Je tiens à remercier CL Richards Zawacki l&#39;aide de conceptualiser la conception expérimentale et aider avec des matériaux, A. Pessier de l&#39;aide instituant un régime de traitement et l&#39;autopsie des analyses, MWH Chatfield, MJ Robak, K. Tasker et D. Lenger de l&#39;aide à la laboratoire et de soutien et M. McFadden et P. Harlow au zoo de Taronga pour l&#39;utilisation de leur espace pendant le tournage. Le projet a été financé en partie par la Louisiane Département de la faune et de la pêche grâce à la subvention de la Louisiane Commission de l&#39;éducation de l&#39;environnement décerné à LA Brannelly et par Life Technologies.

Approbation éthique: Cette étude et ses méthodes ont été approuvés par l&#39;utilisation des animaux et des Soins de la commission de l&#39;Université de Tulane (protocole n ° 0407.). Une. Test pour Bd infection <ol><li> Pistonnage pour Bd <ol><li> animaux écouvillon avec un coton-tige stérile cinq fois sur la ventrale, cinq fois sur chaque cuisse, de chaque côté, et chaque membre pour un total de 45 coups. Le décolmatage est une méthode non invasive d&#39;échantillonnage pour Bd sur la peau des amphibiens. </li><li> Tournez doucement l&#39;écouvillon pendant et entre les courses pour assurer la plus grande quantité d&#39;ADN est recueillie sur le coton. </li><li> tampons de magasins dans 1,5 ml microtubes à -20 ° C. </li></ol></li><li> Extraction de l&#39;ADN et analyse qPCR <ol><li> Extraire l&#39;ADN génomique à partir des écouvillons en utilisant un kit d&#39;isolement d&#39;ADN disponible dans le commerce, conformément aux instructions d&#39;un tissu animal. </li><li> Analyser l&#39;ADN extrait en utilisant la PCR quantitative en temps réel (qPCR) suivant Boyle <em&gt; et al 17, avec les modifications suivantes.: </li><li> Diluer les échantillons d&#39;extraction d&#39;ADN à 1:10 avec de l&#39;eau doublement désionisée. </li><li> Ajouter 0,7 pi d&#39;albumine de sérum bovin (BSA) afin d&#39;empêcher l&#39;inhibition de la PCR. </li><li> Inclure un contrôle positif interne dans chaque échantillon afin de s&#39;assurer inhibition de la PCR n&#39;a pas influé sur les résultats. </li><li> Échantillons fonctionnent avec un contrôle positif et négatif et une série de normes de dilution pour estimer zoospore (infection) charge. </li></ol></li></ol> 2. Inoculation <ol><li> Culture Bd <ol><li> Préparer un bouillon de tryptone en ajoutant 10 g de tryptone à 1 L d&#39;osmose inverse ou de l&#39;eau désionisée, à l&#39;autoclave (121 ° C pendant 40 min) et on le laisse refroidir. Une fois refroidi à 23 ° C, ajouter 1 ml de pénicilline-streptomycine. </li><li> Cultivez souche Bd (JEL411 de J. Longcore, isolé de Guabal, Panama d&#39;une Phyllomedusa lémurien) dans un bouillon de tryptone pendant sept jours à 23 ° C. </li></ol></li><li> Transférer la broth culture tryptone et des plaques d&#39;agar. <ol><li> Pour fabriquer des plaques, ajouter 10 g de tryptone et 10 g d&#39;agar à 1 L d&#39;osmose inverse ou de l&#39;eau déminéralisée et autoclave (121 ° C pendant 40 min). </li><li> Une fois le mélange à l&#39;autoclave est suffisamment froid au toucher, mais avant qu&#39;il ne se fige, verser dans des boîtes de Pétri, 1/4-1/3 plein, dans une hotte à flux laminaire. </li><li> Lorsque la gélose a figé et complètement refroidi, ajouter 0,5 ml de bouillon Bd de chaque plaque et répartir uniformément autour de la plaque. Laisser sécher. </li><li> Une fois les plaques sont inoculées avec des zoospores Bd, plaques d&#39;étanchéité avec Parafilm. </li><li> Laisser les plaques à incuber à 23 ° C pendant cinq à sept jours. Vérifiez zoospore mouvement pour assurer la viabilité avant l&#39;inoculation. </li></ol></li><li> plaques d&#39;inondation pour la solution d&#39;inoculation zoospore <ol><li> Après les cinq à sept jours de Bd incubation et la croissance, inonder