Nous remercions sincèrement les membres du laboratoire Pal pour l&#39;aide à la préparation de cette manifestation. Cette étude a été soutenue par PHS subventions AI076684 et AI080615 de NIH / NIAID.

1. Microinjection de la tique Ixodes scapularis nymphes 1. Préparation des aiguilles <ol><li> Fabriquer des aiguilles de microinjection plusieurs reprises par le chauffage et en tirant une des tubes de verre mm capillaire (instruments de précision du monde) dans un dispositif en verre micropipette extracteur (Narishige). Retirer délicatement les tubes capillaires fragiles. </li><li> Boutique tiré aiguilles (avec la pointe dirigée vers le haut) sur une bande adhésive dans une boîte de Pétri. </li></ol> 2. Préparation B. burgdorferi <ol><li> Cultivez B. burgdorferi dans les milieux de culture BSK 7 jusqu&#39;à ce que, à une concentration d&#39;environ 10 7 cellules par ml. Spirochètes sont comptés sous un microscope en champ sombre par l&#39;utilisation d&#39;un comptage Petroff-Hausser chambre (Hausser scientifique). </li><li> Pellet B. burgdorferi par centrifugation à 3000 xg pendant 10 minutes à température ambiante. </li><li> Retirer le surnageant et complètement resuspendre le culot dans BSK médias doucement en passant par une micropointe stériles 200 uL d&#39;une concentration finale de 10 9 cellules par ml. Puisque les cellules sont remises en suspension à haute densité cellulaire, et pourrait s&#39;agglomérer, la suspension cellulaire doit être utilisé pour la microinjection immédiatement. </li></ol> 3. Tiques Préparation <ol><li> Placez claire, ruban adhésif double-face sur une lame de verre. </li><li> Travailler sur un tapis collant, retirez soigneusement les tiques nymphes du conteneur à l&#39;aide d&#39;une petite brosse. </li><li> Placez le nombre requis de tiques pour être injecté sur le ruban adhésif, la face ventrale en place, dans la même direction. </li></ol> 4. Tiques par injection <ol><li> Charge 5 uL de la B. la culture burgdorferi dans l&#39;aiguille tiré capillaire en utilisant un 20 uL microloader pointe de la pipette (Eppendorf). Inspectez l&#39;aiguille des bulles d&#39;air piégées et recharger l&#39;aiguille avec une culture bactérienne, si nécessaire. </li><li> Voir la tique sous le microscope binoculaire de dissection et de se concentrer sur la zone de la tique ouverture anale. Toucher doucement le bout de l&#39;aiguille capillaire afin de briser le tube dont le diamètre est légèrement plus petit que celui de l&#39;ouverture anale de la tique, formant l&#39;aiguille de microinjection. Cette étape est essentielle car toute tentative d&#39;injecter les tiques avec une pointe d&#39;aiguille non appropriée entraîne la mort des blessures et possibles de la tique injecté. </li><li> Placez les tiques immobilisé sous le microscope binoculaire de dissection et de se concentrer sur l&#39;ouverture anale, qui est couvert par deux plaques anales mobiliers. En utilisant une pince fine, toucher doucement et appliquer une pression très légère à une zone près de l&#39;ouverture anale. Cela permettra la séparation des plaques anales et l&#39;ouverture des pores anaux qui se connecte à l&#39;intestin. Insérez soigneusement la pointe de l&#39;aiguille légèrement dans l&#39;ouverture anale par l&#39;ouverture forcée des plaques anales. Insertion de l&#39;aiguille doit être maintenue à un minimum, car la pointe de verre peut endommager l&#39;intestin postérieur semi qui se connecte au rectum. L&#39;utilisation d&#39;un microinjecteur équipé d&#39;un contrôle automatisé du pied (Eppendorf), injecter B. solution burgdorferi utilisant les paramètres suivants: 1 000 hectopascals (hPa) la pression d&#39;injection, temps d&#39;injection 0,2 secondes et 8 compensation de pression hPa. Chaque graduation reçoit une injection unique. </li><li> Après microinjection, les tiques doivent se comporter similaire à l&#39;état pré-injection. Par exemple, la tique doit ramper dans la réponse aux stimuli. </li><li> Nous accordons habituellement les tiques de récupérer pendant 48 heures dans une chambre environnementale fixée à 24 ° C avec 16 heures / 8 heures de lumière / obscurité régime de photopériode et de 95% d&#39;humidité. Si une chambre n&#39;est pas disponible, les tiques injectées pourraient être stockés à température ambiante dans des conditions humides dans un contenant hermétique, comme une chambre régulière dessicateur. Si nécessaire, le temps de récupération des tiques pourrait également être réduite à quelques heures avant utilisé pour l&#39;alimentation sur des souris. </li></ol> 2. B. Localisation burgdorferi par immunofluorescence confocale 1. Tiques de dissection <ol><li> Placez une goutte de tampon phosphate salin (PBS) sur une lame de verre propre pour la dissection et l&#39;autre sur une lame de poly-L-lysine couché (Sigma) pour la microscopie. Préparer une autre lame de verre avec du ruban adhésif double-face. </li><li> Retirer la tique du récipient