Vorremmo ringraziare Elisabeth Guillemet, Christophe Buisson e Ludovic Bridoux per un&#39;eccellente assistenza tecnica. Siamo molto in debito con Sylvie Salamitou e Sinda Fedhila per la configurazione iniziale del sistema.

1. Allevamento insetti L&#39;intero ciclo da uovo all&#39;ultimo larve instar dura circa 5 settimane a 25 ° C. Uno o 2 settimane supplementari sono necessari per ottenere farfalle adulte. <ol><li> Inserire almeno 100 pupe o di nuova fusione adulto G. farfalle mellonella in un 5 litri maglia metallica gabbia. Farfalle maschio misurare 10 a 15 mm. La falena maschio adulto è beige con la luce debole e macchie scure. Donna misura farfalle di circa 20 mm. Le femmine sono più scure dei maschi con un marrone / grigio. </li><li> Sospendere due pacchetti di quattro strati di carta nella gabbia per la deposizione delle uova. Dopo 2 giorni, la femmina adulta si depongono le uova sul bordo tra le carte. </li><li> Due volte a settimana, posizionare le uova di carta confezioni in nuove scatole di allevamento di plastica con griglie per permettere all&#39;aria di circolare, contenente polline e cera d&#39;api. Le uova si schiudono in circa 3 giorni e piccole larve iniziano a sviluppare nutrendosi di cera e polline. In alternativa, le larve possono essere posizionati su una dieta artificiale consisting di una miscela di 500 g di miele liquido, glicerina 400 g, 100 g di lievito di birra essiccato, farina di frumento 250 g, 200 g di latte scremato in polvere e 400 g polenta. Per fornire il rapporto adeguato di cibo per larva, regolarmente separare le larve in scatole nuove e riempire il cibo ogni due giorni. </li><li> Le larve sono allevati durante le sei fasi dello stadio larva. Ogni stadio è caratterizzato dalla slittamento della capsula testa di larve. Quando le larve raggiungono l&#39;ultima tappa prima impupamento, smettono di alimentazione e iniziare a costruire un bozzolo di seta leggera. </li><li> Nelle loro bozzoli protettivi, le larve si riposerà e si trasformano in pupe. Vermi di cera rimangono in fase di pupa per una o due settimane per emergere in falene adulte. Le falene adulte non bere né mangiare. L&#39;accoppiamento avviene e il ciclo di vita dei vermi di cera &#39;ricomincia. </li><li> Per uniformare il saggio patologia, prendere le larve durante l&#39;ultimo stadio larvale. Durante questa fase si fermano alimentazione, spostare il coperchio della scatola di allevamento e iniziare a produrre la seta. Questo stage dura circa 5 giorni. </li></ol> 2. Selezione insetti per l&#39;infezione <ol><li> Selezionare ultimo larve instar, che sono 2-3 cm di lunghezza e 180-250 mg di peso, 24 ore prima di infezioni e metterle in una scatola vuota per farli morire di fame. </li><li> Rimuovere il bozzolo di seta attorno al nascente larve. </li></ol> 3. Preparazione batterica <ol><li> Preparare sospensioni batteriche Bacillus per ottenere concentrazioni finali che vanno da 10 4 unità formanti colonie (ufc) / ml a 10 8 cfu / ml per iniezione intra haemocoelic e da 3x10 6 cfu / ml a 1x10 8 cfu / ml per ingestione (la ufc per ottenere un LD 50 dipende dalla specie batterica). Nel nostro caso, B. cereus è coltivato in Luria-Bertani brodo (LB, 10 g / l triptone, 5 g / l di estratto di lievito, 10 g / l NaCl) a 37 ° C sotto agitazione fino all&#39;entrata in fase stazionaria, corrispondente alla incurvation della crescita curva. Misurare le OD (600 nmtra 1 e 2 di B. cereus) e usarlo per valutare la concentrazione batterica, utilizzando una curva di titolazione precedentemente fatto. Batteri raccolto per centrifugazione a 5000 xg per 10 min a temperatura ambiente e risospendere in tampone fosfato isotonico (PBS: 1M KH 2 PO 4, 1M K 2 HPO 4, 5M NaCl, pH 7,2). Ogni larva riceverà 10 ml di sospensione batterica alla concentrazione desiderata. </li><li> In alternativa, sospensione di spore può essere preparato per l&#39;infezione. Ottenere spore coltivando i batteri nella sporulazione mezzo HCT (5 g / L triptone, 2 g / L caseina idrolizzato, 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 a 30 ° C per 3 giorni. Harvest spore mediante centrifugazione (10.000 xg, 15 min), e lavare due volte in acqua distillata sterile. Risospendere il pel sporelasciate in acqua distillata sterile e calore per 15 min a 78 ° C per rimuovere residui batteri vegetativi. Numerare la sospensione da placcatura diluizioni seriali