המחברים מודים לק Kissa לדיונים מועילים ולמתן Lipo-clodronate ול &#39;Ramakrishnan למתנה הנדיבה של pTEC27 וpTEC15 המאפשרים ביטוי של tdTomato וואסאבי, בהתאמה. חלק צורות עבודה זו של הפרויקטים של הסוכנות הלאומית למחקר הצרפתי (ANR ZebraFlam-10-MIDI-009 וDIMYVIR ANR-13-BSV3-007-01) ותכנית המסגרת השביעית של הקהילה האירופית (FP7-אנשים-2011-ITN) על פי הסכם מענק לא. PITN-GA-2011-289209 להדרכה ראשונית רשת FishForPharma מארי קירי-. אנו מאחלים גם להודות לאיגוד גרגורי Lemarchal וVaincre La Mucoviscidose (RF20130500835) למימון CM דופון.

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</ol>

The authors have nothing to disclose.

דג הזברה התפתחה לאחרונה כמערכת מודל חוליות מצוינת ללימוד הדינמיקה של זיהום חיידקים באמצעות שדה רחב והדמיה confocal בזמן אמת 36. השילוב של מתלים התפזרו mycobacterial (פרוטוקול 2.2) יחד עם שיטות הזרקת מיקרו (פרוטוקול 4) מאפשר זיהומים לשחזור מערכתיים, ולאחר מכן ניטור והדמיה של ?...

