Die Autoren bedanken sich bei Labormitglieder für Zebrafisch Pflege und Wartung zu danken. Diese Arbeit wurde vom National Institutes of Health, National Research Service Award A155397 EA Harvie und NIH R01GM074827 Anna Huttenlocher unterstützt.

<ol>
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The authors declare that they have no competing financial interests.

Die Infektion Verfahren verwendet hier ist nützlich für die Untersuchung der Immunantwort des Wirts zu einer anfänglich lokalisierten Infektion bei 2-3 dpf Embryonen und Larven. Der Fokus eines Entzündungsreiz, wie Infektion, in einem geschlossenen Hohlraum, wie der Ohrbläschen ermöglicht die Untersuchung von Neutrophilen und Makrophagen-Chemotaxis und Phagozytose. Eine Einschränkung des Injizierens von Bakterien in das Ohrbläschen ist, daß die Fähigkeit von Neutrophilen, effizient phagozytieren Bakterien in m...

Streptococcus iniae ist ein großer Wasserkrankheitserreger, der in der Lage ist, die systemische Krankheiten bei den Fischen und Menschen 1 ist. Während S. iniae ist für große Verluste in der Aquakultur-Industrie zuständig ist, ist es auch ein Potenzial Zoonoseerreger, der Lage verursacht Erkrankung bei immun menschlichen Wirten mit klinischen Erkrankungen ähnlich denen von anderen Streptokokken menschlichen Erregern verwendet wird. Aufgrund ihrer Ähnlichkeit mit menschlicher Krankheitserreger, ist es wichtig, S. studieren iniae Pathogenese der Erkrankung in Zusammenhang mit einem natürlichen Wirt. Ein erwachsenen Zebrafisch-Modell von S. iniae Infektion ergab, robust Infiltration von Leukozyten an das Host-lokalisierten Ort der Infektion sowie eine schnelle Zeit bis zum Tod Gastgeber, eine Zeit zu kurz, um das adaptive Immunsystem 7 einzubeziehen. Um zu gewinnen eine eingehende in der angeborenen Immunantwort auf S. betrachten iniae Infektion in vivo ist es erforderlich, ein Modell zu zugänglicher n verwendenon-invasive Echtzeit-Bildgebung. Die Larven Zebrafisch hat eine Reihe von Vorteilen, die es immer attraktiver Wirbeltiermodell zur Untersuchung Wirt-Pathogen-Interaktionen zu machen. Zebrafische sind relativ preiswert und einfach zu bedienen und zu halten im Vergleich zu Säugermodellen. Adaptive Immunität ist nicht funktionell reifen, bis 4-6 Wochen nach der Befruchtung, aber Larven haben eine hoch konservierte Wirbel angeborenen Immunsystems mit Ergänzung, Toll-like Rezeptoren, Zytokine und Neutrophilen und Makrophagen, die mit antimikrobiellen Funktionen, einschließlich Phagozytose und Respiratory Burst 2-6, 8-11. Darüber hinaus ist die genetische Lenkbarkeit und der optischen Transparenz der embryonalen und larvalen Stadien der Entwicklung ermöglichen die Erzeugung von stabilen transgenen Linien mit fluoreszenzmarkierten Immunzellen macht es möglich, Wirt-Pathogen-Wechselwirkungen in Echtzeit in vivo zu untersuchen. Die Erzeugung dieser transgenen Linien mit einem Photokonvertierbare Protein wie Dendra2 ermöglicht die Verfolgung einzelner Wirtszelle Herkunft und Schicksal im Laufe der Infektion 12. Bei der Entwicklung eines Zebrafisch-Larven-Infektionsmodell, wird der gewählte Standort der Mikroinjektion Feststellung der Infektion zunächst lokal oder systemisch. Der systemische