Nous remercions sincèrement le Dr Mustafa Khokha, Yale University, permettant l’utilisation de ses ressources de laboratoire. Nous sommes reconnaissants à M. Wu Ming-Jie pour assistance technique excellente. EF est un enquêteur HHMI. Ce travail a été soutenu par un don du John Monsky et Jennifer Weis Monsky Lyme Disease Research Fund.

<ol>
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Les auteurs n’ont aucun conflit d’intérêt à divulguer.

Il s’agit de la première utilisation d’une technique d’hybridation (WMISH) ensemble-mount sur place pour étudier la flore microbienne d’un arthropod vecteur d’agents pathogènes. Notre protocole est une adaptation de celui utilisé pour étudier le développement chez la drosophile et les embryons de grenouille25,26. Entier-Montez RNA in situ hybridation a été couramment utilisée pour localiser la transcription des gènes...

Humaine et animale des pathogènes transmis par les arthropodes vecteurs sont trouvent dans le monde entier et représentent environ 20 % des maladies infectieuses dans le monde1, mais efficace et n’existe pas de vaccin sécuritaire contre la plupart de ces agents pathogènes. Notre compréhension de l’importance du rôle des micro-organismes commensaux, symbiotiques et pathogènes, collectivement connus comme le microbiome2, en modulant et façonner la santé de presque tous les métazoaires3 est en expansion. Il est maintenant évident que les arthropodes vecteurs d’agents pathogènes hébergent également le microbiote intestinal et auraient dû être divulgués ces micro-organismes associés à vecteur d’influencer divers vecteurs pathogènes4,5. Le microbiome arthropod se compose des eubactéries, archées, les virus et les microbes eucaryotes comme les protozoaires, nématodes et champignons6. Cependant, recherche prédominant mis l’accent a sur eubactéries due, en partie, à la disponibilité de gènes marqueurs et bases de données bibliographiques pour identifier des membres bactériens spécifiques.
En mettant l’accent sur les Ixodes scapularis, la tique vectrice de plusieurs agents pathogènes humains, dont Borrelia burgdorferi7, l’agent causal de la maladie de Lyme, l’optimisation d’une technique de visualisation microbienne visait à améliorer notre compréhension des tiques microbiote intestinal dans le cadre d’interactions pathogène-vecteur. Il reste plusieurs questions à trancher dans le domaine de microbiome tique. L’intestin est le site de la première rencontre élargie entre la tique et le pathogène entrant dans le cadre des pathogènes horizontalement transférés ; par conséquent, comprendre le rôle de vecteur microbiote intestinal en modulant les interactions pathogène-vecteur révélera aperçu significatif. Tiques ont un mode unique de la digestion du repas de sang, où le traitement de la farine de sang composants prend place intracellulairement8. La lumière du tube digestif sert apparemment comme récipient pour contenir le repas de sang comme les flux de la tique et assimilation et digestion des éléments nutritifs s’ensuivent tout au long des jours d’alimentation et continuent après réplétion. Les agents pathogènes acquis par la tique pendant l’alimentation entrer la lumière du tube digestif ainsi que les farines de sang et la lumière devient donc un site principal d’interactions entre la tique, pathogène et la microflore résidente. Comme digestion produit par le biais de la réplétion et de la tique Ixodes mue, le tube digestif subit des changements structuraux et fonctionnels9. La composition et l’organisation spatiale des bactéries de l’intestin est également susceptible de varier de concert avec le milieu changeant de tube digestif. Par conséquent, il est important de comprendre l’architecture des bactéries résidentes dans l’intestin de la tique pour bien comprendre l’interaction des tiques, pathogène et microbiote intestinal.
