Nous tenons à remercier Elisabeth Guillemet, Christophe Buisson et Ludovic Bridoux pour l&#39;assistance technique excellente. Nous sommes très reconnaissants à Sylvie Salamitou et Sinda Fedhila pour la configuration initiale du système.

1. Élevage des insectes Le cycle entier de l&#39;oeuf à larves du dernier dure environ 5 semaines à 25 ° C. Un ou 2 semaines supplémentaires sont nécessaires pour obtenir les papillons adultes. <ol><li> Placez au moins 100 nymphes ou adultes nouvellement fusionnée G. papillons mellonella dans un 5-litres cage grillagée. Papillons mâles mesurent 10 à 15 mm. Le papillon mâle adulte est de couleur beige avec une lumière faible et des marques sombres. Femme papillons mesure environ 20 mm. Les femelles sont plus sombres que les hommes avec une couleur marron / gris. </li><li> Suspendre deux paquets de quatre couches de papier dans la cage pour la ponte. Après 2 jours, la femelle adulte pondent des œufs sur le bord entre les documents. </li><li> Deux fois par semaine, placer les paquets d&#39;œufs en papier dans de nouvelles boîtes d&#39;élevage en plastique avec des grilles pour laisser l&#39;air circuler, contenant du pollen et de cire d&#39;abeille. Les oeufs éclosent au bout de 3 jours et les petites larves commencent à se développer en se nourrissant de la cire et de pollen. Alternativement, les larves peuvent être placés sur une alimentation artificielle consisting d&#39;un mélange de 500 g de miel liquide, 400 g de glycérine, 100 g de levure de bière séchée, 250 g de farine de blé, 200 g de poudre de lait écrémé et 400 g de polenta. Pour fournir ratio approprié de nourriture par larves, régulièrement séparer les larves dans de nouvelles boîtes et remplir les plats tous les deux jours. </li><li> Larves sont élevées au cours des six étapes du stade de larve. Chaque stade est caractérisé par le glissement de la capsule de la tête de larves. Lorsque les larves atteignent le dernier stade avant la nymphose, elles cessent de se nourrir et de commencer à construire un cocon de soie légère. </li><li> Dans leurs cocons protecteurs, les larves se reposer et se transforment en chrysalides. Vers de cire restent dans le stade de pupe pendant une à deux semaines pour émerger dans papillons adultes. Les papillons adultes ne boire ni manger. L&#39;accouplement a lieu et le cycle de vie des vers de cire »recommence. </li><li> Pour normaliser le dosage pathologie, prendre les larves au dernier stade larvaire. Au cours de cette étape, ils cessent de se nourrir, se déplacer sur le couvercle de la boîte de l&#39;élevage et de commencer à produire de la soie. Ce stage dure environ 5 jours. </li></ol> 2. Sélection des insectes de l&#39;infection <ol><li> Sélectionnez dernier stade larvaire, qui sont 2-3 cm de long et de 180 à 250 mg en poids, 24 heures avant l&#39;infection et les mettre dans une boîte vide de les affamer. </li><li> Retirez le cocon de soie naissante autour des larves. </li></ol> 3. Préparation bactérienne <ol><li> Préparer les suspensions bactériennes de Bacillus pour obtenir des concentrations finales allant de 10 4 unités formant colonies (UFC) / ml à 10 8 UFC / ml pour injection intra haemocoelic et de 3x10 6 UFC / ml à 1x10 8 UFC / ml pour l&#39;ingestion (ufc pour obtenir l&#39; une DL 50 dépend des espèces bactériennes). Dans notre cas, B. cereus est cultivé en milieu Luria-Bertani Broth (LB, 10 g / l de tryptone, 5 g d&#39;extrait de levure / l, 10 g / l de NaCl) à 37 ° C sous agitation jusqu&#39;à ce que l&#39;entrée en phase stationnaire, ce qui correspond à l&#39;incurvation de la croissance courbe. Mesurer les DO 600 nm (entre 1 et 2 pour B. cereus) et l&#39;utiliser pour évaluer la concentration bactérienne, en utilisant une courbe de titrage déjà fait. Récolte des bactéries par centrifugation à 5000 xg pendant 10 min à température ambiante et remettre en suspension dans un tampon phosphate salin (PBS: 1M KH 2 PO 4, 1M K 2 HPO 4, 5 M de NaCl, pH 7,2). Chaque larve recevra 10 ul de suspension bactérienne à la concentration souhaitée. </li><li> Alternativement, suspension de spores peuvent être préparés à l&#39;infection. Obtenir des spores en cultivant les bactéries dans le milieu de sporulation HCT (5 g / l de tryptone, 2 g / l d&#39;hydrolysat de caséine, 12,5 mM de K 2 HPO 4, 12,5 mM MgSO 4, 0,05 mM de MnSO 4, 1,2 mM de ZnSO 4, 1,2 mM Fe 2 (SO 4) 3, 0,5% de H 2 SO 4, 25 mM de CaCl 2) 20 à 30 ° C pendant 3 jours. Spores de récolte par centrifugation (10.000 xg, 15 min), et laver deux fois dans de l&#39;eau distillée stérile. Reprendre le pel sporeslaisser dans l&#39;eau distillée stérile et de la chaleur pendant 15 min à 78 ° C pour