Wir möchten Elisabeth Guillemet, Christophe Buisson und Ludovic Bridoux für hervorragende technische Unterstützung danken. Wir sind zu großem Dank verpflichtet Sylvie Salamitou und Sinda Fedhila für die erste System-Setup.

Ein. Insect Aufzucht Der gesamte Zyklus vom Ei bis zum letzten Larvenstadium dauert ca. 5 Wochen bei 25 ° C. Ein oder zwei zusätzliche Wochen sind nötig, um erwachsenen Schmetterlinge zu erhalten. <ol><li> Zeigen mindestens 100 Puppen oder neu fusionierten erwachsenen G. mellonella Schmetterlinge in einem 5-Liter-wire-mesh Käfig. Männlich Schmetterlinge messen 10 bis 15 mm. Der erwachsene männliche Motte ist beige mit schwachen hellen und dunklen Markierungen. Weibliche Schmetterlinge Maß um 20 mm. Die Weibchen sind dunkler als die Männchen mit einem braun / grau. </li><li> Suspend zwei Packungen vierschichtigen Papier in den Käfig für Eiablage. Nach 2 Tagen wird das erwachsene Weibchen Eier auf der Kante zwischen den Papieren lag. </li><li> Zweimal in der Woche, legen Sie die Ei-Papier Packs in neue Kunststoff-Aufzucht-Boxen mit Gittern zu lassen Luft zirkulieren, die Pollen und Bienenwachs. Eier brüten in ungefähr 3 Tage und kleine Larven beginnen, indem auf Wachs und Pollen zu entwickeln. Alternativ können Larven auf einer künstlichen Diät consistin platziert werdeng einer Mischung aus 500 g flüssiger Honig, 400 g Glycerin, 100 g getrocknete Bierhefe, 250 g Weizenmehl, 200 g Magermilchpulver und 400 g Polenta. Um eine angemessene Verhältnis der mit Lebensmitteln pro Larve bieten regelmäßig trennen die Larven in neue Boxen und füllt die Lebensmittel alle zwei Tage. </li><li> Larven werden in den sechs Etappen der Larve Stadion aufgezogen. Jedes Stadion wird vom Schlupf des Kopfkapsel der Larven ist. Wenn Larven die letzte Stufe zu erreichen vor der Verpuppung, stoppen sie Fütterung und dem Bau einer leichte Seide Kokon. </li><li> In ihrer schützenden Kokons, werden die Larven Ruhe und verwandeln sich in Puppen. Wachs Würmer verbleiben im Puppenstadium für ein bis zwei Wochen, in Falter entstehen. Die Falter nicht trinken noch essen. Die Paarung erfolgt und das Wachs Würmer Lebenszyklus beginnt von neuem. </li><li> Um die Pathologie Assay zu standardisieren, nehmen die Larven im letzten Larvenstadium. Während dieser Phase diese Fütterung zu stoppen, an der Abdeckung der Aufzucht zu verschieben und starten, um Seide zu produzieren. Diese stage dauert ca. 5 Tage. </li></ol> 2. Insect Selection für Infektionsforschung <ol><li> Wählen letzten Larvenstadium, die 2-3 cm lang und 180-250 mg im Gewicht sind, 24 Stunden vor Infektionen und steckte sie in ein leeres Feld, um sie verhungern. </li><li> Entfernen Sie die entstehenden Seidenkokon um die Larven. </li></ol> 3. Bakterielle Zubereitung <ol><li> Planen Bacillus Bakteriensuspensionen um Endkonzentrationen im Bereich von 10 4 koloniebildenden Einheiten (KBE) / ml bis 10 8 KBE / ml für intra haemocoelic Injektion und von 3x10 6 CFU / ml bis 1x10 erhalten 8 KBE / ml bei Einnahme (die cfu zu erhalten ein LD 50 hängt von den bakteriellen Spezies). In unserem Fall, B. cereus wird in Luria-Bertani broth Medium (LB, 10 g / l Trypton, 5 g / l Hefeextrakt, 10 g / l NaCl) bei 37 ° C unter Rühren bis zum Eintritt in die stationäre Phase, entsprechend der Einwärtskrümmung des Wachstums Kurve. Messen Sie die OD 600 nm (zwischen 1 und 2 für B. cereus) und es verwenden, um bakterielle Konzentration zu bewerten, mit einem bisherigen Titrationskurve. Ernte Bakterien durch Zentrifugation bei 5.000 xg für 10 min bei Raumtemperatur und resuspendieren in phosphatgepufferter Kochsalzlösung (PBS: 1 M KH 2 PO 4, 1 M K 2 HPO 4, 5 M NaCl, pH 7,2). Jede Larve wird 10 ul Bakteriensuspension in der gewünschten Konzentration erhalten. </li><li> Alternativ können Sporensuspension für eine Infektion vorbereitet werden. Sporen erhalten durch Kultivieren der Bakterien in dem Sporulationsmedium HCT (5 g / l Trypton, 2 g / L Caseinhydrolysat, 12,5 mM K 2 HPO 4, 12,5 mM MgSO4, 0,05 mM MnSO 4, 1,2 mM ZnSO 4, 1,2 mM Fe 2 (SO 4) 3, 0,5% H 2 SO 4, 25 mM CaCl 2) 20 bis 30 ° C für 3 Tage. Ernte Sporen durch Zentrifugation (10.000 xg, 15 min) und waschen zweimal in sterilem destilliertem Wasser. Resuspendieren spore pelreinlassen sterilem destilliertem Wasser und Wärme für 15 min bei 78 ° C, um die verbleibenden vegetativen Bakterien zu entfernen. Numerate die