Techniques moléculaires pour décrire le microbiote hôte associés à systématiquement utilisent les parallèles séquençage haut débit stratégies10 amplifier et de séquencer l’ADN ribosomique 16 s bactérienne (ADNr). Ces stratégies de séquençage contournent la nécessité d’obtenir des cultures axéniques de bactéries spécifiques et fournir une description détaillée de tous les membres bactériennes représentées dans l’échantillon. Néanmoins, ces stratégies sont confondus par l’incapacité à distinguer des contaminations environnementales des résidents de bona fide . En outre, lors de l’évaluation d’échantillons, comme les tiques, qui sont de petite taille et donc contiennent de l’ADN de microbiote spécifiques faible rendements, la probabilité de l’amplification des contaminants environnementaux est accrue11 et se traduit par l’interprétation ambiguë de composition de microbiome. Caractérisation fonctionnelle en conjonction avec la visualisation de certaines bactéries viables, par conséquent, sera essentielle pour définir et pour discerner le microbiome du coutil temporellement et spatialement. Pour atteindre cet objectif, nous avons profité de l’entier-Montez RNA in situ hybridation. Cette technique est couramment utilisée pour évaluer les profils d’expression génique dans les organes et les embryons12,13,14 et permet une analyse semi-quantitative de l’expression sur l’ensemble de l’échantillon d’intérêt. Cela diffère des traditionnels in situ hybridation des techniques qui utilisent des sections de tissu et nécessitent souvent une analyse approfondie du matériel sectionné avec un assemblage informatique pour prédire l’expression en entier organes15. Tandis que l’ensemble monture désigne généralement tout organismes12, entier-Montez désigne ici les boyaux entiers et organes. Les avantages d’utiliser l’approche de l’ARN entier-montent en situ hybridation afin d’évaluer l’architecture des tiques microbiote intestinal sont multiples. L’intestin de la tique est composé de 7 paires de diverticules, chaque paire variant en taille16. Les différences fonctionnelles, si il en est, parmi ces diverticules, ne sont pas compris dans le contexte de la biologie de la tique, cocher microbiote ou tique-pathogen interactions. Manipulations de l’intestin qui rompent les diverticules de l’intestin écarteraient microbiote présent dans la lumière du tube digestif ou ceux qui sont associés sans serrer le tube digestif et aboutir à une interprétation erronée de la localisation spatiale du microbiote. Fluorescence-étiquetée RNA in situ hybridation a été utilisée plus tôt pour examiner la tique gut transcriptions17 en fixant et en ouvrant les diverticules gut individuels afin d’assurer l’hybridation de la sonde et de localiser de b. burgdorferi transcriptions en sectionnant la paraffine entier tiques18. Ces deux approches nécessitent des manipulations des tissus tique avant l’hybridation qui influeraient sur l’architecture de microflore intestinale.
Dans ce rapport, nous décrivons en détail le protocole afin d’examiner la tique viable microbiote intestinal entier-montent en situ hybridation (WMISH). L’utilisation de l’ensemble de montage RNA in situ hybridation permet une compréhension globale de la présence et l’abondance des bactéries de l’intestin spécifique dans les différentes régions de l’intestin et peut stimuler de nouvelles connaissances sur la biologie gut tick dans le contexte de la colonisation de l’agent pathogène et la transmission. En outre, l’utilisation de sondes d’ARN dirigée contre des ARN bactérien spécifiques permet la détection de bactéries viables dans l’intestin de la tique.