chaque plaque avec 5 ml d&#39;eau du robinet d&#39;âge et permettent zoospores d&#39;entrer les médias pendant 10 min. </li><li> Verser le suspe de zoosporesnsion dans un récipient portable. </li><li> Estimer la concentration de zoospores avec un hématimètre. </li><li> Diluer le mélange à une concentration de 1 x 10 6 par 3 ml avec de l&#39;eau du robinet vieilli. </li></ol></li><li> Inoculer des animaux <ol><li> Dans individuels de 50 ml conteneurs, inoculer chaque animal en versant 3 ml de mélange d&#39;inoculum sur son ventrale. Laisser inoculum supplémentaire de s&#39;écouler dans le fond du récipient d&#39;inoculation. </li><li> Chaque animal restera dans le récipient d&#39;inoculation pendant 24 heures afin d&#39;assurer l&#39;infection. </li><li> Après 24 h de l&#39;inoculation, les animaux de terrariums retourner désinfectés individuels. </li><li> Laisser infection de construire pour deux semaines. Confirmer l&#39;infection avec la tige et les résultats de qPCR, et commencer le traitement. </li><li> Pour les contrôles Bd-négatifs, inondations Bd sans plaques d&#39;agar avec 5 ml âgés de l&#39;eau du robinet, et ensemencer maquette les animaux en utilisant les mêmes méthodes que ci-dessus. </li></ol></li></ol> 3. Schéma de traitement <ol><li> Prepare bain de traitement <ol><li> Ajouter 2,50 ml itraconazole aqueuse (1% Sporanox Solution orale) à une solution de L amphibiens Ringer (0,0025% de l&#39;itraconazole - la plus faible concentration de traitement efficace). <ol><li> Solution la recette de Amphibian sonnerie: 1 L d&#39;osmose inverse ou de l&#39;eau déminéralisée, 6,6 g de chlorure de sodium, 0,15 g de chlorure de potassium, 0,15 g de chlorure de calcium, et 0,2 g de bicarbonate de sodium. Solution autoclave (121 ° C pendant 40 minutes) et laisser refroidir à température ambiante. </li><li> Pour 0,01% de la concentration de traitement itraconazole, ajouter 10,00 ml itraconazole aqueuse (1% Sporanox Solution orale) à la solution de 1 L amphibiens sonnerie </li><li> Pour 0,005% la concentration de traitement itraconazole, ajouter 5,00 ml itraconazole aqueuse (1% Sporanox Solution orale) à la solution de 1 L amphibiens Ringer. </li></ol></li><li> Pour les contrôles Bd positifs et négatifs Bd, baigner les animaux dans une solution de Ringer amphibiens sans itraconazole. </li></ol></li><li> Traitement &lt;ol&gt; <li> Placez amphibiens individuels désinfectés 50 ml conteneurs avec 35 ml de la solution de traitement. </li><li> S&#39;assurer que les animaux sont entièrement couverts pendant cinq minutes en remuant doucement le récipient. </li><li> Animaux Retour à terrariums désinfectés. Répétez l&#39;opération pour six jours. </li></ol></li><li> Assurer le succès du traitement <ol><li> Les animaux de laboratoire pour l&#39;infection Bd sur le traitement de jour commence. </li><li> Les animaux de laboratoire pour l&#39;infection Bd une semaine après la fin du traitement, et une fois par semaine pendant quatre semaines pour assurer infection a disparu. </li></ol></li></ol> 4. Bd comme Biohazard <ol><li> Désinfecter individuel hebdomadaire de terrariums. <ol><li> Baignez les terrariums et de traitement des conteneurs dans un bain d&#39;eau de Javel solution à 10%. </li><li> Rincer chaque conteneur blanchi deux fois à l&#39;eau courante. </li><li> Laisser les cartouches à sécher pendant 24 heures avant de les réutiliser. </li></ol></li><li> Porter des gants en nitrile propres lors de la manipulation des animaux pour éviter la contamination croiséeBd de l&#39;agent pathogène. Changer de gants entre chaque manipulation des animaux. </li><li> Désinfecter tous les déchets liquides avant leur élimination en apportant la solution à 10% l&#39;eau de Javel. </li><li> Autoclave tous les déchets solides qui est potentiellement exposée au Bd avant l&#39;élimination. </li></ol>