Suspension durch Ausplattieren serielle Verdünnungen auf LB-Agarplatten. Jede Larve wird 10 ul Sporensuspension in der gewünschten Konzentration erhalten. </li></ol> 4. Cry Toxin Vorbereitung <ol><li> Bereiten Sie die Cry1C Toxin aus dem B. thuringiensis Stamm 407 mit dem Plasmid pHTF31C 21, die das Gen kodiert Cry1C transformiert. Kultur der Stamm in 100 ml HCT-Medium bei 30 ° C für 72 Stunden mit Antibiotika (Erythromycin 10mg/ml) in voller Sporulation, Toxin crystal Produktion und Befreiung in den Kulturüberstand zu ermöglichen. </li><li> Entschlacken Kristalle aus der Überstand auf 72% -79% Saccharose-Gradienten. Fügen Sie zunächst 17 ml 79% Saccharose in einer 40 ml Tube. Vorsichtig 17 ml 72% Saccharose auf dem 79% Saccharose. Schließlich schließen Schicht 5-6 ml des 10X konzentrierter sporulierten Kultur und das Rohr. Zentrifugieren für 14 h bei 20,000 × g, 4 ° C, um die Kristalle von den Sporen getrennt. Sammle die Kristalle bei der Gradient-Schnittstelle. Resuspendieren des Kristalls Pellet in 25 ml kaltem sterilem Wasser zu waschen. Zentrifuge bei 8.000 xg für 20 min. Dieser Schritt wird dreimal wiederholt, um alle Saccharose Kleben an den Kristallen zu entfernen. Resuspendieren die Kristalle in 5 ml sterilem Wasser und beobachten unter dem Mikroskop, um die Reinheit zu bewerten. </li><li> Bewerten Toxinprotein Konzentration durch klassische Bradford Färbung auf Kristall-Lösung in 50 mM NaOH presolubilized. Toxin Kristalle können bei -20 ° C bis zur Verwendung gelagert werden. </li></ol> 5. Injector Vorbereitung <ol><li> Um die genaue Injektionsvolumen steuern, verwenden Sie eine automatisierte Spritzenpumpe (zB KD Scientific KDS 100) mit dem Setup auf 1 ul / sec (Abbildung 1A). </li><li> Füllen Sie eine 1 ml Spritze mit 300 ul Wasser oder die bakterielle Lösung (1 Spritze pro Bedingung) für die Infektion von 20-25 Larven. Entfernen Sie die Luftblasen durch vorsichtiges tapping der Spritze, dann fügen Sie eine 0,45 x 12 mm Nadel an der Spritze. </li><li> Legen Sie die Spritze in den Injektor. </li><li> Führen Sie eine leere Injektion von 10 ul in einem leeren Rohr, um das injizierte Volumen steuern. </li></ol> 6. Insect Intrahaemocoelic Injection <ol><li> Legen Sie die Insekten manuell zwischen Daumen und Zeigefinger. Dann führen Sie die Nadel im Injektor in der Larve Haut (Epidermis) (1C) fixiert. </li><li> Halten Sie das Insekt an Ort und Stelle während der Injektion die 10 ul bakterieller Lösung (Sporen oder vegetativen Bakterien). </li><li> Entfernen Sie vorsichtig das Insekt von der Nadel. </li><li> Legen Sie die infizierten Insektenzellen in einer kleinen Petrischale (5 cm Durchmesser), 5 Larven pro Gericht. </li><li> Infect mindestens 20 Larven für jede experimentelle Bedingung. </li><li> Die Schalen werden bei 37 ° C im Brutschrank für 48 Stunden. </li></ol> 7. Insect Zwangsernährung (Verschlucken) <ol><li> In einem 2 ml Eppendorf-Röhrchen, mischen 0,2 ug / &amp; mu; l Cry1C Toxin entweder mit Sporen oder vegetative Zellsuspension in Endkonzentrationen im Bereich von 3x10 4 bis 1x10 7 je 10 ul erhalten. </li><li> Füllen Sie eine 1 ml Spritze mit einer 30G, 25 mm Injektionsnadel mit 300 ul des bakteriellen Cry1C Toxinlösung für die Infektion von 20-25 Larven. Entfernen Sie die Luftblasen aus der Spritze. </li><li> Legen Sie die Insekten manuell, so dass der Mündung der Nadel fixiert im Injektor (1D) und Kraft-füttern mit 10 ul der bakteriellen Lösung unter Verwendung der automatischen Spritzenpumpe betritt. </li><li> Infect mindestens 20 Larven für jede experimentelle Bedingung. </li><li> Legen Sie die infizierten Insektenzellen in einem kleinen 5 cm Petrischale (5 Larven pro Gericht). Die Schalen werden bei 37 ° C im Brutschrank für 48 Stunden. </li></ol> 8. Recording Insektenmortalität <ol><li> Nach der Zwangsernährung oder Injektion Versuche, halten die Larven in der Petrischale ohne Nahrung bei 25 ° C oder 37 ° C. Überprüfen Larven mortality regelmäßig über einen 48-Stunden-Zeitraum. Tote Larven sind inert und in der Regel schwarz werden (Abbildung 1B). </li><li> Mortality Daten können analysiert mit Hilfe des Log-Probit Programm 14 werden. Dieses Programm testet die Linearität der Dosis Sterblichkeit Kurven und bietet letalen Dosen (LD 50). Alternativ können auch andere Programme, wie Prism (Graph Pad), Analyse Überleben oder Mortalität Daten verwendet werden. </li></ol>