su piastre di agar LB. Ogni larva riceverà 10 pl di sospensione di spore alla concentrazione desiderata. </li></ol> 4. Cry Preparazione Tossina <ol><li> Preparare la tossina Cry1C dal B. ceppo thuringiensis 407 trasformato con il plasmide pHTF31C 21 portando la Cry1C gene codificante. Coltura del ceppo in mezzo HCT 100 ml a 30 ° C per 72 ore con antibiotici (eritromicina 10mg/ml) per consentire sporulazione completa, cristallo produzione della tossina e liberazione nel supernatante di coltura. </li><li> Purificare cristalli dal supernatante su un gradiente di saccarosio 72% -79%. Prima aggiungere 17 ml di 79% saccarosio in una provetta 40 ml. Attentamente strato 17 ml di saccarosio 72% sopra il 79% di saccarosio. Infine, strato 5-6 ml della coltura concentrato 10X sporigeni e chiudere il tubo. Centrifugare la provetta per 14 ore a 20,000 xg, a 4 ° C, per separare i cristalli dalle spore. Raccogliere i cristalli all&#39;interfaccia gradiente. Risospendere il pellet di cristallo in 25 ml di acqua fredda sterile per lavarlo. Centrifugare a 8000 xg per 20 min. Questo passaggio viene ripetuto tre volte per rimuovere tutti saccarosio aderiscano alle cristalli. Risospendere i cristalli in 5 ml di acqua sterile ed osservare al microscopio per valutare la purezza. </li><li> Valutare la concentrazione di proteine ​​tossina mediante colorazione classica Bradford sulla soluzione a cristalli presolubilized in 50 mM NaOH. Cristalli tossina può essere conservato a -20 ° C fino all&#39;uso. </li></ol> 5. Iniettore Preparazione <ol><li> Per controllare il volume esatto di iniezione, utilizzare una pompa automatizzato siringa (es Scientific KDS KD 100) con velocità di installazione a 1 ml / sec (Figura 1A). </li><li> Riempire una siringa da 1 ml ipodermica con 300 microlitri di acqua o la soluzione batterica (1 siringa per condizione) per l&#39;infezione di 20-25 larve. Rimuovere le bolle con cura tapping la siringa, quindi fissare un x 0.45 12 ferri alla siringa. </li><li> Posizionare la siringa nell&#39;iniettore. </li><li> Effettuare un&#39;iniezione vuoto di 10 microlitri in un tubo vuoto per controllare il volume iniettato. </li></ol> 6. Insetto iniezione Intrahaemocoelic <ol><li> Posizionare l&#39;insetto manualmente tra il pollice e l&#39;indice. Quindi inserire l&#39;ago fissato l&#39;iniettore nella pelle larva (cuticola) (Figura 1C). </li><li> Tenere l&#39;insetto in posizione durante l&#39;iniezione di 10 ul di soluzione batterica (batteri spore o vegetativa). </li><li> Rimuovere con cautela l&#39;insetto dall&#39;ago. </li><li> Posizionare l&#39;insetto infetto in una piccola scatola di Petri (5 cm di diametro), 5 larve per piatto. </li><li> Infect almeno 20 larve per ogni condizione sperimentale. </li><li> Porre le capsule a 37 ° C in un incubatore per 48 ore. </li></ol> 7. Forza alimentazione degli insetti (Ingestione) <ol><li> In una provetta Eppendorf da 2 ml, miscelare 0,2 mg / m &amp;u; l di tossina Cry1C sia con sospensione cellulare spore o vegetativa ottenere concentrazioni finali che vanno da 3x10 a 1x10 4 7 per 10 pl. </li><li> Riempire una siringa da 1 ml con una 30G, 25 ago ipodermico mm con 300 microlitri della soluzione batterica-Cry1C tossina per l&#39;infezione di 20-25 larve. Rimuovere le bolle dalla siringa. </li><li> Posizionare l&#39;insetto manualmente in modo che la sua bocca entra l&#39;ago fissato l&#39;iniettore (Figura 1D), e forzarlo a mangiare con 10 pl della soluzione batterica utilizzando la pompa automatizzato siringa. </li><li> Infect almeno 20 larve per ogni condizione sperimentale. </li><li> Posizionare l&#39;insetto infetto in un piccolo 5 centimetri Petri (5 larve per piatto). Porre le capsule a 37 ° C in un incubatore per 48 ore. </li></ol> 8. Registrazione Insetto Mortalità <ol><li> Dopo gli esperimenti di ingozzatura o iniezione, mantenere le larve nella scatola di Petri senza cibo a 25 ° C o 37 ° C. Controllare larvale mortality regolarmente per un periodo di 48 ore. Larve morte sono inerti e di solito diventa nero (Figura 1B). </li><li> I dati di mortalità possono essere analizzati utilizzando il log-probit programma 14. Questo programma collauda la linearità delle curve di mortalità dose e fornisce dosi letali (LD 50). In alternativa, altri programmi, come Prism (grafico Pad), analizzando la sopravvivenza o dati di mortalità possono essere utilizzati. </li></ol>