éliminer les bactéries végétatives restantes. Numerate la suspension en étalant des dilutions en série sur des plaques d&#39;agar LB. Chaque larve recevra 10 ul de suspension de spores à la concentration souhaitée. </li></ol> 4. Préparation toxines Cry <ol><li> Préparer la toxine Cry1C de la B. 407 thuringiensis souche transformée par le plasmide pHTF31C 21 portant le gène codant pour Cry1C. Culture de la souche dans 100 ml milieu HCT à 30 ° C pendant 72 heures avec des antibiotiques (érythromycine 10mg/ml) afin de permettre la sporulation complète, la production de cristaux de toxine et de la libération dans le surnageant de culture. </li><li> Purifier cristaux à partir du surnageant sur un gradient de saccharose à 79% de 72%. D&#39;abord, ajoutez 17 ml de saccharose à 79% en un tube de 40 ml. Soigneusement couche 17 ml de sucrose 72% au-dessus de la saccharose 79%. Enfin, la couche de 5-6 ml de la culture sporulée 10X plus concentré et fermer le tube. Centrifuger le tube pendant 14 heures à 20,000 xg, 4 ° C, pour séparer les cristaux à partir des spores. Recueillir les cristaux à l&#39;interface du gradient. Reprendre le culot de cristal dans 25 ml d&#39;eau stérile froide pour le laver. Centrifuger à 8000 xg pendant 20 min. Cette étape est répétée trois fois pour éliminer toute saccharose coller aux cristaux. Remettre en suspension les cristaux dans 5 ml d&#39;eau stérile et observer au microscope pour évaluer la pureté. </li><li> Évaluer la concentration en protéines de toxines par coloration classique Bradford sur la solution de cristal presolubilized dans 50 mM de NaOH. Cristaux de toxine peut être conservé à -20 ° C jusqu&#39;à utilisation. </li></ol> 5. Préparation injecteur <ol><li> Pour contrôler le volume d&#39;injection précis, utiliser une pompe à seringue automatisée (par exemple KD Scientific KDS 100) avec une vitesse de configuration à 1 l / sec (figure 1A). </li><li> Remplir une seringue de 1 ml hypodermique avec 300 pl d&#39;eau ou de la solution bactérienne (1 seringue par condition) pour l&#39;infection de 20-25 larves. Retirer les bulles en prenant soin de tapping la seringue, puis fixer une aiguille 0,45 x 12 mm à la seringue. </li><li> Placer la seringue dans l&#39;injecteur. </li><li> Effectuer une injection de 10 ul vide dans un tube vide pour contrôler le volume injecté. </li></ol> 6. Insect injection Intrahaemocoelic <ol><li> Placez l&#39;insecte manuellement entre le pouce et l&#39;index. Ensuite, insérer l&#39;aiguille fixée dans l&#39;injecteur dans la peau larve (cuticule) (figure 1C). </li><li> Gardez l&#39;insecte en place lors de l&#39;injection de 10 ul de solution bactérienne (bactéries ou spores végétatives). </li><li> Retirez délicatement l&#39;insecte de l&#39;aiguille. </li><li> Placez l&#39;insecte infecté dans une petite boîte de Petri (5 cm de diamètre), 5 larves par boîte. </li><li> Infecter au moins 20 larves pour chaque condition expérimentale. </li><li> Placez les plats à 37 ° C dans un incubateur pendant 48 heures. </li></ol> 7. Le gavage des insectes (ingestion) <ol><li> Dans un tube Eppendorf de 2 ml, mélanger 0,2 pg / m &amp;u; l de toxine Cry1C soit avec suspension cellulaire des spores ou végétative pour obtenir des concentrations finales allant de 3x10 4 à 1x10 7 pour 10 pl. </li><li> Remplir une seringue de 1 ml avec une 30G, 25 mm aiguille hypodermique avec 300 ul de la solution de toxine bactérienne-Cry1C pour l&#39;infection de 20-25 larves. Retirer les bulles de la seringue. </li><li> Placez l&#39;insecte manuellement de sorte que sa bouche entre l&#39;aiguille fixée dans l&#39;injecteur (figure 1D), et la force-feed avec 10 ul de la solution bactérienne à l&#39;aide de la pompe à seringue automatisée. </li><li> Infecter au moins 20 larves pour chaque condition expérimentale. </li><li> Placez l&#39;insecte infecté dans une petite boîte de Petri de 5 cm (5 larves par boîte). Placez les plats à 37 ° C dans un incubateur pendant 48 heures. </li></ol> 8. La mortalité des insectes d&#39;enregistrement <ol><li> Après des expériences de gavage ou par injection, gardez les larves dans la boîte de Pétri sans nourriture à 25 ° C ou 37 ° C. Vérifiez larves mortality régulièrement sur une période de 48 heures. Larves mortes sont inertes et se tournent généralement noir (figure 1B). </li><li> Les données de mortalité peuvent être analysées à l&#39;aide du programme log-Probit 14. Ce programme teste la linéarité des courbes dose-mortalité et fournit des doses létales (DL 50). Alternativement, d&#39;autres programmes, tels que Prism (Graph Pad), l&#39;analyse de la survie ou de données sur la mortalité peuvent être utilisés. </li></ol>