Blutinfektionen in die Schwanzvene oder Duct von Cuvier werden am häufigsten verwendet, um mikrobielle Krankheitserreger im Zebrafisch zu untersuchen und sind nützlich für das Studium Interaktionen zwischen Wirt und mikrobiellen Zellen, Zytokin-Antworten, und die Unterschiede in der Virulenz zwischen Erregerstämme. Für langsamere Wachstum von Mikroorganismen kann eine frühe Einspritzung in den Dottersack von Embryos im 16-1,000 Zellstadium für die Mikroinjektion eines langsam wachsenden Mikroorganismus gefunden, zwischen werden verwendet werden, um eine systemische Infektion 13,14 zu erzeugen, mit der optimalen Entwicklungsstadium die 16 bis 128 Zellstadium 15. Allerdings Dottersack Injektionen von vielen Mikroben in späteren Phasen der Entwicklung sind in der Regel tödlich Host zu t zu seiner aufgrund Gastgeber an den nährstoffreichen Umgebung für Mikroben und der Mangel an infiltrierenden Leukozyten 16-18. Eine lokalisierte Infektion führt in der Regel gerichtete Wanderung von Leukozyten in Richtung der Infektionsstelle, die leicht mit nicht-invasive Abbildungs ​​quantifiziert werden kann. Diese Art der Infektion kann für die Präparation der Mechanismen, die Leukozyten-Migration sowie Untersuchung verschiedener Zugvögel und phagozytischen Funktionen verschiedener Leukozytenpopulationen vermitteln können. Lokalisierte Infektionen sind auch nützlich bei der Prüfung Unterschiede in der Virulenz zwischen Bakterienstämme sowie Studium Mikrobe Invasionsmechanismen seit physischen Host Barrieren müssen für eine lokalisierte Infektion gekreuzt systemischen zu werden werden. Zebrafische sind typischerweise bei Temperaturen von 25-31 ° C 19 angehoben wird, sie können aber auch bei Temperaturen von bis zu 34-35 ° C für die Untersuchung der Invasivität von bestimmten Pathogenen beim Menschen strengen Temperaturanforderungen aufrechterhalten werdenVirulenz 20, 21. Viele verschiedene Standorte wurden verwendet, um eine anfänglich lokalisierten bakteriellen Infektionen, einschließlich der Hinterhirn Ventrikel 22, dorsalen Schwanzmuskel 18, Perikardhöhle 23 und Ohrbläschen (Ohr) 5, 16, 24 zu erzeugen. Es hat sich jedoch gezeigt, dass die Injektion von Bakterien in den Ruten Muskel Gewebeschädigung und Entzündung unabhängig von den Bakterien, die bei der Untersuchung Leukozytenreaktion 13 Ergebnisse verzerren können verursachen gefunden. Obwohl weniger Schaden mit Injektion in die Hinterhirn verbunden, und obwohl es zunächst frei von Leukozyten in jungen Embryonen ist das Hinterhirn Ventrikel stetig gewinnt mehr Immunzellen im Laufe der Zeit als Mikroglia Wohnsitz zu nehmen. Das Hinterhirn Ventrikel ist auch ein schwieriger Ort zum Bild. Der Ohrbläschen ist ein geschlossener Hohlraum, ohne direkten Zugang zu dem Gefäßsystem 25, 26. Es ist in der Regel frei von leukocytes, aber Leukozyten an das Ohrbläschen als Antwort auf Entzündungsreize wie Infektion rekrutiert werden. Es ist auch ein bevorzugter Ort der Mikroinjektion von Bakterien in Zebrabärbling Alter von 2-3 Tagen nach der Befruchtung (dpf) wegen der Einfachheit der Bildverarbeitung und die Visualisierung der Injektion. Daher haben wir uns für das Ohrbläschen wie unsere Website von lokalisierten bakteriellen Infektion.