<ol>
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Keine Interessenskonflikte erklärt.

Der Einsatz von Insekten und vor allem das Larvenstadium, wie Infektionen Modelle für mehrere Erreger, wird immer häufiger. Ein Modell der Wahl für einige Aspekte ist Drosophila (die Fliege Modell) und Erwachsene Larvenstadium 1,2 verwendet. Die Lepidoptera G. mellonella wurde auch hauptsächlich zum Testen bakteriellen Virulenz durch Injektion verwendet. Der Vorteil tolerieren höheren Temperaturen (über 37 ° C) als Drosophila (maximal 25 ° C) ist wichtig, wenn Säuger Errege...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> Wachs und Pollen </td><td> La Ruche Roanaise </td><td> 303000 </td><td> Jede Honigproduzent </td></tr><tr><td> Automatisierte Spritzenpumpe </td><td> KD Scientific </td><td> KDS 100 </td><td></td></tr><tr><td> Spritze 1 ml </td><td> Terumo </td><td> BS 01T </td><td></td></tr><tr><td> Needle 0,45 x 12 mm </td><td> Terumo </td><td> NN 2613R </td><td></td></tr><tr><td> Petrischale 5 cm </td><td> VWR </td><td> 89000-300 </td><td></td></tr><tr><td> Needle 30G, 25 mm hypodermic </td><td> Burkard Mfg Co. Ltd </td><td> PDE0005 </td><td></td></tr></tbody></table>

the insect galleria mellonella as a powerful infection model to investigate bacterial pathogenesis