<ol>
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Aucun conflit d&#39;intérêt déclaré.

L&#39;utilisation d&#39;insectes et en particulier au stade larvaire, comme des modèles d&#39;infection de plusieurs agents pathogènes, devient fréquente. Un modèle de choix pour certains aspects est la drosophile (mouche du modèle) utilisés comme les adultes et 1,2 stade larvaire. Le lépidoptère insectes G. mellonella a également été utilisé principalement pour tester la virulence bactérienne par injection. L&#39;avantage de tolérer des températures plus élevées (supérieur...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td> Cire et de pollen </td><td> La Ruche Roanaise </td><td> 303000 </td><td> Tout producteur de miel </td></tr><tr><td> Pompe à seringue automatique </td><td> KD Scientific </td><td> KDS 100 </td><td></td></tr><tr><td> Seringue de 1 ml </td><td> Terumo </td><td> BS 01T </td><td></td></tr><tr><td> Aiguille 0,45 x 12 mm </td><td> Terumo </td><td> NN 2613R </td><td></td></tr><tr><td> Boîte de Petri de 5 cm </td><td> VWR </td><td> 89000-300 </td><td></td></tr><tr><td> 30G aiguille, 25 mm hypodermique </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

L&#39;étude de la virulence bactérienne nécessite souvent un modèle animal approprié. Modèles de mammifères d&#39;infection sont coûteux et peuvent soulever des questions éthiques. L&#39;utilisation d&#39;insectes comme des modèles d&#39;infection constitue une alternative intéressante. Comparé à d&#39;autres hôtes non vertébrés modèle comme les nématodes, les insectes ont un système relativement avancé des défenses antimicrobiennes et sont donc plus susceptibles de produire des informations pertinentes pour le processus d&#39;infection chez les mammifères. Comme les mammifères, les insectes possèdent un complexe système immunitaire inné 1. Les cellules de l&#39;hémolymphe sont capables de phagocyter ou d&#39;encapsuler des envahisseurs microbiens et réponses humorales comprennent la production inductible de lysozyme et petits peptides antibactériens 2,3. En outre, les analogies se retrouvent entre les cellules épithéliales des insectes larvaires estomacs et les cellules intestinales de l&#39;appareil digestif de mammifères. Enfin, plusieurs composantes de base essentielles pour le processus d&#39;infection bactérienne telle que l&#39;adhérence cellulaire, la résistance à lapeptides antimicrobiens, la dégradation des tissus et de l&#39;adaptation au stress oxydatif sont susceptibles de jouer un rôle important dans les deux insectes et les mammifères 1. Ainsi, les insectes sont des outils polyvalents pour l&#39;identification et la caractérisation des facteurs de virulence impliqués dans les infections microbiennes mammifères. Les larves de la teigne plus grande Galleria mellonella ont été montré pour fournir un aperçu utile de la pathogenèse d&#39;un large éventail d&#39;infections microbiennes, y compris les mammifères fongiques (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) et des bactéries pathogènes, telles que Staphylococcus aureus, Proteus vulgaris , Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes ou Enterococcus faecalis 4-7. Quelles que soient les espèces bactériennes, les résultats obtenus avec des larves infectées Galleria par injection directe à travers la cuticule toujours en corrélation avec ceux de similar des études de mammifères: les souches bactériennes qui sont atténuées dans les modèles de mammifères montrent moins virulents dans la Galleria, et des souches responsables de graves infections humaines sont également très virulent dans le modèle Galleria 8-11. L&#39;infection orale de la Galleria est beaucoup moins utilisées et d&#39;autre composés, comme des toxines spécifiques, sont nécessaires pour atteindre la mortalité. G. mellonella larves présentent plusieurs avantages techniques: elles sont relativement importantes (dernier stade larvaire avant la nymphose ya environ 2 cm de long et un poids de 250 mg), ce qui permet l&#39;injection de doses définies de bactéries, ils peuvent être élevés à différentes températures (20 ° études C à 30 ° C) et l&#39;infection peut être effectuée entre 15 ° C et au-dessus de 37 ° C 12,13, ce qui permet d&#39;expériences qui imitent un environnement de mammifère. En outre, l&#39;élevage d&#39;insectes est facile et relativement pas cher. Infection des larves permet de surveiller la virulence bactérienne par plusieurs moyens, includincalcul g de LD 50 14, mesure de la survie de la bactérie 15,16 et l&#39;examen du processus d&#39;infection 17. Ici, nous décrivons l&#39;élevage des insectes, couvrant toutes les étapes de la vie de G. mellonella. Nous fournissons un protocole détaillé de l&#39;infection par deux voies d&#39;inoculation: haemocoelic orale et intra. Le modèle bactérienne utilisée dans ce protocole est Bacillus cereus, un agent pathogène à Gram positif impliquées dans gastro-intestinal ainsi que dans d&#39;autres locaux ou systémiques sévères infections opportunistes 18,19.

