Ringraziamo sinceramente i membri del laboratorio Pal per l&#39;assistenza con la preparazione di questa manifestazione. Questo studio è stato supportato da PHS concede AI076684 e AI080615 dal NIH / NIAID.

1. Microiniezione di ninfale zecche Ixodes scapularis 1. Aghi preparazione <ol><li> Fabbricare aghi microiniezione diversi dal riscaldamento e tirando 1 tubi di vetro capillari mm (Strumenti di precisione World) in un dispositivo di vetro micropipetta estrattore (Narishige). Rimuovere con attenzione i tubi capillari fragili. </li><li> Conservare tirato aghi (con punta rivolta verso l&#39;alto) sul nastro adesivo in una capsula di Petri. </li></ol> 2. Preparazione B. burgdorferi <ol><li> Crescere B. burgdorferi in BSK cultura mediatica 7 fino ad una concentrazione di circa 10 7 cellule per ml. Spirochete sono contati con un microscopio a campo oscuro con l&#39;uso di una camera di conteggio Petroff-Hausser (Hausser scientifica). </li><li> Pellet B. burgdorferi mediante centrifugazione a 3.000 xg per 10 minuti a temperatura ambiente. </li><li> Rimuovere il surnatante e risospendere completamente il pellet in BSK mezzi delicatamente passando attraverso un microtip 200 ul sterile ad una concentrazione finale di 10 9 cellule per ml. Dal momento che le cellule sono risospese ad alta densità cellulare, e potrebbero raggrupparsi insieme, la sospensione cellulare deve essere utilizzato per microiniezione immediatamente. </li></ol> 3. Preparare le zecche <ol><li> Collocazione chiara, su due lati del nastro adesivo su un vetrino. </li><li> Lavorare su un tappetino, rimuovere con attenzione le zecche ninfale dal contenitore con un pennello di piccole dimensioni. </li><li> Collocare il numero di zecche da iniettare sul nastro adesivo, lato ventrale in su, verso la stessa direzione. </li></ol> 4. Zecche iniettando <ol><li> Di carico 5 microlitri della B. cultura burgdorferi in l&#39;ago tirato capillare utilizzando un 20 l microloader puntale (Eppendorf). Controllare l&#39;ago bolle d&#39;aria intrappolate e ricaricare l&#39;ago con coltura batterica, se necessario. </li><li> Mostra il segno di spunta sotto il microscopio binoculare da dissezione e concentrarsi sulla zona anale apertura della zecca. Delicatamente toccare la punta dell&#39;ago capillare al fine di rompere il tubo in cui il diametro è leggermente inferiore a quello di apertura anale della zecca, formando l&#39;ago microiniezione. Questo passaggio è fondamentale, come ogni tentativo di iniettare le zecche con un non adeguato punta dell&#39;ago provoca la morte e possibili lesioni della zecca iniettato. </li><li> Posizionare le zecche immobilizzato sotto il microscopio binoculare da dissezione e mettere a fuoco l&#39;apertura anale, che è coperto da due piastre mobili anale. Utilizzando una pinza sottile, toccare delicatamente e applicare una pressione molto lieve a qualsiasi area nei pressi di apertura anale. Questo permetterà la separazione delle placche anali e apertura del poro anale che si collega l&#39;intestino. Inserire con attenzione la punta dell&#39;ago leggermente in apertura anale attraverso l&#39;apertura forzata delle piastre anale. L&#39;inserimento dell&#39;ago dovrebbe essere ridotto al minimo come la punta di vetro potrebbe danneggiare il hindgut semitrasparente che si connette al retto. Utilizzando un microinjector dotato di comando a pedale automatico (Eppendorf), iniettare B. burgdorferi soluzione utilizzando i seguenti parametri: 1.000 ettopascal (hPa) pressione di iniezione, iniezione di 0,2 secondi di tempo e 8 compensazione della pressione hPa. Ogni tick riceve una sola iniezione. </li><li> In seguito a microiniezione, le zecche dovrebbero comportarsi simile a quello pre-iniezione di stato. Ad esempio, il segno di spunta dovrebbe strisciare in risposta agli stimoli. </li><li> Noi di solito permettono di recuperare le zecche per 48 ore in una camera climatica impostata a 24 ° C con 16 ore / 8 ore di luce / buio regime di fotoperiodo e il 95% di umidità. Se una camera non è disponibile, zecche iniettato può essere conservato a temperatura ambiente in condizioni di umidità all&#39;interno di un contenitore a tenuta stagna, come una camera di essiccatore regolare. Se necessario, i tempi di recupero delle zecche potrebbe anche essere ridotto a poche ore prima utilizzata per l&#39;alimentazione sui topi. </li></ol> 2. B. Localizzazione burgdorferi da microscopia di immunofluorescenza confocale 1. Zecche dissezione <ol><li> Mettere una goccia di tampone fosfato (PBS) su un vetrino pulito per la dissezione e l&#39;altro su una poli-L-lisina scivolo rivestito (Sigma) per microscopia. Preparare un altro vetrino con doppio nastro adesivo. </li><li> Togliere il segno di spunta dal contenitore e luogo due lati del nastro adesivo con un pennello di piccole