<table><tbody><tr><td>Sefar NITEX Nylon Mesh, 110 micron</td><td>Amazon</td><td>03-110/47</td><td></td></tr><tr><td>pGEM-T Easy Vector System</td><td>Promega</td><td>A1360</td><td></td></tr><tr><td>Digoxygenin-11-UTP</td><td>Roche</td><td>1209256910</td><td></td></tr><tr><td>dNTP</td><td>New England Biolabs&amp;nbsp;</td><td>N0447S</td><td></td></tr><tr><td>DNAse I(RNAse-free)</td><td>New England Biolabs</td><td>M0303S</td><td></td></tr><tr><td>HiScribe SP6 RNA synthesis kit</td><td>New England Biolabs</td><td>E2070S</td><td></td></tr><tr><td>HiScribe T7 High Yield RNA Synthesis Kit</td><td>New England Biolabs</td><td>E2040S</td><td></td></tr><tr><td>Water, RNase-free, DEPC-treated</td><td>American Bioanalytical</td><td>AB02128-00500</td><td></td></tr><tr><td>EDTA, 0.5M, pH 8.0</td><td>American Bioanalytical</td><td>AB00502-01000</td><td></td></tr><tr><td>Formaldehyde, 37%</td><td>JT Baker</td><td>2106-01</td><td></td></tr><tr><td>Formamide</td><td>American Bioanalytical</td><td>AB00600-00500</td><td></td></tr><tr><td>EGTA</td><td>Sigma Aldrich</td><td>E-4378</td><td></td></tr><tr><td>DPBS, 10X</td><td>Gibco</td><td>14300-075</td><td></td></tr><tr><td>Tween-20</td><td>Sigma Aldrich</td><td>P1379-25ML</td><td></td></tr><tr><td>Proteinase K</td><td>Sigma Aldrich</td><td>3115879001</td><td></td></tr><tr><td>Triethanolamine HCl</td><td>Sigma Aldrich</td><td>T1502-100G</td><td></td></tr><tr><td>Acetic anhydride</td><td>Sigma Aldrich</td><td>320102-100ML</td><td></td></tr><tr><td>Paraformaldehyde</td><td>ThermoScientific/Pierce</td><td>28906</td><td></td></tr><tr><td>SSC, 20X</td><td>American Bioanalytical</td><td>AB13156-01000</td><td></td></tr><tr><td>RNA from torula yeast</td><td>Sigma Aldrich</td><td>R3629-5G</td><td></td></tr><tr><td>Heparin, sodium salt</td><td>Sigma Aldrich</td><td>H3393-10KU</td><td></td></tr><tr><td>Denhardt&#039;s Solution, 50X</td><td>Sigma Aldrich</td><td>D2532-5ML</td><td></td></tr><tr><td>CHAPS hydrate</td><td>Sigma Aldrich</td><td>C3023-1G</td><td></td></tr><tr><td>RNase A</td><td>Sigma Aldrich</td><td>10109142001</td><td></td></tr><tr><td>RNase T1</td><td>ThermoScientific&amp;nbsp;</td><td>EN0541</td><td></td></tr><tr><td>Maleic acid</td><td>Sigma Aldrich</td><td>M0375-100G</td><td></td></tr><tr><td>Blocking reagent</td><td>Sigma Aldrich</td><td>11096176001</td><td></td></tr><tr><td>Anti-Digoxigenin-AP, Fab fragments</td><td>Sigma Aldrich</td><td>11093274910</td><td></td></tr><tr><td>Levamisol hydrochloride</td><td>Sigma Aldrich</td><td>31742-250MG</td><td></td></tr><tr><td>Chromogenic substrate for alkaline phosphatase</td><td>Sigma Aldrich</td><td>11442074001</td><td></td></tr><tr><td>Bouin&#039;s solution</td><td>Sigma Aldrich</td><td>HT10132-1L</td><td></td></tr><tr><td>Hydrogen peroxide</td><td>Mallinkrodt Baker, Inc</td><td>2186-01</td><td></td></tr><tr><td>Single stranded RNA ladder</td><td>Ambion -Millenium</td><td>AM7151</td><td></td></tr><tr><td>&amp;nbsp;#11 High-Carbon steel blades&amp;nbsp;</td><td>C and A Scientific Premiere</td><td>#11-9411</td><td></td></tr><tr><td>Thermocycler</td><td>BioRad, CA</td><td>1851148</td><td></td></tr><tr><td>Spectrophotometer</td><td>ThermoScientific&amp;nbsp;</td><td>NanoDrop 2000C</td><td></td></tr><tr><td>Orbital shaker</td><td>VWR</td><td>DS-500E Digital Orbital shaker</td><td></td></tr><tr><td>Shaking water bath</td><td>BELLCO Glass, Inc</td><td>Hot Shaker-7746-12110</td><td></td></tr><tr><td>Gel&amp;nbsp; documentation system</td><td>BioRad</td><td>Gel Doc XR+ Gel documentation system</td><td></td></tr><tr><td>Bright-field Microscope&amp;nbsp;</td><td>Nikon</td><td>NikonSM2745T</td><td></td></tr><tr><td>Bright-field Microscope&amp;nbsp;</td><td>Zeiss</td><td>AXIO Scope.A1</td><td></td></tr><tr><td>Dissection microscope&amp;nbsp;</td><td>Zeiss</td><td>STEMI 2000-C</td><td></td></tr><tr><td>&amp;nbsp;Light box</td><td>VWR</td><td>102097-658</td><td></td></tr><tr><td>PCR purification kit&amp;nbsp;</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>Image capture software</td><td>Zeiss</td><td>Zen lite</td><td></td></tr><tr><td>Image editing software&amp;nbsp;</td><td>Adobe&amp;nbsp;</td><td>Adobe Photoshop CS4 version 11.0</td><td></td></tr><tr><td>Image analysis software</td><td>National Institutes of Health</td><td>ImageJ-NIH /imagej.nih.gov/ij/</td><td></td></tr><tr><td>Automation compatible instrumentation</td><td>Intavis Bioanalytical Instruments, Tubingen, Germany).&amp;nbsp;</td><td>Intavis, Biolane HT1.16v</td><td></td></tr></tbody></table>