Das Studium der bakteriellen Virulenz erfordert oft einen geeigneten Tiermodell. Mammalian Modelle Infektion sind kostspielig und können ethische Probleme aufwerfen. Die Verwendung von Insekten-Infektion Modelle bietet eine wertvolle Alternative. Im Vergleich zu anderen nicht-Vertebraten Modell Hosts wie Nematoden, Insekten haben ein relativ fortgeschrittenes System der antimikrobiellen Abwehr und sind somit eher einschlägigen Informationen zu den Säugetieren Infektion herzustellen. Wie bei Säugetieren besitzen Insekten eine komplexe angeborene Immunsystem 1. Zellen in der Hämolymphe der Lage sind, phagozytierenden oder Einkapseln mikrobielle Invasoren und humorale Reaktionen gehören die induzierbare Produktion von Lysozym und kleine antibakterielle Peptide 2,3. Darüber hinaus werden Analogien zwischen den Epithelzellen von Insekten Larven Mitteldärmen und Darmzellen von Säugetieren Verdauungssystem gefunden. Schließlich mehreren Basiskomponenten essentiell für die bakterielle Infektion Verfahren wie Zelladhäsion, Resistenz gegenantimikrobielle Peptide, Gewebeabbau und Anpassung an oxidativem Stress sind wahrscheinlich in beiden Insekten und Säugern 1 wichtig. Somit sind Insekten polyvalenten Werkzeuge für die Identifizierung und Charakterisierung mikrobieller Virulenzfaktoren in Säugetier Infektionen beteiligt. Larven des größeren Wachsmotte Galleria mellonella ist gezeigt worden, um eine nützliche Einblick in die Pathogenese einer Vielzahl von mikrobiellen Infektionen, einschließlich Säuger-Pilz (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) und bakterieller Pathogene, wie Staphylococcus aureus, Proteus vulgaris bereitzustellen , Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes oder Enterococcus faecalis 4-7. Unabhängig von den Bakterienarten, die Ergebnisse mit Galleria Larven durch direkte Injektion durch die Kutikula infiziert konsistent mit denen von simi korrelieren erhaltenlar Säugetieren Studien: Bakterienstämme, die gedämpft in Säugetierzellen Modelle sind zu demonstrieren niedrigerer Virulenz in der Galleria und Stämme verursacht schwere Infektionen beim Menschen sind auch sehr virulent in der Galleria-Modell 8-11. Oral Infektion Galleria ist viel weniger genutzt und zusätzliche Verbindungen, wie bestimmte Toxine, sind erforderlich, um die Sterblichkeit zu erreichen. G. mellonella Larven vorhanden mehrere technische Vorteile: Sie sind relativ groß (letzten Larvenstadium vor der Verpuppung sind etwa 2 cm lang und Gewicht 250 mg), so dass die Injektion von definierten Dosen von Bakterien, sie können bei verschiedenen Temperaturen gehalten werden (20 ° C bis 30 ° C) und Infektionsstudien kann zwischen 15 ° C werden über 37 ° C 12,13 durchgeführt, so dass Versuche, das eine Säuger-Umgebung nachahmen. Darüber hinaus ist Insekt Aufzucht leicht und relativ billig. Infektion der Larven ermöglicht die Überwachung bakteriellen Virulenz durch verschiedene Mittel, including Berechnung der LD 50 14, Messung der Überlebensrate der Bakterien 15,16 und Prüfung der Infektion 17. Hier beschreiben wir die Aufzucht der Insekten, die alle Lebensphasen von G. mellonella. Wir bieten Ihnen ein detailliertes Protokoll der Infektion auf zwei Wegen der Impfung: mündliche und intra haemocoelic. Die bakterielle Modell in diesem Protokoll ist Bacillus cereus, einem Gram-positive Erreger in gastrointestinalen sowie in andere schwere lokale oder systemische opportunistische Infektionen 18,19 verwickelt.