dimensioni. </li><li> Visualizza e mettere a fuoco il segno di spunta al microscopio dissezione. Inserire una lama affilata rasoio sul segno di spunta tra le coppie prima e la seconda di gambe. Premere con decisione taglio il segno di spunta in due pezzi che permette l&#39;accesso all&#39;addome. Immediatamente, immergere l&#39;addome in una goccia di PBS. </li><li> Utilizzando pinze molto fine, afferrare l&#39;esoscheletro dorsale e ventrale intorno al sito del taglio. Estrarre con cautela lo scudo dorsale alto e lontano dalla zecca di esporre il diverticoli intestinali bruno colore. Fare attenzione a tenere le zecche dissezione sotto il PBS in ogni momento. </li><li> Il semi-trasparente panino ghiandole salivaridles si trovano su entrambi i lati della regione anteriore dell&#39;intestino, e può essere rimosso, a questo punto, se lo si desidera. Tenendo l&#39;esoscheletro rimanere al loro posto con una pinza, estrarre con cautela l&#39;intestino fuori dall&#39;addome. </li><li> Pulire delicatamente l&#39;intestino rimuovendo i tessuti intrappolati (ad esempio, trachea). </li><li> Utilizzando la punta di una pinza sottile, trasferire rapidamente l&#39;intestino zecca in una goccia di PBS su un poli-L-lisina vetrino. Separare l&#39;intestino in pezzi più piccoli con lame fine o premendo delicatamente con la punta di una pinza sottile. Con attenzione aspirare l&#39;eccesso di PBS in tutto il tessuto intestinale. </li><li> Lasciare i tessuti dell&#39;intestino di aria secca a temperatura ambiente. </li><li> Fissare l&#39;intestino tick immergendo in acetone per 10 minuti. Lasciare asciugare a temperatura ambiente. Le diapositive possono essere conservati a questo passaggio per mesi a -20 ° C in un contenitore a tenuta stagna. </li></ol> 2. Colorazione <ol><li> Utilizzando la carta velina, rimuovere l&#39;umidità in eccesso in tutto il tessuto e disegna un cerchio intorno l&#39;intestino tick secco da un pap-penna o un qualsiasi dispositivo di rivestimento idrofobo barriera. Ciò contribuirà a mantenere le soluzioni colorazione durante le fasi di incubazione successive. </li><li> Coprire l&#39;intestino barrare con una o poche gocce di tampone di bloccaggio (0,05% di Tween-20, siero di capra al 5% in PBS) per 30 minuti a temperatura ambiente. Il siero utilizzato per bloccare dipende dalla fonte di animale ospite per l&#39;anticorpo. Non lasciare che il vetrino si asciughi da questo punto in avanti. </li><li> Lentamente aspirare il tampone di bloccaggio. Incubare l&#39;intestino con primario adeguato e / o soluzioni anticorpo secondario. Noi usiamo isotiocianato di fluoresceina (FITC) - marcato anti-B. burgdorferi anticorpi (Kirkegaard &amp; Perry Laboratories) alla diluizione 1:100 in tampone bloccante per 1 ora a temperatura ambiente. Coprire il recipiente con un foglio di alluminio per limitare l&#39;esposizione alla luce. </li><li> Rimuovere la soluzione di anticorpi da aspirazione delicata. Incubare l&#39;intestino con un colorante fluorescente ai tessuti zecca etichetta, ad esempio 4 &#39;,6-diamidino-2-fenilindolo (DAPI) o ioduro di propidio. Usiamo normalmente 20 mg / ml di ioduro di propidio (Sigma) in PBS per 5 min. a temperatura ambiente. </li><li> Lavare 3 volte con 0,05% di Tween-20 in PBS. </li><li> Montare il vetrino in glicerolo tamponata contenente un reagente Antifade come Slowfade (Invitrogen) e coprire accuratamente con coprioggetto di vetro. Le diapositive possono essere conservati a questo passaggio per qualche mese a 4 ° C all&#39;interno di un contenitore a tenuta stagna. Immagine e localizzare B. burgdorferi al microscopio confocale. </li></ol> 3. Rappresentante Risultati Posizione di un tick ninfale per microiniezione e un&#39;immagine che rappresenta B. localizzazione burgdorferi nell&#39;intestino zecca è presentato in Figura 1. <img src="/files/ftp_upload/2544/2544fig1.jpg" alt="Figura 1" /> Figura 1. Microiniezione e localizzazione di B. burgdorferi nell&#39;intestino zecca. (A) Vista ventrale di una ninfa I. tick scapularis posizionato per microiniezione. Anal apertura (freccia) con ago microiniezione inserito (freccia) viene mostrata sotto ingrandimento nel riquadro. Una pinza fine è utilizzato per applicare una leggera pressione sul corpo, che ha permesso la separazione delle piastre anale e apertura del poro anale per microiniezione. L&#39;ago viene riempito con una soluzione di Coomassie Brilliant Blue per migliorare la visibilità. (B) I risultati rappresentativi di immunofluorescenza confocale immagini di B. burgdorferi all&#39;interno dell&#39;intestino tick. Regione anteriore di un diverticolo intestinale viene mostrato. Nuclei Gut e spirochete (freccia) sono etichettati con ioduro di propidio (colore rosso) o FITC-coniugato anti-B. burgdorferi (colore verde), rispettivamente. Bar = 20 micron.