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L&#39;uso di insetti e in particolare la fase larvale, come modelli di infezione per i patogeni più, sta diventando frequente. Un modello di scelta per alcuni aspetti è Drosophila (il modello di volo) utilizzato come adulti e 1,2 stadio larvale. L&#39;insetto lepidotteri G. mellonella stato anche utilizzato principalmente per saggiare la virulenza batterica per iniezione. Il vantaggio di tollerare temperature più elevate (oltre i 37 ° C) rispetto Drosophila (massimo 25 ° C) è ...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Cera e polline </td><td> La Ruche Roanaise </td><td> 303000 </td><td> Ogni produttore di miele </td></tr><tr><td> Siringa pompa automatica </td><td> KD scientifico </td><td> KDS 100 </td><td></td></tr><tr><td> Siringa da 1 ml </td><td> Terumo </td><td> BS 01T </td><td></td></tr><tr><td> Ago 0,45 x 12 mm </td><td> Terumo </td><td> NN 2613R </td><td></td></tr><tr><td> Petri 5 centimetri </td><td> VWR </td><td> 89000-300 </td><td></td></tr><tr><td> Ago 30G, 25 millimetri ipodermico </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

Lo studio della virulenza batterica richiede spesso un modello animale adatto. Modelli mammiferi di infezione sono molto costosi e possono sollevare questioni etiche. L&#39;uso di insetti come modelli di infezione fornisce una valida alternativa. Rispetto ad altri non vertebrati host modello come nematodi, insetti hanno un sistema relativamente avanzato di difesa antimicrobici e sono quindi più in grado di produrre informazioni rilevanti per il processo di infezione dei mammiferi. Come mammiferi, insetti possiedono un complesso sistema immunitario innato 1. Le cellule della emolinfa sono in grado di phagocytosing o riassume invasori microbici, e le risposte umorali includono la produzione di peptidi antibatterici inducibile lisozima e piccole 2,3. Inoltre, si riscontrano analogie tra le cellule epiteliali di insetto midguts larvali e cellule intestinali dei sistemi digestivi mammiferi. Infine, alcuni componenti di base essenziali per il processo di infezione batterica, come adesione cellulare, resistenzapeptidi antimicrobici, il degrado dei tessuti e l&#39;adattamento allo stress ossidativo sono suscettibili di essere importante in entrambi i insetti e mammiferi 1. Così, gli insetti sono strumenti polivalenti per l&#39;identificazione e la caratterizzazione di fattori di virulenza microbici coinvolti nelle infezioni mammiferi. Larve di falena maggiore della cera Galleria mellonella hanno dimostrato di fornire una visione utile nella patogenesi di una vasta gamma di infezioni fungine microbiche inclusi mammiferi (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) e batteri patogeni, quali Staphylococcus aureus, Proteus vulgaris , Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes o Enterococcus faecalis 4-7. Indipendentemente dalla specie batteriche, i risultati ottenuti con larve Galleria infettato da iniezione diretta attraverso la cuticola costantemente correlate con quelle di similar studi mammiferi: ceppi batterici che sono attenuati nei modelli mammiferi dimostrano minore virulenza in Galleria, e le tensioni che causano gravi infezioni umane sono altamente virulenti nel modello Galleria 8-11. Infezione orale della Galleria è molto meno usato e ulteriori composti, come le tossine specifiche, sono necessari per raggiungere la mortalità. G. mellonella larve presentano diversi vantaggi tecnici: sono relativamente grandi (ultima larve instar prima pupation sono circa 2 cm e peso 250 mg), consentendo così l&#39;iniezione di dosi prestabilite di batteri, possono essere allevati a diverse temperature (20 ° studi C a 30 ° C) e l&#39;infezione può essere condotta tra 15 ° C al di