abscessus Mycobacterium הוא הפתוגן מתעוררים שגורם ספקטרום רחב של תסמונות קליניות בבני אדם. אלה כוללים זיהומים עוריים, כמו גם זיהומי ריאות כרוניים קשים, בעיקר נתקלו במדוכאי חיסון וחולי סיסטיק פיברוזיס 1,2,3,4. מ &#39; abscessus גם נחשב למינים צומחים במהירות גדול mycobacterial אחראים לזיהומי nosocomial וiatrogenic בבני אדם. יתר על כן, לאחרונה פורסם מספר מחקרים הדגישו את האפשרות שמ &#39; abscessus יכול לחצות את מחסום דם-המוח ולגרום לנגעים חשובים במערכת העצבים המרכזית (CNS) 5,6. למרות היותו מגדל מהיר, מ &#39; תערוכות abscessus גם כמה תכונות פתוגניים הקשורים לאלה של שחפת Mycobacterium, כולל היכולת לשתוק במשך שנים בתוך מבני granulomatous וליצור נגעי caseous בריאות 7. יותר מדאיג היא סן הנמוכהsitivity של מ &#39; abscessus לאנטיביוטיקה, טיוח זיהומים אלה קשים מאוד לטיפול מוביל לשיעור כישלון טיפולי משמעותי 8,9. האיום החשוב של מין זה הוא בעיקר התנגדותו הפנימית לאנטיביוטיקה, אשר היא דאגה מרכזית במוסדות בריאות ציבוריים 10 ונגד להשתלת ריאה 11. מציג abscessus מ morphotypes מושבה (R) חלק (S) או מחוספס שיובילו לתוצאות קליניות שונות. בניגוד למתח S, יש לי חיידקי R נטייה לגדול קצה לקצה, שהוביל לחבל או כמו חוט-מבנה 12,13. מספר מחקרים עצמאיים המבוססים על מודלים או סלולריים או בעלי חיים גילו את הפנוטיפ היפר-האלימות של R morphotype 14,15. ממחקרים אפידמיולוגיים, במקרים החמורים ביותר של מ &#39; זיהומי ריאות abscessus מופיעים להיות מזוהים עם R גרסאות 16 שהגרסה היחידה שכבר ראה להתמיד במשך שנים במארח נגוע 3. הבדל morphotype מסתמך על הנוכחות (בS) או הפסד (בR) של glycopeptidolipids הקשורים משטח (GPL) 12. עם זאת, בשל המגבלות המובנות של הדגמים סלולריים / בעלי החיים הזמינים המשמש כיום ללמוד מ &#39; זיהום abscessus, הידע שלנו על אירועי pathophysiological של גרסאות R או S נותר מעורפל. זיהום של עכברים חיסוניים מוסמכת באמצעות נתיבים תוך ורידי או תרסיס מוביל להתישבות ארעית, המעכב את השימוש בעכברים ללמוד זיהומים מתמשכים ולin vivo בדיקת רגישות לסמים 17. לכן, פיתוח מודלים של בעלי חיים ניתנים למניפולציה של תגובת המארח הוא אתגר גדול. בהקשר זה, מודלים שאינם יונקים של זיהום פותחו לאחרונה, כולל דרוזופילה melanogaster 18 שמציעה מספר יתרונות כגון עלות, מהירות וo קבילות האתיתVer מודל העכבר. מודל דג הזברה (Danio rerio) של זיהום גם נחקר לדמיין, על ידי הדמיה לא פולשנית, ההתקדמות והכרונולוגיה של מ &#39; זיהום abscessus בחי 19. חשוב לציין, הוכחה של קונספט הוקמה גם כדי להדגים את התאמתה לin vivo הערכות אנטיביוטיות נגד מ &#39; abscessus 17,20. דג הזברה הייתה בשימוש נרחב בשני העשורים האחרונים ללמוד את יחסי הגומלין בין פתוגנים שונים והמערכת החיסונית מארח 21. ההצלחה הגוברת של מודל חוליות חלופה זו מסתמכת על הזדמנויות גדולות וייחודיות שהניעו ומאומתים השימוש בו להבנה טובה יותר של זיהומים נגיפיים וחיידקיים רבים 19,22,23,24,25,26,27,28,29. בניגוד למרבית המודלים של בעלי החיים האחרים, עוברי דג הזברה הם שקופים אופטי, המאפשרים הדמיה הקרינה בלתי פולשני 30. זה חהשעות הובילו ללמוד מ &#39; abscessus נגוע עוברי דג הזברה עם פרטים חסרי תקדים, שהגיע לשיא עם התיאור של cording תאי, המייצג דוגמא לגמישות מורפולוגיים בקטריאלי. Cording מייצג מנגנון חדש של חתרנות של מערכת החיסון ומנגנון מפתח קידום פתוגנזה של מ &#39;החריף זיהום abscessus 19. דו&quot;ח זה מתאר כלים ושיטות חדשים באמצעות עובר דג הזברה לפענח את תכונות pathophysiological של מ &#39; abscessus זיהום וללמוד את יחסי הגומלין ההדוקים בין מערכת החיסון המולדת החיידקים ו. ראשית, פרוטוקול microinjection מפורט הכולל עיבוד של הבידוד החיידקים, הכנת עובר, וזיהום כשלעצמו, מוצג. שיטות מותאמות במיוחד כדי להעריך מ &#39; ארסיות abscessus על ידי מדידת פרמטרים שונים, כגון הישרדות מארח וניטל חיידקים, מוצגות. דגש מיוחד יינתן לאופןכדי לפקח, ברמת spatiotemporal, הגורל והתקדמות של הזיהום ואת התגובה החיסונית מארח למ &#39; abscessus באמצעות מיקרוסקופ וידאו. יתר על כן, כדי לחקור את התרומה ואת התפקיד של מקרופאגים במ &#39; abscessus זיהום, שיטות להפקת מקרופאגים מדולדלים עוברים (תוך שימוש בגישות או genetically- או מבוססות כימי) מתוארים. לבסוף, פרוטוקולים כדי להמחיש את האינטראקציות הספציפיות עם מקרופאגים או נויטרופילים או באמצעות עוברים קבועים או חיים מתועדים. מטרת דו&quot;ח זה היא לעורר מחקרים נוספים כדי לשפוך אור חדש לתוך מ &#39; מנגנוני abscessus אלימות ובמיוחד את התפקיד של cording בהקמת תהליך זיהום חריף ובלתי מבוקר.