<table><tbody><tr><td>1.7 ml Eppendorfs</td><td>MidSci</td><td>AVSS1700</td><td></td></tr><tr><td>14 ml Falcon tube</td><td>BD Falcon</td><td>352059</td><td></td></tr><tr><td>27 G x &amp;frac12; in. needle</td><td>BD Biosciences</td><td>305109</td><td></td></tr><tr><td>96-well Plate</td><td>Corning Incorporated</td><td>3596</td><td></td></tr><tr><td>Agar</td><td>BD Biosciences</td><td>214030</td><td></td></tr><tr><td>CellTracker Red</td><td>Molecular Probes, Invitrogen</td><td>C34552</td><td></td></tr><tr><td>CNA agar</td><td>Dot Scientific, Inc</td><td>7126A</td><td></td></tr><tr><td>Disposable transfer pipets</td><td>Fisher Scientific</td><td>13-711-7m</td><td></td></tr><tr><td>Dissecting scope</td><td>Nikon</td><td>SMZ745</td><td></td></tr><tr><td>DMSO</td><td>Sigma Aldrich</td><td>D2650</td><td></td></tr><tr><td>Ethanol 200 proof</td><td>MDS</td><td>2292</td><td></td></tr><tr><td>Fine tweezers</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>Gel comb</td><td>VWR</td><td>27372-482</td><td>4.2 mm width, 1.5 mm thick</td></tr><tr><td>Glass bottom dishes</td><td></td><td></td><td>Custom made by drilling a 16 - 18 mm hole in the center of a 35 mm tissue culture dish bottom and placing a 22 mm round #1 coverslip in the hole and sealing with a thin layer of Norland Optical Adhesive 68 cured by UV light.</td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>G33-4</td><td></td></tr><tr><td>High melt agarose</td><td>Denville Scientific, Inc.</td><td>CA3510-6</td><td></td></tr><tr><td>Hydrogen peroxide</td><td>Fisher Scientific</td><td>H325</td><td></td></tr><tr><td>Laser scanning confocal microscope</td><td>Olympus</td><td>with FV-1000 system</td><td></td></tr><tr><td>Low melt agarose</td><td>Fisher</td><td>BP165-25</td><td></td></tr><tr><td>Magnetic stand</td><td>Tritech (Narishige)</td><td>GJ-1</td><td></td></tr><tr><td>Microinjection system</td><td>Parker</td><td>Picospritzer III</td><td></td></tr><tr><td>Microloader pipet tips</td><td>Eppendorf</td><td>930001007</td><td></td></tr><tr><td>Micromanipulator</td><td>Tritech (Narishige)</td><td>M-152</td><td></td></tr><tr><td>Micropipette puller</td><td>Sutter Instrument Company</td><td>Flaming/Brown P-97</td><td></td></tr><tr><td>Nanodrop spectrophotmeter</td><td>Thermo Scientific</td><td>ND-1000</td><td></td></tr><tr><td>N-Phenylthiourea (PTU)</td><td>Sigma Aldrich</td><td>P7629</td><td></td></tr><tr><td>Paraformaldheyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Petri dishes</td><td>Fisher Scientific</td><td>FB0875712</td><td>100 x 15 mm</td></tr><tr><td>Phenol</td><td>Sigma Aldrich</td><td>P-4557</td><td></td></tr><tr><td>Phenol Red</td><td>Ricca Chemoical Company</td><td>572516</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Fisher Scientific</td><td>BP665-1</td><td></td></tr><tr><td>Potassium hydroxide</td><td>Sigma Aldrich</td><td>P-6310</td><td></td></tr><tr><td>Pronase</td><td>Roche</td><td>165921</td><td></td></tr><tr><td>Protease peptone</td><td>Fluka Biochemika</td><td>29185</td><td></td></tr><tr><td>Small cell culture dish</td><td>Corning Incorporated</td><td>430165</td><td>35 x 10 mm</td></tr><tr><td>Sudan Black</td><td>Sigma Aldrich</td><td>S2380</td><td></td></tr><tr><td>Thin wall glass capillary injection needles</td><td>World Precision Instruments, Inc.</td><td>TW100-3</td><td></td></tr><tr><td>Todd Hewitt</td><td>Sigma Aldrich/Fluka Analytical</td><td>T1438</td><td></td></tr><tr><td>Tricaine (ethyl 3-aminobenzoate)</td><td>Argent Chemical Laboratory/Finquel</td><td>C-FINQ-UE-100G</td><td></td></tr><tr><td>Triton X-100</td><td>Fisher Scientific</td><td>BP151-500</td><td></td></tr><tr><td>Tween 20</td><td>Fisher Scientific</td><td>BP337-500</td><td></td></tr><tr><td>Yeast extract</td><td>Fluka Biochemika</td><td>92144</td><td></td></tr></tbody></table>

non-invasive imaging of the innate immune response in a zebrafish larval model of streptococcus iniae infection