et le placer sur un ruban adhésif double-face en utilisant une petite brosse. </li><li> Voir et se concentrer la tique dans le microscope de dissection. Placer une lame de rasoir sur la tique entre la première et seconde paires de pattes. Appuyez fermement couper la tique dans deux pièces permettant l&#39;accès à l&#39;abdomen. Immédiatement, submerger l&#39;abdomen dans une goutte de PBS. </li><li> En utilisant des pinces très fines, prenez l&#39;exosquelette dorsale et ventrale autour du site de la coupe. Retirez soigneusement le bouclier dorsal haut et loin de la tique exposant les diverticules intestinales brunâtre. Veillez à garder les tiques dissection sous le PBS, à tout moment. </li><li> Le chignon semi-translucide glande salivairedles sont situés de chaque côté de la région antérieure de l&#39;intestin, et peut être retirée à ce stade, si désiré. Tenir l&#39;exosquelette restant en place avec une pince, tirez délicatement sur le tube digestif à partir de l&#39;abdomen. </li><li> Nettoyez délicatement l&#39;intestin en éliminant tous les tissus piégés (par exemple, la trachée). </li><li> Avec la pointe d&#39;une pince fine, transférer rapidement l&#39;intestin cocher dans une goutte de PBS sur une lame de verre poly-L-lysine. Séparez les intestins en petits morceaux à l&#39;aide de fines lames ou appuyant doucement avec le bout de pinces fines. Aspirer délicatement l&#39;excès de PBS dans le tissus du tube digestif. </li><li> Laisser le tissus du tube digestif à l&#39;air sec à température ambiante. </li><li> Fixer l&#39;intestin tique par immersion dans l&#39;acétone pendant 10 minutes. Laisser sécher à l&#39;air à température ambiante. Les diapositives peuvent être conservés à cette étape pour les mois à -20 ° C dans un contenant hermétique. </li></ol> 2. Coloration <ol><li> Utiliser du papier de soie, enlever l&#39;excès d&#39;humidité autour du tissu et dessinez un cercle autour de l&#39;intestin tic séchée par un pap-stylo ou tout autre dispositif de barrière hydrophobe doublure. Cela aidera à retenir les solutions de coloration au cours des étapes d&#39;incubation ultérieures. </li><li> Couverture de l&#39;intestin tique avec un ou quelques gouttes de tampon de blocage (0,05% de Tween-20, du sérum de chèvre 5% dans du PBS) pendant 30 minutes à température ambiante. Le sérum utilisé pour le blocage dépend de la source de l&#39;animal hôte pour l&#39;anticorps. Ne pas laisser la lame sécher à partir de ce moment. </li><li> Lentement aspirer le tampon de blocage. Incuber le tube digestif avec des primaires appropriés et / ou solutions d&#39;anticorps secondaire. Nous utilisons l&#39;isothiocyanate de fluorescéine (FITC) - anticorps anti-B burgdorferi anticorps (Kirkegaard &amp; Perry Laboratories) à une dilution 1:100 dans du tampon de blocage pendant 1 heure à température ambiante. Couvrez le récipient avec du papier aluminium pour limiter l&#39;exposition à la lumière. </li><li> Retirer la solution d&#39;anticorps par aspiration douce. Incuber le tube digestif avec un colorant fluorescent aux tissus cochez l&#39;étiquette, tel que 4 &#39;,6-diamidino-2-phénylindole (DAPI) ou de l&#39;iodure de propidium. Nous utilisons normalement 20 mg / ml d&#39;iodure de propidium (Sigma) dans du PBS pendant 5 min. à température ambiante. </li><li> Laver 3 fois avec 0,05% de Tween-20 dans du PBS. </li><li> Montez le glisser dans le glycérol tamponnée contenant un réactif Antifade tels que Slowfade (Invitrogen) et soigneusement couvrir avec une lamelle de verre. Les diapositives peuvent être conservés à cette étape pour quelques mois à 4 ° C dans un contenant hermétique. Image et de localiser B. burgdorferi sous un microscope confocal. </li></ol> 3. Les résultats représentatifs Position d&#39;une tique nymphe pour la microinjection et une image représentant B. localisation burgdorferi dans l&#39;intestin tique est présenté dans la Figure 1. <img src="/files/ftp_upload/2544/2544fig1.jpg" alt="Figure 1" /> Figure 1. Microinjection et la localisation de B. burgdorferi dans l&#39;intestin des tiques. (A) Vue ventrale d&#39;une nymphe I. Cochez scapularis positionné pour la microinjection. Anal ouverture (flèche) avec une aiguille insérée microinjection (flèche) est montré sous un grossissement de l&#39;encart. Une pince fine est utilisé pour appliquer une légère pression sur le corps, ce qui a permis la séparation des plaques anales et l&#39;ouverture de l&#39;anus pour la microinjection. L&#39;aiguille est remplie avec une solution de bleu de Coomassie pour améliorer la visibilité. (B) Les résultats représentatifs de l&#39;imagerie confocale par immunofluorescence de B. burgdorferi dans les intestins de tiques. Région antérieure d&#39;un diverticule intestinal est montré. Noyaux Gut et spirochètes (flèche) sont marqués avec de l&#39;iodure de propidium (couleur rouge) ou FITC conjugué anti-B. burgdorferi (couleur verte), respectivement. Barre = 20 um.