<ol>
	<li>Steere, A. C., Coburn, J., Glickstein, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emergence+of+Lyme+disease.">The emergence of Lyme disease.</a> J Clin Invest. 113, 1093-1101 (2004).</li><li>Barthold, S. W., Diego, C., Philipp, M. T., Samuels, D. S., Radolf, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Borrelia, Molecular Biology, Host Interaction and Pathogenesis. , 353-405 (2010).</li><li>Pal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OspC+facilitates+Borrelia+burgdorferi+invasion+of+Ixodes+scapularis+salivary+glands.">OspC facilitates Borrelia burgdorferi invasion of Ixodes scapularis salivary glands.</a> J Clin Invest. 113, 220-230 (2004).</li><li>Yang, X. F., Pal, U., Alani, S. M., Fikrig, E., Norgard, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+for+OspA/B+in+the+life+cycle+of+the+Lyme+disease+spirochete.">Essential role for OspA/B in the life cycle of the Lyme disease spirochete.</a> J Exp Med. 199, 641-648 (2004).</li><li>Zhang, X., Yang, X., Kumar, M., Pal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BB0323+function+is+essential+for+Borrelia+burgdorferi+virulence+and+persistence+through+tick-rodent+transmission+cycle.">BB0323 function is essential for Borrelia burgdorferi virulence and persistence through tick-rodent transmission cycle.</a> J Infect Dis. 200, 1318-1330 (2009).</li><li>Pal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TROSPA,+an+Ixodes+scapularis+receptor+for+Borrelia+burgdorferi.">TROSPA, an Ixodes scapularis receptor for Borrelia burgdorferi.</a> Cell. 119, 457-468 (2004).</li><li>Barbour, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+cultivation+of+Lyme+disease+spirochetes.">Isolation and cultivation of Lyme disease spirochetes.</a> Yale J Biol Med. 57, 521-525 (1984).</li><li>Narasimhan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+Ixodes+scapularis+anticoagulation+by+using+RNA+interference.">Disruption of Ixodes scapularis anticoagulation by using RNA interference.</a> Proc Natl Acad Sci U S A. 101, 1141-1146 (2004).</li><li>Narasimhan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tick+antioxidant+facilitates+the+Lyme+disease+agent's+successful+migration+from+the+mammalian+host+to+the+arthropod+vector.">A tick antioxidant facilitates the Lyme disease agent's successful migration from the mammalian host to the arthropod vector.</a> Cell Host Microbe. 2, 7-18 (2007).</li><li>Broadwater, A. H., Sonenshine, D. E., Hynes, W. L., Ceraul, S., DeSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glass+capillary+tube+feeding:+a+method+for+infecting+nymphal+Ixodes+scapularis+(Acari:+Ixodidae)+with+the+Lyme+disease+spirochete+Borrelia+burgdorferi.">Glass capillary tube feeding: a method for infecting nymphal Ixodes scapularis (Acari: Ixodidae) with the Lyme disease spirochete Borrelia burgdorferi.</a> J Med Entomol. 39, 285-292 (2002).</li><li>Policastro, P. F., Schwan, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+infection+of+Ixodes+scapularis+larvae+(Acari:+Ixodidae)+by+immersion+in+low+passage+cultures+of+Borrelia+burgdorferi.">Experimental infection of Ixodes scapularis larvae (Acari: Ixodidae) by immersion in low passage cultures of Borrelia burgdorferi.</a> J Med Entomol. 40, 364-370 (2003).</li><li>Fuente, d. e. l. a., Kocan, J., M, K., Almazan, C., Blouin, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+for+the+study+and+genetic+manipulation+of+ticks.">RNA interference for the study and genetic manipulation of ticks.</a> Trends Parasitol. 23, 427-433 (2007).</li></ol>