visualization of microbiota in tick guts by whole-mount in situ hybridization

Maladies infectieuses transmises par des arthropodes vecteurs continuent de poser une menace significative pour la santé humaine dans le monde entier. Les pathogènes responsables de ces maladies, n’existent pas isolément lorsqu’ils colonisent le vecteur ; plutôt, ils s’engagent sans doute dans leurs interactions avec les microorganismes résidents dans la lumière du tube digestif. Le vecteur intestinal a été démontré à jouer un rôle important dans la transmission d’agents pathogènes pour plusieurs maladies à transmission vectorielle. Si les bactéries résidant dans l’intestin de la tique Ixodes scapularis , le vecteur de plusieurs agents pathogènes humains, y compris de Borrelia burgdorferi, influent sur la transmission de la tique d’agents pathogènes n’est pas déterminé. Nous avons besoin de méthodes permettant de caractériser la composition des bactéries associées à l’intestin de la tique afin de faciliter une meilleure compréhension des interactions entre les espèces potentielles dans l’intestin de la tique. L’utilisation entier-montent en situ hybridation pour visualiser des ARN transcrits associées à certaines espèces bactériennes permet pour la collecte de données qualitatives concernant l’abondance et la distribution de la microflore dans le tissu intact. Cette technique permet d’analyser les changements dans le milieu de la microflore intestinale au cours de la tique d’alimentation et peut également être appliquée pour analyser l’expression des gènes de la tique. Coloration de toute tique tripes rendement d’informations sur la distribution spatiale brute des ARN dans le tissu cible sans la nécessité d’une reconstruction tridimensionnelle et est moins affectée par la contamination de l’environnement, qui confond souvent basée sur le séquençage Méthodes fréquemment utilisées pour l’étude des communautés microbiennes complexes. Dans l’ensemble, cette technique est un outil précieux qui permet de mieux comprendre les interactions pathogène-vecteur-microbiote et leur rôle dans la transmission de la maladie.

La mesure et estimation de la qualité des sondes d’ARN sont essentiels avant de commencer la coloration. In vitro l’efficacité de transcription dépend fortement de la quantité et la qualité de la matrice d’ADN. Régulièrement, nous avons visualisé les sondes d’ARN sur un gel de formaldéhyde pour vérifier la pureté et la quantité de sonde généré par les réactions de la transcription. Les sondes doivent apparaître sous forme de bandes lumineuses, discrets (<...

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Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Visualisation du microbiote dans Guts Tick par hybridation entier-Montez In Situ

Nous présentons ici un protocole pour spatialement et temporellement évaluer la présence de la microflore viable dans les tripes de tique à l’aide d’une approche modifiée entier-Montez l’hybridation in situ.

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these ...

visualization-microbiota-tick-guts-whole-mount-situ

Research

JoVE Journal

Genetics

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

9.5K vues.  Yale University School of Medicine. Nous présentons ici un protocole pour spatialement et temporellement évaluer la présence de la microflore viable dans les tripes de tique à l’aide d’une approche modifiée entier-Montez l’hybridation in situ.

Vidéo: Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

In Situ Étiquetage de l&#39; ADN mitochondrial de réplication chez la drosophile adulte Ovaires par EdU Coloration

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

Recherche

Génétique

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Optogénétiques mutagenèse aléatoire Utilisation Histone-miniSOG dans<em&gt; C. elegans</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

Détection de Inter-chromosomiques Aberrations Stable par Fluorescence Multiple<em&gt; In Situ</em&gt; Hybridation (mFISH) et Spectral caryotype (SKY) chez des souris irradiées