<ol>
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P., Mendelson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A Manual For Control of Infectious Diseases in Amphibian Survival Assurance Colonies and Reintroduction Programs. , (2009).</li><li>Page, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloramphenicol+1.+Hazards+of+use+and+the+current+regulatory+environment.">Chloramphenicol 1. Hazards of use and the current regulatory environment.</a> Aus. Vet. J. 68 (1), 1-2 (1991).</li><li>Yoon, H. -. J., Seo, M. H., Kim, J. -. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pharmaceutical formulations for itraconazole. , (2005).</li><li>Garner, T. W. J., Garcia, G., Carroll, B., Fisher, M. 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L&#39;auteur déclare aucun intérêt financier concurrents.

En utilisant ce protocole, nous avons pu réduire en toute sécurité et efficacement la dose et du temps de traitement de traitement pour l&#39;itraconazole Bd des amphibiens. La plus faible dose de traitement et le temps (0,0025% pendant six jours) est un traitement efficace pour I. subclinique infectés nebulifer. L&#39;efficacité des deux une dose plus faible et le temps de traitement de démontrer la nécessité d&#39;essais cliniques de traitement pour les traitements de l&#39;infection...

Amphibiens connaissent actuellement le plus grand déclin de la biodiversité de tous les taxons de vertébrés 1. Chytridiomycose, une maladie de la peau causée par les champignons dendrobatidis pathogène Batrachochytrium chytrides (BD), est l&#39;une des principales causes de ces déclins dramatiques 2 Bd est la pire pathogène sur dossier à entraîner la perte de la biodiversité:. Qu&#39;il infecte plus de 600 espèces d&#39;amphibiens et a ont provoqué une baisse de plus de 300 espèces, dont pas moins de 200 espèces sont devenues danger critique d&#39;extinction ou éteintes 2,3. La chytridiomycose amphibie n&#39;est pas l&#39;hôte spécifique et il n&#39;est pas clair quelles sont les qualités de l&#39;hôte permet à l&#39;infection Bd de progresser provoquer une maladie grave. Chytridiomycose provoque la mort en perturbant les canaux ioniques dans la peau, entraînant une perte d&#39;électrolyte et l&#39;insuffisance cardiaque 4. En outre, Bd surnageant (contenant aucun zoospores Bd) provoque une réponse immunitaire chez des têtards 5 leading chercheurs croient Bd produit un sous-produit toxique 6. Parce que de nombreuses espèces sont en déclin rapide, les colonies d&#39;assurance captives et les programmes d&#39;élevage sont importantes mesures de conservation, et peut-être la seule bouée de sauvetage pour certaines espèces. Établir et maintenir des colonies libres maladie est un défi pour les installations de gestion des amphibiens. Les méthodes actuelles de chimiothérapie pour le traitement des infections Bd, bien qu&#39;efficaces, peuvent être nuisibles pour les animaux qui sont traités, et aucune méthode unique de traitement a été un succès à travers les espèces et classes d&#39;âge de 7 amphibiens. Peu d&#39;essais cliniques de traitements pour l&#39;infection Bd ont été réalisées 7. Les trois méthodes les plus couramment utilisées sont le traitement thérapie par la chaleur, le chloramphénicol et itraconazole. la thérapie de chaleur est une option de traitement nonchemotherapeutic qui semble avoir peu d&#39;effets secondaires, peuvent être utilisés sur plusieurs étapes de la vie, et est généralementallié efficace. Dans un essai de traitement, Chatfield et Richards Zawacki 8 ont trouvé que 96% des animaux traités a dégagé infections après avoir été logé à 30 ° C pendant 10 jours. Bien que cet essai n&#39;était pas à 100% de réussite, il fournit une base pour d&#39;autres pour évaluer la thérapie de chaleur pour leur propre espèce. Thérapie par la chaleur, mais efficace pour de nombreuses espèces, n&#39;est pas pratique pour de nombreuses espèces alpines qui ne peuvent pas rester à des températures élevées pendant une période de temps prolongée. Chloramphénicol, un antibactérien, a été utilisée avec succès pour traiter Bd chez de nombreuses espèces, et inhibe la croissance fongique in vitro. Comme antibactérien, le chloramphénicol n&#39;attaque pas spécifiquement le champignon mais a été connu pour provoquer l&#39;apoptose dans les cellules eucaryotes 9. Il a été spéculé que la thérapie de chloramphenicol provoque l&#39;apoptose dans la peau, en tuant des sporanges intracellulaire, bien que le mécanisme d&#39;action exact n&#39;est pas connu. Bien qu&#39;aucune étude clinique a été menée, et doncdose et la durée du traitement n&#39;ont pas été évalués, les protocoles de traitement actuels exigent généralement immersion pour deux à quatre semaines 10,11, ce qui en fait un traitement pratique pour les amphibiens terrestres. Chloramphénicol a également été restreinte dans certains pays à usage vétérinaire, car l&#39;exposition humaine a été associée à une forme rare d&#39;anémie aplasique 12. Itraconazole, un antifongiques azolés, est le plus largement utilisé pour le traitement de l&#39;infection Bd des amphibiens en captivité. Le protocole le plus utilisé est les animaux de bain dans une solution itraconazole 0,01% pour cinq minutes par jour pendant 11 jours consécutifs. Ce protocole a été dérivé de normes utilisées pour l&#39;administration de mammifères, et, tout en étant efficace pour les amphibiens, il n&#39;est pas basé sur les essais cliniques chez les amphibiens 11,13. Ce protocole peut entraîner des effets secondaires négatifs dans certaines espèces, en particulier dans le genre Rana et des têtards et métamorphes dernières 11.Têtard traitement a été couronnée de succès en utilisant une concentration beaucoup plus faible (0,0005%) de l&#39;itraconazole, mais était associée à une dépigmentation 14. Cette concentration a été mis à l&#39;essai même sur métamorphes récentes et a constaté à l&#39;échec à traiter une infection 15. Parce qu&#39;il ya risque d&#39;effets secondaires négatifs avec le traitement itraconazole, certains praticiens ont utilisé la moitié de la concentration habituelle (0,005%) pour 11 jours consécutifs et trouvé ceci pour être réussi à guérir les infections Bd 16. La présente étude est un résumé d&#39;un essai clinique de traitement utilisant l&#39;itraconazole pour traiter l&#39;infection Bd 15. Le protocole décrit une tentative de trouver la dose la plus faible et le traitement du temps qui est à la fois sûr et efficace pour traiter les animaux infectés Bd avec l&#39;itraconazole. Les animaux utilisés dans cette étude étaient récentes métamorphes de crapauds de côte du golfe, Incilius nebulifer.