Nessun conflitto di interessi dichiarati.

Qui si dimostrano una microiniezione procedura basata per l&#39;infezione rapida ed efficiente dei ninfale zecche Ixodes con il batterio patogeno B. burgdorferi. Abbiamo anche descrivere una procedura di immunofluorescenza confocale per il rilevamento di B. burgdorferi nell&#39;intestino zecca in situ. Anche se la nostra manifestazione coinvolge intestino ninfale, procedure simili sono applicabili anche per le altre fasi di sviluppo delle zecche, come larve o gli adulti 8,9. Tuttavi...

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<td>Glass capillary tubes</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>TW100F-4</td>
<td>&nbsp;</td>
<tr>
<td>Vertical glass puller</td>
<td>&nbsp;</td>
<td>Narishige</td>
<td>PC-10</td>
<td>&nbsp;</td>
<tr>
<td>Petroff-Hausser counting chamber</td>
<td>&nbsp;</td>
<td>Hausser scientific</td>
<td>3900</td>
<td>&nbsp;</td>
<tr>
<td>Microloader pipette tips</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>930001007</td>
<td>&nbsp;</td>
<tr>
<td>Femtojet microinjector</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>920010504</td>
<td>&nbsp;</td>
<tr>
<td>Foot control FemtJet</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>920005098</td>
<td>&nbsp;</td>
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<td>Phosphate buffered saline</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP665-1</td>
<td>Filter-sterilized</td>
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methods for rapid transfer and localization of lyme disease pathogens within the tick gut

La malattia di Lyme è causata da infezione con il patogeno spirocheta Borrelia burgdorferi, che è mantenuto in natura da una zecca-roditore ciclo di infezione 1. Un zecche modello murino 2 è stato sviluppato per studiare la malattia di Lyme in laboratorio. Mentre zecche ingenuo può essere infettato con B. burgdorferi nutrendole sui topi infetti, il processo di muta impiega diverse settimane o mesi per essere completata. Pertanto, lo sviluppo di tecniche di zecca più rapido ed efficace l&#39;infezione, come una procedura di microiniezione-based, è uno strumento importante per lo studio della malattia di Lyme 3,4. La procedura richiede ore solo per generare zecche infette e consente di controllare l&#39;erogazione di quantità uguali di spirochete in una coorte di zecche. Ciò è particolarmente importante come la generazione di B. burgdorferi zecche infette dal processo di alimentazione naturale usando topi non riesce a garantire tasso di infezione del 100% e, potenzialmente, si traduce in variazione di carico patogeno tra zecche nutriti. Inoltre, la microiniezione può essere usato per infettare le zecche con B. burgdorferi isola nei casi in cui un ceppo attenuato non è in grado di stabilire l&#39;infezione nei topi e quindi non può essere acquisita naturalmente dalle zecche 5. Questa tecnica può essere utilizzata anche per fornire una varietà di altri materiali biologici in zecche, per esempio, gli anticorpi specifici o di RNA a doppio filamento 6. In questo articolo, ci dimostrerà la microiniezione di zecche ninfale con in vitro-grown B. burgdorferi. Ci sarà anche descrivere un metodo per la localizzazione di agenti patogeni della malattia di Lyme nell&#39;intestino zecca con microscopia confocale immunofluorescenza.