Injection intra haemocoelic de bactéries dans G. mellonella s&#39;est avéré très utile pour l&#39;identification des facteurs de virulence de nombreuses traitant de dommages aux tissus et la résistance aux facteurs immunitaires innés de plusieurs agents pathogènes humains. A titre d&#39;exemple, la figure 2A représente la mortalité des insectes après l&#39;injection de doses différentes de B. bactéries cereus (type sauvage et des souches mutantes) 22. <s...

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

Orale et intra infection haemocolic des larves de la teigne de la cire plus<em&gt; Galleria mellonella</em&gt; Est décrite. Cet insecte peut être utilisée pour étudier les facteurs de virulence de entomopathogène ainsi que de mammifères bactéries opportunistes. L&#39;élevage des insectes, des méthodes d&#39;infection et des exemples de<em&gt; In vivo</em&gt; L&#39;analyse sont décrits.

L&#39;insecte<em&gt; Galleria mellonella</em&gt; Comme un modèle d&#39;infection puissant pour étudier la pathogenèse bactérienne

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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Immunology and Infection

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

31.6K vues.  INRA, Micalis UMR1319, France. Orale et intra infection haemocolic des larves de la teigne de la cire plus Galleria mellonella Est décrite. Cet insecte peut être utilisée pour étudier les facteurs de virulence de entomopathogène ainsi que de mammifères bactéries opportunistes. L'élevage des insectes, des méthodes d'infection et des exemples de In vivo L'analyse sont décrits.

Vidéo: 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

Dans la formation de biofilms in vitro dans une diapositive Chambre de 8 puits

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

Recherche

Immunologie et infection

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.

En utilisant la luciférase aux infections bactériennes chez les souris image

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.

Présentation de contrainte de cisaillement dans l&#39;étude de l&#39;adhésion bactérienne

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.

La salive, les glandes salivaires, et Collection hémolymphe de tiques Ixodes scapularis

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.

Un test visuel pour surveiller T6SS à médiation bactérienne concurrence

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.

Utilisation d&#39;<em&gt; Galleria mellonella</em&gt; Comme un organisme modèle pour l&#39;étude<em&gt; Legionella pneumophila</em&gt; Maladies infectieuses

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.

Imagerie RICL sécrétion d&#39;enquêter sur l&#39;infection cellulaire par le pathogène bactérien<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.

Poisson zèbre comme modèle pour évaluer le potentiel tératogène de 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 ...

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.

Le Madagascar blatte comme Alternative non-mammifère modèle Animal pour étudier la Virulence, pathogenèse et l’efficacité du médicament

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.

Un modèle de Culture primaire de cellules bursite de poulet Ex Vivo pour étudier la bursite infectieuse Virus pathogenèse

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