<table border="0" cellpadding="0" cellspacing="0" width="503">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">0.1% Aqueous Itraconazole, Sporonox</td>
			<td style="width:147px;">Janssen Pharmaceutica Inc.</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">NaCl</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">S640-500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">KCl</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP366-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">CaCl2</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP510-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">NaHCO3</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:177px;">BP328-500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Sterile cotton swabs</td>
			<td style="width:147px;">Medical and Wire Equipment</td>
			<td style="width:177px;">MW113</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">1.5 ml micro tubes</td>
			<td style="width:147px;">Sarstedt</td>
			<td style="width:177px;">73.703.217</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:179px;">Qiagen DNEasy Blood and Tissue Kit</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:177px;">69581</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">7500 Real-time PCR</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">4351104</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">VIC IPC</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">4308323</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">BSA</td>
			<td style="width:147px;">Applied Biosystems</td>
			<td style="width:177px;">AM2618</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Agar</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">30391023</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Tryptone</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">Q10029</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Penicillin-streptomycin</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:177px;">15070-063</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Petri Dish</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:177px;">CLS430589</td>
		</tr>
	</tbody>
</table>

reduced itraconazole concentration and durations are successful in treating batrachochytrium dendrobatidis infection in amphibians

Amphibiens connaissent la plus forte baisse d&#39;une catégorie de vertébrés et la principale cause de ce déclin est un champignon pathogène, dendrobatidis Batrachochytrium (BD), ce qui provoque la chytridiomycose, maladie. Colonies d&#39;assurance captives sont importantes dans le monde entier pour les espèces d&#39;amphibiens menacées et peuvent être la seule bouée de sauvetage pour ceux qui en menace critique d&#39;extinction. Le maintien de maladies colonies libres est une priorité des gestionnaires captifs, encore des traitements sûrs et efficaces pour toutes les espèces et à travers les étapes de la vie n&#39;ont pas été identifiés. Le traitement chimiothérapeutique le plus utilisé est l&#39;itraconazole, même si la dose utilisée peut être nocif pour certains individus et d&#39;espèces. Nous avons effectué un essai de traitement clinique pour déterminer si une dose plus faible et plus sûr mais efficace de l&#39;itraconazole peut être trouvé pour guérir les infections Bd. Nous avons trouvé que, en réduisant la concentration de 0,01 à 0,0025% le traitement et la réduction de la durée de traitement de 11-6 Jours de 5 min des thermes, grenouilles pourraient être guéris de l&#39;infection Bd avec moins d&#39;effets secondaires et de la mortalité associée au traitement moins.