Watch this Scientific Journal Video about Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut at JoVE.com

Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut

Malattia di Lyme studi di ricerca spesso richiedono la generazione di zecche infette dal patogeno Borrelia burgdorferi, un processo che richiede in genere diverse settimane. Qui si dimostrano una microiniezione procedura basata infezione zecca che può essere compiuto entro poche ore. Abbiamo anche dimostrare un metodo di immunofluorescenza per la localizzazione in situ di B. burgdorferi in zecche.

Metodi per il trasferimento rapido e localizzazione di agenti patogeni malattia di Lyme All&#39;interno della Gut Tick

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1.  ...

methods-for-rapid-transfer-localization-lyme-disease-pathogens-within

Research

JoVE Journal

Immunology and Infection

11.5K Views.  University of Maryland. Malattia di Lyme studi di ricerca spesso richiedono la generazione di zecche infette dal patogeno Borrelia burgdorferi, un processo che richiede in genere diverse settimane. Qui si dimostrano una microiniezione procedura basata infezione zecca che può essere compiuto entro poche ore. Abbiamo anche dimostrare un metodo di immunofluorescenza per la localizzazione in situ di B. burgdorferi in zecche.

Video: Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut

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 ...

Ricerca

Immunologia e infezioni

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Creazione di un liquido coperto Cultura polarizzata Airway epiteliali umane Calu-3 celle per studio cellulare risposta dell&#39;ospite agli agenti patogeni delle vie respiratorie<em&gt; In vitro</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Lyme disease is the most commonly-reported vector-borne disease in North America. The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks. Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

Alimentazione di zecche su animali per trasmissione e Xenodiagnosis a Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the  attachment and blood feeding of Ixodes species ticks on ...

Rescue of Recombinant Newcastle Disease Virus from cDNA

Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.

Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

Salvataggio di virus ricombinante malattia di Newcastle da cDNA

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

Metodologia per lo studio del orizzontale Gene Transfer in<em&gt; Staphylococcus aureus</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

Rapido, sicuro e semplice manuale sul comodino estrazione dell'acido nucleico per la rilevazione del Virus in campioni di sangue intero

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. While the direct fluorescent antibody (DFA) test or the direct rapid immunohistochemical test (DRIT) are most commonly utilized for the antigen detection, both tests require fresh and/or frozen tissues for impressions on slides prior to the antigen detection using antibodies. If samples are collected and fixed in formalin, neither test is optimal for the antigen detection, however, testing can be performed by conventional immunohistochemistry (IHC) after embedding in paraffin blocks and sectioning. With this IHC method, tissues are stained with anti-rabies antibodies, sections are deparaffinized, antigen retrieved by partial proteolysis or other methods, and incubated with primary and secondary antibodies. Antigens are stained using horseradish peroxidase / amino ethyl carbazole and counterstained with hematoxylin for the visualization using a light microscope. In addition to the specific antigen detection, formalin fixation offers other advantages like the determination of histological changes, relaxed conditions for specimen storage and transport (under ambient temperatures), ability to test retrospective cases and improved biological safety through the inactivation of infectious agents.

Here, we present an immunohistochemistry test protocol for the detection of rabies virus antigen as an alternative diagnostic test for formalin-fixed tissues.

Test immunoistochimico per il rilevamento dell'antigene del lyssavirus da tessuti fissati con formalina

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. ...

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic infection. The development of chronic P. aeruginosa infections is associated with a decline in lung function and increased morbidity. Therefore, there is a great interest in elucidating the reasons for the failure to eradicate P. aeruginosa with antibiotic therapy which occurs in approximately 10-40% of pediatric patients. One of many factors that can affect host clearance of P. aeruginosa and antibiotic susceptibility is variations in spatial organization (such as aggregation or biofilm formation) and polysaccharide production. Therefore, we were interested in visualizing the in situ characteristics of P. aeruginosa within the sputum of CF patients. A tissue clearing technique was applied to sputum samples after embedding the samples into a hydrogel matrix to retain the 3D structures relative to host cells. After tissue clearing, fluorescent labels and dyes were added to allow visualization. Fluorescence in situ hybridization was performed for the visualization of bacterial cells, binding of fluorescently labeled anti-Psl-antibodies for the visualization of the exopolysaccharide and DAPI staining to stain host cells to obtain structural insight. These methods allowed for the high-resolution imaging of P. aeruginosa within the sputum of CF patients via confocal laser scanning microscopy.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.