<table><tbody><tr><td>BBL MGIT PANTA</td><td>BD Biosciences</td><td>245114</td><td></td></tr><tr><td>Bovine Serum Albumin&amp;nbsp;</td><td>Euromedex</td><td>04-100-811-E</td><td></td></tr><tr><td>Catalase from Bovine Liver&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C40</td><td></td></tr><tr><td>Difco Middlebrook 7H10 Agar</td><td>BD Biosciences</td><td>262710</td><td></td></tr><tr><td>Difco Middlebrook 7H9 Broth</td><td>BD Biosciences</td><td>271310</td><td></td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)</td><td>Sigma-Aldrich</td><td>A5040</td><td></td></tr><tr><td>Oleic Acid</td><td>Sigma-Aldrich</td><td>O1008</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Delta Microscopie</td><td>15710</td><td></td></tr><tr><td>Phenol Red</td><td>Sigma-Aldrich</td><td>319244</td><td></td></tr><tr><td>Tween 80</td><td>Sigma-Aldrich</td><td>P4780</td><td></td></tr><tr><td>Agar</td><td>Gibco Life Technologie</td><td>30391-023</td><td></td></tr><tr><td>Low melting agarose</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Instant Ocean Sea Salts&amp;nbsp;</td><td>Aquarium Systems Inc</td><td></td><td></td></tr><tr><td>Borosilicate glass capillaries&amp;nbsp;</td><td>Sutter instrument Inc</td><td>BF100-78-10</td><td>1 mm O.D. X 0.78 mm I.D.</td></tr><tr><td>Micropipette puller device&amp;nbsp;</td><td>Sutter Instrument Inc</td><td></td><td>Flamming/Brown Micropipette Puller p-87</td></tr><tr><td>Microinjector</td><td>Tritech Research&amp;nbsp;</td><td></td><td>Digital microINJECTOR, MINJ-D</td></tr><tr><td>Tweezers</td><td>Sciences Tools inc</td><td></td><td>Dumont # M5S&amp;nbsp;</td></tr><tr><td>Microloader Tips</td><td>Eppendorf</td><td></td><td></td></tr></tbody></table>

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.

למרות שניתן להזריק אתרים אנטומיים שונים 32, זריקות וריד הזנב משמשים לעתים קרובות כדי ליצור זיהום מערכתי לניתוחים שלאחר מכן, כולל ניסויי הישרדות, נחישות נטל חיידקים, פעילות phagocytosis או היווצרות חוט. זריקות בשרירי הזנב משמשות להעריך את הגיוס של מקרופאגים באתר ההזרק...

Watch this Scientific Journal Video about Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos at JoVE.com

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.

פענוח ופתוגנזה הדמיה והשתרגות<em&gt; Abscessus Mycobacterium</em&gt; בעוברי דג הזברה

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

deciphering-imaging-pathogenesis-cording-mycobacterium-abscessus

Research

JoVE Journal

Immunology and Infection

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

10.2K Views.  Université Montpellier. 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. 

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

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

ב גיבוש biofilm חוץ גופית ב 8-Slide גם לשכת

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

Microbead השרשה בעובר דג הזברה

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Developmental Biology

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.

זיהום של עוברים דג הזברה עם פתוגנים חיידקיים תאיים

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

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.

הדמיה לא פולשנית של פטרת מופצת הזחלים דג הזברה

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

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

הקמה ואופטימיזציה של הגדרת תפוקה גבוהה ללמוד<em&gt; סטפילוקוקוס epidermidis</em&gt; ו<em&gt; Marinum Mycobacterium</em&gt; זיהום כמודל לגילוי תרופות

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

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.

מודל העובר דג זברה, ויזואליזציה Vivo, ניתוח Intravital של Biomaterial-הקשורים Staphylococcus aureus זיהום

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

Bioengineering

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

מידול שחפת ב דג נגוע זברה בוגרת התחממות עולמית

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

Visualization of Macrophage Lytic Cell Death During Mycobacterial Infection in Zebrafish Embryos via Intravital Microscopy

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune system during early development. The Zebrafish Mycobacterium marinum (M. marinum) infection model has been well-established in studying host immune response against mycobacterial infection. It has been suggested that different macrophage cell death types will lead to the diverse outcomes of mycobacterial infection. Here we describe a protocol using intravital microscopy to observe macrophage cell death in zebrafish embryos following M. marinum infection. Zebrafish transgenic lines that specifically label macrophages and neutrophils are infected via intramuscular microinjection of fluorescently labeled M. marinum in either the midbrain or the trunk. Infected zebrafish embryos are subsequently mounted on low melting agarose and observed by confocal microscopy in X-Y-Z-T dimensions. Because long-term live imaging requires using low laser power to avoid photobleaching and phototoxicity, a strongly expressing transgenic is highly recommended. This protocol facilitates the visualization of the dynamic processes in vivo, including immune cell migration, host pathogen interaction, and cell death.