Die aquatische Erreger Streptococcus iniae, ist seit mehr als 100 Millionen Dollar in jährlichen Verluste für die Aquakultur zuständig und in der Lage ist, die systemische Erkrankung in beiden Fische und Menschen. Ein besseres Verständnis der S. iniae Pathogenese der Erkrankung erfordert eine entsprechende Modellsystem. Die genetische Lenkbarkeit und die optische Transparenz der frühen Entwicklungsstadien von Zebrabärbling ermöglichen die Erzeugung und nicht-invasive Abbildung von transgenen Linien mit fluoreszenzmarkierten Immunzellen. Das adaptive Immunsystem nicht voll funktionsfähig, bis mehrere Wochen nach der Befruchtung, aber Zebrafisch-Larven haben eine konservierte Wirbel angeborenen Immunsystems mit beiden Neutrophilen und Makrophagen. Somit ist die Erzeugung eines Larveninfektionsmodell ermöglicht die Untersuchung des spezifischen Beitrags der angeborenen Immunität bei der Kontrolle S. iniae Infektion. Die Website der Mikroinjektion wird geprüft, ob eine Infektionsystemische oder zunächst lokalisiert. Hier präsentieren wir unsere Protokolle für Ohrbläschen Injektion von Zebrafisch im Alter von 2-3 Tagen nach der Befruchtung sowie unsere Methoden zur Fluoreszenz konfokale Abbildung der Infektion. Eine lokalisierte Infektion Website ermöglicht die Beobachtung der ersten Mikroben-Invasion, Rekrutierung von Wirtszellen und die Verbreitung der Infektion. Unsere Ergebnisse unter Verwendung des Zebrafisch-Larven-Modell von S. iniae Infektion zeigen, daß Zebrafisch kann verwendet werden, um die unterschiedlichen Beiträge von Aufnahme Neutrophilen und Makrophagen in lokalisierten bakteriellen Infektionen zu untersuchen. Darüber hinaus wird beschrieben, wie Photomarkierung von Immunzellen können einzelne Wirtszelle Schicksal im Verlauf der Infektion zu verfolgen.

Mikroinjektion von S. iniae in das Ohrbläschen (Abbildung 1 und 2) zu einer Reaktion zunächst lokalisiert Host. Wenn korrekt injiziert, sollten die Bakterien nur in der Ohrbläschen und nicht in das umliegende Gewebe oder Blut zu erkennen. Dies kann bei der Mikroinjektion mit Phenolrot-Farbstoff (1A) sichtbar gemacht werden. Alternativ, wenn markierten Bakterien injiziert werden, einen schnellen Scan des infizierten Larven sofort nach der Injektion kann best?...

Watch this Scientific Journal Video about Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection at JoVE.com

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

Nicht-invasive Bildgebung von der angeborenen Immunantwort in einem Zebrafisch-Larven Modell<em&gt; Streptococcus iniae</em&gt; Infektions

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

non-invasive-imaging-innate-immune-response-zebrafish-larval-model

Research

JoVE Journal

Immunology and Infection

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

10.7K Aufrufe.  University of Wisconsin-Madison. Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle. 

Serie: Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

Live-Imaging von angeborenen Immun und Präkanzeröse Zell-Interaktionen unter Verwendung eines induzierbaren Gal4 / UAS Expression System in Zebrafisch-Larven Haut

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

Forschung

Entwicklungsbiologie

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

Die Infektion von Zebrafisch-Embryos mit Intrazelluläre bakterielle Krankheitserreger

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Immunologie und Infektion

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Nicht-invasive Bildgebung von disseminierter Candidiasis in Zebrafischlarven

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

Gründung der Larven Zebrafisch als Tier-Modell zu untersuchen Trypanosomen Trypanosoma Motilität In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

Die Quantifizierung der Atem Burst Antwort als Indikator für Angeborene Immunsystem Gesundheit in Zebrafisch

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

Entschlüsselung und Imaging Pathogenese und Cording von<em&gt; Mycobacterium abscessus</em&gt; In Zebrafischembryonen