Visualizzazione di Pseudomonas aeruginosa all'interno dell'espettorato di pazienti affetti da fibrosi cistica

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic ...

Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi

Introducing foreign DNA into the spirochete Borrelia burgdorferi has been almost exclusively accomplished by transformation using electroporation. This process has notably lower efficiencies in the Lyme disease spirochete relative to other, better-characterized Gram-negative bacteria. The rate of success of transformation is highly dependent upon having concentrated amounts of high-quality DNA from specific backgrounds and is subject to significant strain-to-strain variability. Alternative means for introducing foreign DNA (i.e., shuttle vectors, fluorescent reporters, and antibiotic-resistance markers) into B. burgdorferi could be an important addition to the armamentarium of useful tools for the genetic manipulation of the Lyme disease spirochete. Bacteriophage have been well-recognized as natural mechanisms for the movement of DNA among bacteria in a process called transduction. In this study, a method has been developed for using the ubiquitous borrelial phage &#966;BB-1 to transduce DNA between B. burgdorferi cells of both the same and different genetic backgrounds. The transduced DNA includes both borrelial DNA and heterologous DNA in the form of small shuttle vectors. This demonstration suggests a potential use of phage-mediated transduction as a complement to electroporation for the genetic manipulation of the Lyme disease spirochete. This report describes methods for the induction and purification of phage &#966;BB-1 from B. burgdorferi, the use of this phage in transduction assays, and the selection and screening of potential transductants.

Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi

The ability of bacteriophage to move DNA between bacterial cells makes them effective tools for the genetic manipulation of their bacterial hosts. Presented here is a methodology for inducing, recovering, and using &#966;BB-1, a bacteriophage of Borrelia burgdorferi, to transduce heterologous DNA between different strains of the Lyme disease spirochete.

Manipolazione genetica mediata dai fagi della malattia di Lyme Spirochete Borrelia burgdorferi

Introducing foreign DNA into the spirochete Borrelia burgdorferi has been almost exclusively accomplished by transformation using electroporation. This ...

Quantificazione degli anticorpi neutralizzanti indotti dal vaccino nelle varianti di SARS-CoV-2

Application of Ha-CoV-2 Pseudovirus for Rapid Quantification of SARS-CoV-2 Variants and Neutralizing Antibodies

The coronavirus disease 2019 pandemic (COVID-19) has highlighted the need for rapid assays to accurately measure the infectivity of emerging SARS-CoV-2 variants and the effectiveness of vaccine-induced neutralizing antibodies against viral variants. These assays are essential for pandemic surveillance and validating vaccines and variant-specific boosters. This manuscript demonstrates the application of a novel hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2) for quick quantification of SARS-CoV-2 variant infectivity and vaccine-induced neutralizing antibodies to viral variants. Ha-CoV-2 is a SARS-CoV-2 virus-like particle consisting of viral structural proteins (S, M, N, and E) and a fast-expressing RNA genome derived from an alphavirus, Semliki Forest Virus (SFV). Ha-CoV-2 also contains both green fluorescent protein (GFP) and luciferase reporter genes that allow for quick quantification of viral infectivity. As an example, the infectivity of the SARS-CoV-2 Delta (B.1.617.2) and the Omicron (B.1.1.529) variants are quantified, and their sensitivities to a neutralizing antibody (27VB) are also measured. These examples demonstrate the great potential of Ha-CoV-2 as a robust platform for rapid quantification of SARS-CoV-2 variants and their susceptibility to neutralizing antibodies.

Autore in primo piano: Un sistema di virus pseudotipo per la valutazione delle sottovarianti di Omicron e degli anticorpi neutralizzanti nella ricerca su SARS-CoV-2

This protocol describes the application of a novel hybrid alphavirus-SARS-CoV-2 pseudovirus (Ha-CoV-2) as a platform for rapid quantification of infectivity of SARS-CoV-2 variants and their sensitivity to neutralizing antibodies.

Applicazione dello pseudovirus Ha-CoV-2 per la quantificazione rapida delle varianti di SARS-CoV-2 e degli anticorpi neutralizzanti

The coronavirus disease 2019 pandemic (COVID-19) has highlighted the need for rapid assays to accurately measure the infectivity of emerging SARS-CoV-2 ...