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1. A tick-borne murine model 2 has been developed to study Lyme disease in the laboratory. While na&iacute;ve ticks can be infected with B. burgdorferi by feeding them on infected mice, the molting process takes several weeks to months to complete. Therefore, development of more rapid and efficient tick infection techniques, such as a microinjection-based procedure, is an important tool for the study of Lyme disease 3,4. The procedure requires only hours to generate infected ticks and allows control over the delivery of equal quantities of spirochetes in a cohort of ticks. This is particularly important as the generation of B. burgdorferi infected ticks by the natural feeding process using mice fails to ensure 100% infection rate and potentially results in variation of pathogen burden amongst fed ticks. Furthermore, microinjection can be used to infect ticks with B. burgdorferi isolates in cases where an attenuated strain is unable to establish infection in mice and thus can not be naturally acquired by ticks 5. This technique can also be used to deliver a variety of other biological materials into ticks, for example, specific antibodies or double stranded RNA 6. In this article, we will demonstrate the microinjection of nymphal ticks with in vitro-grown B. burgdorferi. We will also describe a method for localization of Lyme disease pathogens in the tick gut using confocal immunofluorescence microscopy.

Lyme disease research studies often require generation of ticks infected with the pathogen Borrelia burgdorferi, a process that typically takes several weeks. Here we demonstrate a microinjection-based tick infection procedure that can be accomplished within hours. We also demonstrate an immunofluorescence method for in situ localization of B. burgdorferi within ticks.

Méthodes de transfert rapide et la localisation des agents pathogènes dans l&#39;intestin de Lyme Tick

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1.  ...

Immunologie et infection

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Lyme disease is the most commonly-reported vector-borne disease in North America. The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks. Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

Alimentation des tiques sur les animaux pour la transmission et Xénodiagnostic dans la recherche la maladie de Lyme

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the  attachment and blood feeding of Ixodes species ticks on ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

Visualisation de Candida albicans dans le tractus gastro-intestinal murine utilisant l'hybridation fluorescente in Situ

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota and the vertebrate gut. The spatial landscape of the gut microbiota is tightly controlled by physical features - intestinal mucus, crypts, and folds - and is affected by host-controlled properties such as pH, oxygen availability, and immune factors. These properties limit the ability of commensal microbes and pathogens alike to colonize the gut stably. At the micron-scale, microbial organization determines the close-range interactions between different microbes as well as the interactions between microbes and their host. These interactions then affect large-scale organ function and host health.
This protocol enables the visualization of the gut microbiota spatial organization from distances between cells to organ-wide scales. The method is based on fixing gut tissues while preserving intestinal structure and mucus properties. The fixed samples are then embedded, sectioned, and stained to highlight specific bacterial species through fluorescence in situ hybridization (FISH). Host features, such as mucus and host cell components, are labeled with fluorescently labeled lectins. Finally, the stained sections are imaged using a confocal microscope utilizing tile-scan imaging at high magnification to bridge the micron to centimeter length scales. This type of imaging can be applied to intestinal sections from animal models and biopsies from human tissues to determine the biogeography of the microbiota in the gut in health and disease.

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

This streamlined protocol details a workflow to detect and image bacteria in complex tissue samples, from fixing the tissue to staining microbes with fluorescent in situ hybridization.

Visualisation des interactions entre le microbiote intestinal et l’hôte par hybridation in situ par fluorescence, coloration par lectine et imagerie

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota ...

Suivi d’agents pathogènes d’organe entier par quantification par rapporteur fluorescent

High&#45;Resolution Three&#45;Dimensional Whole&#45;Organ Tomography of Microbial Infections

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen cells within this diverse environment significantly affects their stress levels, responses, fate, and contribution to the overall progression of the disease and treatment failure. However, due to the technical difficulties in locating &#181;m-sized pathogen cells within cm-sized host organs, this area of research has been relatively unexplored. Here, we present a method for addressing this challenge. We employ serial two-photon tomography and AI-enhanced image analysis to locate individual Salmonella cells throughout the entire spleen, liver lobes, and whole lymph nodes of infected mice. Using fluorescent reporters and in vivo antibody administration, the replication rate of single Salmonella cells, their local interaction with specific immune cells, and bacterial responses to antibiotics can be determined. These methodologies open avenues for a comprehensive examination of infections, their prevention, and treatment within the three-dimensional tissue context.

Pleins feux sur l’auteur : Démêler les réponses bactériennes aux antibiotiques et au système immunitaire dans les tissus

Here, we describe a procedure that enables organ-wide detection of pathogenic bacteria during infection and quantification of fluorescent reporter activities.

Tomographie tridimensionnelle à haute résolution des infections microbiennes

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen ...

Visualisation du microbiote dans Guts Tick par hybridation entier-Montez In Situ

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