<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>

Ici nous démontrons une procédure basée sur la micro-injection d&#39;infection rapide et efficace de la tique Ixodes nymphes avec la bactérie pathogène B. burgdorferi. Nous décrivons également une procédure d&#39;immunofluorescence confocale pour la détection de B. burgdorferi dans l&#39;intestin tic in situ. Bien que notre démonstration implique l&#39;intestin nymphes, des procédures similaires sont également applicables à d&#39;autres stades de développement des tiques, comme l...

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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>
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methods for rapid transfer and localization of lyme disease pathogens within the tick gut

La maladie de Lyme est causée par une infection par le spirochète pathogène Borrelia burgdorferi, qui est maintenu dans la nature par un cycle d&#39;infection chez les rongeurs tic-1. Un modèle murin tiques 2 a été développé pour étudier la maladie de Lyme dans le laboratoire. Alors que les tiques peuvent être naïfs infectés par B. burgdorferi en les nourrissant sur ​​des souris infectées, le processus de mue prend plusieurs semaines à plusieurs mois à compléter. Par conséquent, le développement des plus rapides et plus efficaces les techniques infection des tiques, comme une procédure basée sur la micro-injection, est un outil important pour l&#39;étude de la maladie de Lyme 3,4. La procédure nécessite quelques heures seulement de générer des tiques infectées et permet de contrôler la livraison des quantités égales de spirochètes dans une cohorte de tiques. Ceci est particulièrement important que la génération de B. tiques infectées burgdorferi par le processus d&#39;alimentation naturelle en utilisant des souris ne parvient pas à assurer un taux d&#39;infection de 100% et potentiellement entraîne une variation de la charge de pathogènes chez les tiques nourries. Par ailleurs, la microinjection peut être utilisé pour infecter les tiques avec B. burgdorferi des isolats dans les cas où une souche atténuée est incapable d&#39;établir une infection chez les souris et donc ne peut pas être acquise naturellement par les tiques 5. Cette technique peut également être utilisé pour offrir une variété d&#39;autres matériaux biologiques dans les tiques, par exemple, des anticorps spécifiques ou l&#39;ARN double brin 6. Dans cet article, nous allons démontrer la microinjection de tiques nymphes aux in vitro grandi B. burgdorferi. Nous allons également décrire une méthode pour la localisation de la maladie de Lyme pathogènes dans l&#39;intestin tic utilisant microscopie confocale à immunofluorescence.

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

Des études de Lyme recherche sur les maladies nécessitent souvent de génération de tiques infectées par l&#39;agent pathogène Borrelia burgdorferi, un processus qui prend généralement plusieurs semaines. Ici nous démontrons une procédure basée sur la microinjection tic infection qui peut être accompli en quelques heures. Nous démontrons également une méthode d&#39;immunofluorescence pour la localisation in situ de B. burgdorferi dans les tiques.

Méthodes de transfert rapide et la localisation des agents pathogènes dans l&#39;intestin de Lyme 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 vues.  University of Maryland. Des études de Lyme recherche sur les maladies nécessitent souvent de génération de tiques infectées par l'agent pathogène Borrelia burgdorferi, un processus qui prend généralement plusieurs semaines. Ici nous démontrons une procédure basée sur la microinjection tic infection qui peut être accompli en quelques heures. Nous démontrons également une méthode d'immunofluorescence pour la localisation in situ de B. burgdorferi dans les tiques.

Vidéo: 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.

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 ...

Recherche

Immunologie et infection

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.

L&#39;établissement d&#39;une culture liquide recouvert de Airway humaines épithéliales polarisées cellules Calu-3 pour étudier la réponse cellulaire aux agents pathogènes respiratoires hôte<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.

Alimentation des tiques sur les animaux pour la transmission et Xénodiagnostic dans la recherche la maladie de Lyme

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.

Sauvetage de virus de la maladie de Newcastle recombinant à partir d&#39;ADNc

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.

Méthodologie de l&#39;étude du transfert horizontal de gènes dans<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).

Extraction rapide, sûre et Simple acide nucléique chevet manuelle pour la détection des Virus dans des échantillons de sang total

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 d’immunohistochimie pour la détection de l’antigène du lyssavirus à partir de tissus fixés au for formel

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.

Visualisation de Pseudomonas aeruginosa dans les expectorations de patients atteints de fibrose kystique

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.

Manipulation génétique médiée par phage du spirochète de la maladie de Lyme Borrelia burgdorferi

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

Quantification des anticorps neutralisants induits par le vaccin dans les variants du SRAS-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.

Pleins feux sur l’auteur : Un système de pseudotype de virus pour évaluer les sous-variants d’Omicron et les anticorps neutralisants dans la recherche sur le SRAS-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.

Application du pseudovirus Ha-CoV-2 pour la quantification rapide des variants du SRAS-CoV-2 et des anticorps neutralisants

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