Intra haemocoelic Injektion von Bakterien in G. mellonella ist erwiesen sehr nützlich für die Identifizierung vieler Virulenzfaktoren Umgang mit Gewebebeschädigung und Beständigkeit gegen angeborene Immunsystem Faktoren mehrerer menschlicher Pathogene. Als Beispiel stellt Figur 2A Insektenmortalität nach Injektion von verschiedenen Dosen von B. cereus Bakterien (Wildtyp und Mutanten) 22. 2B stellt die Überlebensrate der Bakterien nach der Infektion <em...

Watch this Scientific Journal Video about The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis at JoVE.com

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

Mündliche und intra haemocolic Infektion der Larven der größeren Wachsmotte<em&gt; Galleria mellonella</em&gt; Beschrieben. Das Insekt kann verwendet werden, um Virulenzfaktoren entomopathogener sowie Säugetierzellen opportunistische Bakterien zu untersuchen. Aufzucht der Insekten, Methoden der Infektion und Beispiele<em&gt; In vivo</em&gt; Analyse beschrieben.

Das Insekt<em&gt; Galleria mellonella</em&gt; Als leistungsfähiges Infektion Modell zur bakterielle Pathogenität Untersuchen

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

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JoVE Journal

Immunology and Infection

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

31.6K Aufrufe.  INRA, Micalis UMR1319, France. Mündliche und intra haemocolic Infektion der Larven der größeren Wachsmotte Galleria mellonella Beschrieben. Das Insekt kann verwendet werden, um Virulenzfaktoren entomopathogener sowie Säugetierzellen opportunistische Bakterien zu untersuchen. Aufzucht der Insekten, Methoden der Infektion und Beispiele In vivo Analyse beschrieben.

Serie: The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

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

In-vitro-Bildung von Biofilmen in einem 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. ...

Forschung

Immunologie und Infektion

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Mit Luciferase to Image Bakterielle Infektionen bei Mäusen

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Introducing Schubspannung in der Studie der bakteriellen Adhäsion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Speichel, Speicheldrüse, und Hämolymphe Abholung Ixodes scapularis Zecken

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

Ein Visual Assay zur T6SS-vermittelte bakterielle Wettbewerb überwachen

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

Die Verwendung von<em&gt; Galleria mellon</em&gt; Als Modellorganismus zum Studium<em&gt; Legionella pneumophila</em&gt; Infektion

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

Imaging InlC Sekretion to Cellular-Infektion durch den bakteriellen Erreger untersuchen<em&gt; Listeria monocytogenes</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac&#160;edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.

Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

Zebrafisch als Modell zur Bewertung der teratogene Potenzial von Nitrit

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can be utilized as an alternative animal model for the study of virulence, host-pathogen interaction, innate immune response, and drug efficacy. Details for the rearing, care and breeding of the hissing cockroach are provided. We also illustrate how it can be infected with bacteria such as the intracellular pathogens Burkholderia mallei, B. pseudomallei, and B. thailandensis. Use of the hissing cockroach is inexpensive and overcomes regulatory issues dealing with the use of mammals in research. In addition, results found using the hissing cockroach model are reproducible and similar to those obtained using mammalian models. Thus, the Madagascar hissing cockroach represents an attractive surrogate host that should be explored when conducting animal studies.

We present a protocol to utilize the Madagascar hissing cockroach as an alternative non-mammalian animal model to conduct bacterial virulence, pathogenesis, drug toxicity, drug efficacy, and innate immune response studies.

Der Madagaskar Zischen Schabe als Alternative nicht-Säugetier Tiermodell, Virulenz, Pathogenese und Wirksamkeit von Arzneimitteln zu untersuchen

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Ein Ex-Vivo Huhn Primärkultur Bursitis-Zelle Modell zur Bursitis Infektionskrankheit Virus Pathogenese

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...