
Pascal de Santa Barbara ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
In Situ Hybridization ◽  
Calcium Imaging ◽  
Interstitial Cells Of Cajal ◽  
Interstitial Cell ◽  
Sharp Transition ◽  
Anoctamin 1 ◽  
Cells Of Cajal ◽  
First Time

We reveal a sharp transition from smooth muscle to interstitial cell of Cajal (ICC)-driven motility in the chicken embryo, leading to higher-frequency, more rhythmic contractile waves. We predict the transition to happen between 12 and 14 embryonic wk in humans. We image for the first time the onset of ICC activity in an embryonic gut by calcium imaging. We show the first KIT and anoctamin-1 (ANO1) in situ hybridization micrographs in the embryonic chicken gut.

scholarly journals Comparison of 2 fixatives in the porcine colon for in situ microbiota studies

2019 ◽  
Vol 97 (12) ◽  
pp. 4803-4809 ◽  
Author(s):  
Anna K Blick ◽  
Paula R Giaretta ◽  
Sarah Sprayberry ◽  
Clara Bush-Vadala ◽  
Chad B Paulk ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescent In Situ Hybridization ◽  
Goblet Cells ◽  
Clinical Settings ◽  
Tissue Fixation ◽  
Mucus Layer ◽  
Carnoy’S Solution ◽  
Cell Counts ◽  
Blue Stain

Abstract Fixation is the first step towards preservation of tissues and can impact downstream histological applications. Historically, formalin has been the fixative of choice in both research and clinical settings due to cost, accessibility, and broad applicability. Here, we describe a method for collection of porcine colon, and compare the usage of Carnoy’s solution (CS) to a 10% neutral buffered formalin (NBF) in tissue fixation. Consecutive colon samples were collected from 24 four-wk-old piglets and fixed in CS for 45 min or NBF for 24 h. We measured the thickness of the inner mucus layer using Alcian Blue stain and found thicker inner mucus layers in porcine colons fixed with CS as compared to NBF (P < 0.0001). Carnoy’s solution-fixed colon exhibited greater bacterial cell counts than NBF-fixed colon (P < 0.0022) after labeling with an eubacterial probe in fluorescent in situ hybridization (FISH). No difference was observed between the mucosal height (P = 0.42) and number of goblet cells (P = 0.66) between the 2 fixatives. From this, we concluded CS is more suitable than NBF for the preservation of the mucus layer and the associated mucosal bacteria in the porcine colon without compromising on overall tissue morphology. This study provides a useful sampling and fixation methodology for histology studies in the porcine gastrointestinal tract, and may be beneficial to microbiota, pathology, and nutrition studies.

Sign in / Sign up

Export Citation Format

Share Document