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United States. The innate immune response against C. difficile has been described, but the exact roles of neutrophils and macrophages in CDI are less understood. In the current study, Danio rerio (zebrafish) larvae are used to establish a C. difficile infection model for imaging the behavior and cooperation of these innate immune cells in vivo. To monitor C. difficile, a labeling protocol using a fluorescent dye has been established. A localized infection is achieved by microinjecting labeled C. difficile, which actively grows in the zebrafish intestinal tract and mimics the intestinal epithelial damage in CDI. However, this direct infection protocol is invasive and causes microscopic wounds, which can affect experimental results. Hence, a more noninvasive microgavage protocol is described here. The method involves delivery of C. difficile cells directly into the intestine of zebrafish larvae by intubation through the open mouth. This infection method closely mimics the natural infection route of C. difficile.

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Presented here is a safe and effective method to infect zebrafish larvae with fluorescently labeled anaerobic C. difficile by microinjection and noninvasive microgavage.

Entwicklung eines Larval Zebrafish Infection Model für Clostridioides difficile

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

Mit Zebrabärbling Modelle menschlicher Influenza-A-Virus-Infektionen Virostatika auf Bildschirm und Host-Immun-Zell-Antworten Charakterisieren

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major pathogens in BAI. Current experimental BAI mammalian animal models such as mouse models are costly and time-consuming, and therefore not suitable for high throughput analysis. Thus, novel animal models as complementary systems for investigating BAI in vivo are desired. In the present study, we aimed to develop a zebrafish embryo model for in vivo visualization and intravital analysis of bacterial infection in the presence of biomaterials based on fluorescence microscopy. In addition, the provoked macrophage response was studied. To this end, we used fluorescent protein-expressing S. aureus and transgenic zebrafish embryos expressing fluorescent proteins in their macrophages and developed a procedure to inject bacteria alone or together with microspheres into the muscle tissue of embryos. To monitor bacterial infection progression in live embryos over time, we devised a simple but reliable method of microscopic scoring of fluorescent bacteria. The results from microscopic scoring showed that all embryos with more than 20 colony-forming units (CFU) of bacteria yielded a positive fluorescent signal of bacteria. To study the potential effects of biomaterials on infection, we determined the CFU numbers of S. aureus with and without 10 &#181;m polystyrene microspheres (PS10) as model biomaterials in the embryos. Moreover, we used the ObjectJ project file &#34;Zebrafish-Immunotest&#34; operating in ImageJ to quantify the fluorescence intensity of S. aureus infection with and without PS10 over time. Results from both methods showed higher numbers of S. aureus in infected embryos with microspheres than in embryos without microspheres, indicating an increased infection susceptibility in the presence of the biomaterial. Thus, the present study shows the potential of the zebrafish embryo model to study BAI with the methods developed here.

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

The present study describes a zebrafish embryo model for in vivo visualization and intravital analysis of biomaterial-associated infection over time based on fluorescence microscopy. This model is a promising system complementing mammalian animal models such as mouse models for studying biomaterial-associated infections in vivo.

Zebrafish Embryos Modell für bei Vivo Visualisierung und intravitalen Analyse von Biomaterial-assoziierte Staphylococcus Aureus Infektion

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major ...

Biotechnik

Photoconversion-Based Leukocyte Tracking in Bacterial-Infected Transgenic Zebrafish

Source: Harvie, E. A. et al., <a href="https://app.jove.com/v/52788">Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection.</a>&#160;J. Vis. Exp. (2015)
This video demonstrates the tracking of leukocyte recruitment using transgenic zebrafish larvae expressing photoconvertible green fluorescent protein. Pre-injecting red fluorescent dye-labeled bacteria into the otic vesicle and inducing photoconversion enables observation of leukocytes&#39; migration, phagocytosis, and dispersal throughout the body, providing valuable insights into immune response dynamics.

Photokonversionsbasiertes Leukozyten-Tracking in bakteriell infizierten transgenen Zebrafischen

1. Preparing Microinjection Needles

	Prepare thin wall glass capillary injection needles (1.0 OD/0.75 ID) using a micropipette puller device with the ...

Nicht-invasive Bildgebung von der angeborenen Immunantwort in einem Zebrafisch-Larven Modell

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