sopra di 37 ° C 12,13, consentendo esperimenti che simulano un ambiente di mammifero. Inoltre, l&#39;allevamento degli insetti è facile e relativamente a buon mercato. L&#39;infezione delle larve permette di monitorare virulenza batterica in vari modi, sapcalcolo g di LD 50 14, la misura di sopravvivenza batterica 15,16 e l&#39;esame del processo di infezione 17. Qui, descriviamo l&#39;allevamento degli insetti, che copre tutte le fasi della vita di G. mellonella. Forniamo un dettagliato protocollo di infezione da due vie di inoculazione: haemocoelic orale e intra. Il modello batterica utilizzato in questo protocollo è Bacillus cereus, un patogeno Gram positivi implicati in gastrointestinale e in altre gravi infezioni locali o sistemiche opportunistiche 18,19.

Intra haemocoelic iniezione di batteri in G. mellonella è stato dimostrato molto utile per l&#39;identificazione di fattori di virulenza molti occupano di danno tissutale e resistenza a fattori immuni innate di diversi agenti patogeni umani. Come esempio, la figura 2A rappresenta la mortalità insetto dopo iniezione di varie dosi di B. batteri cereus (wild type e mutanti) 22. figura 2B rappresenta la sopravvivenza dopo l&#39;infezione batterica di ...

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 e infezioni intra haemocolic di larve della tarma della cera maggiore<em&gt; Galleria mellonella</em&gt; È descritto. Questo insetto possono essere utilizzati per studiare i fattori di virulenza di entomopatogeno nonché mammiferi batteri opportunisti. Allevamento degli insetti, i metodi di infezione ed esempi di<em&gt; In vivo</em&gt; L&#39;analisi sono descritti.

L&#39;insetto<em&gt; Galleria mellonella</em&gt; Come un modello di infezione potente per Indagare Patogenesi batterica

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 Views.  INRA, Micalis UMR1319, France. Orale e infezioni intra haemocolic di larve della tarma della cera maggiore Galleria mellonella È descritto. Questo insetto possono essere utilizzati per studiare i fattori di virulenza di entomopatogeno nonché mammiferi batteri opportunisti. Allevamento degli insetti, i metodi di infezione ed esempi di In vivo L'analisi sono descritti.

Video: 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

Nella formazione di biofilm in vitro in un 8-bene scorrere Camera

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

Ricerca

Immunologia e infezioni

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.

Utilizzando luciferasi di infezioni batteriche immagine in topi

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.

L&#39;introduzione di shear stress nello studio di adesione batterica

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.

Saliva, le ghiandole salivari, e Collection emolinfa dalle zecche 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 per il monitoraggio visivo T6SS-mediata Concorso batterica

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.

Uso di<em&gt; Galleria mellonella</em&gt; Come organismo modello per studiare<em&gt; Legionella pneumophila</em&gt; Infezione

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 secrezione per Indagare infezione Cellular dalla batterica patogeni<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.

Zebrafish come modello per valutare il potenziale teratogeno di nitriti

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.

Il Madagascar sibilo scarafaggio come modello alternativo Non mammiferi animali per studiare la virulenza, patogenesi e l'efficacia dei farmaci

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 modello di cultura Bursal-cellula primaria di pollo Ex Vivo per studiare la patogenesi Virus bursite infettiva

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