This protocol describes a technique for visualizing macrophage behavior and death in embryonic zebrafish during Mycobacterium marinum infection. Steps for the preparation of bacteria, infection of the embryos, and intravital microscopy are included. This technique may be applied to the observation of cellular behavior and death in similar scenarios involving infection or sterile inflammation.

ויזואליזציה של מקרופאג Lytic תא המוות במהלך זיהום פטרת העוברים באמצעות מיקרוסקופ אינטרטל

Zebrafish is an excellent model organism for studying innate immune cell behavior due to its transparent nature and reliance solely on its innate immune ...

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal drugs, IA can cause &#62;50% mortality in infected immunocompromised patients. It is crucial to determine both host and pathogen factors that contribute to infection susceptibility and low survival rates in infected patients in order to develop novel therapeutics. Innate immune responses play a pivotal role in recognition and clearance of Aspergillus spores, though little is known about the exact cellular and molecular mechanisms. Reliable models are required to investigate detailed mechanistic interactions between the host and pathogen. The optical clarity and genetic tractability of zebrafish larvae make them an intriguing model&#160;to study host-pathogen interactions of multiple human bacterial and fungal infections in a live and intact host. This protocol describes a larval zebrafish Aspergillus infection model. First, Aspergillus spores are isolated and injected into the zebrafish hindbrain ventricle via microinjection. Then, chemical inhibitors such as immunosuppressive drugs are added directly to the larval water. Two methods to monitor the infection in injected larvae are described, including the 1) homogenization of larvae for colony forming unit (CFU) enumeration and 2) a repeated, daily live imaging setup. Overall, these techniques can be used to mechanistically analyze the progression of Aspergillus infection in vivo and can be applied to different host backgrounds and Aspergillus strains to interrogate host-pathogen interactions.

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

This protocol describes an Aspergillus infection model in zebrafish larvae. Aspergillus spores are microinjected into the hindbrain of larvae, and chemical treatment is used to induce immunosuppression. Infection progression is monitored via a daily imaging setup to monitor fungal growth and immune responses as well as enumeration of live spores by colony forming unit plating.

זיהום זחלי זברה עם נבגי אספרגילוס לניתוח אינטראקציות מארח-פתוגן

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal ...

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

Health monitoring systems are developed and used in zebrafish research facilities because pathogens of Danio rerio such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa have the potential to impair animal welfare and research. The fish are typically analyzed post mortem to detect microbes. The use of sentinels is a suggested way to improve the sensitivity of the surveillance and to reduce the number of animals to sample. The setting of a pre-filtration sentinel tank out of a recirculating system is described. The technique is developed to prevent water pollution and to represent the fish population by a careful selection of age, gender, and strains. In order to use the minimum number of animals, techniques to screen the environment are also detailed. Polymerase Chain Reaction (PCR) on surface sump swabs is used to significantly improve the detection of some prevalent and pathogenic mycobacterial species such as Mycobacterium fortuitum, Mycobacterium haemophilum, and Mycobacterium chelonae. Another environmental method consists of processing the sludge at the bottom of a holding tank or sump to look for P. tomentosa eggs. This is a cheap and fast technique that can be applied in quarantine where a breeding device is submerged into the holding tank of imported animals. Finally, PCR is applied to the sludge sample and A. hydrophila is detected at the sump&#39;s bottom and surface. Generally, these environmental screening techniques applied to these specific pathogens have led to an increased sensitivity compared to the testing of pre-filtration sentinels.

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

This protocol describes the use of sump swabs and sludge analysis of zebrafish systems, which leads to increased detection compared to the sole use of sentinels to detect pathogens such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa. A system to monitor P. tomentosa eggs in quarantine is also proposed.

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