Traitement itraconazole a été considéré comme un succès si tous les animaux étaient Bd négatif quatre semaines après la fin du traitement. Avant le début du traitement, deux semaines après l&#39;inoculation, tous les animaux Bd-inoculées ont été infectés, avec une charge zoospore moyenne de 1,121.7 (± 17,34), tandis que quatre semaines après la fin du traitement, les animaux témoins Bd-positifs avaient la charge d&#39;un moyen de 16,765.12 ( ± 14643,08). <p class="jove_co...

Watch this Scientific Journal Video about Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians at JoVE.com

Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

infection Batrachochytrium dendrobatidis chez les amphibiens peut être éliminé en utilisant une concentration réduite (0,0025%) et de plus courte durée (bains cinq minutes pendant six jours) de l&#39;itraconazole que ce qui est généralement utilisé. Moins de mortalité et moins d&#39;effets secondaires négatifs ont été observés en utilisant ce protocole de traitement modifié.

Réduit la concentration et la durée d&#39;itraconazole et efficace dans le traitement<em&gt; Batrachochytrium dendrobatidis</em&gt; Maladies infectieuses chez les amphibiens

Amphibians are experiencing the greatest decline of any vertebrate class and a leading cause of these declines is a fungal pathogen, Batrachochytrium ...

reduced-itraconazole-concentration-durations-are-successful-treating

Research

JoVE Journal

Immunology and Infection

Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

11.5K vues.  James Cook University.  infection Batrachochytrium dendrobatidis chez les amphibiens peut être éliminé en utilisant une concentration réduite (0,0025%) et de plus courte durée (bains cinq minutes pendant six jours) de l'itraconazole que ce qui est généralement utilisé. Moins de mortalité et moins d'effets secondaires négatifs ont été observés en utilisant ce protocole de traitement modifié. 

Vidéo: Reduced Itraconazole Concentration and Durations Are Successful in Treating Batrachochytrium dendrobatidis Infection in Amphibians

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

Visualisation virus de la dengue grâce à Alexa Fluor Étiquetage

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

Recherche

Immunologie et infection

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

L&#39;utilisation de moyen d&#39;expectoration artificielle pour tester l&#39;efficacité des antibiotiques contre<em&gt; Pseudomonas aeruginosa</em&gt; Dans des conditions plus pertinents à la fibrose kystique du poumon

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia de culture du système nerveux central néonatale et de l&#39;adulte

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

Extraction des protéines totales et 2-D Méthodes électrophorèse sur gel pour<em&gt; Burkholderia</em&gt; Espèces

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophiles Isolement et analyse pour déterminer leur rôle dans le lymphome cellulaire Sensibilité aux agents thérapeutiques

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Isolement de Salmonella typhimurium-contenant des Phagosomes des Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose (Herpestes Auropunctatus) Sera for Use as an Oral Rabies Vaccination Biological Marker

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases reported annually. The control of RABV circulation in wildlife reservoirs is typically accomplished by a strategy of oral rabies vaccination (ORV). Currently no wildlife ORV program exists in Puerto Rico. Research into oral rabies vaccines and various bait types for mongooses has been conducted with promising results. Monitoring the success of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RABV neutralizing antibodies (RVNA) post vaccination. This strategy may be difficult to interpret in areas with an active wildlife ORV program or in areas where RABV is enzootic and background levels of RVNA are present in reservoir species. In such situations, a biomarker incorporated with the vaccine or the bait matrix may be useful. We offered 16 captive mongooses placebo ORV baits containing ethyl-iophenoxic acid (et-IPA) in concentrations of 0.4% and 1% inside the bait and 0.14% in the external bait matrix. We also offered 12 captive mongooses ORV baits containing methyl-iophenoxic acid (me-IPA) in concentrations of 0.035%, 0.07% and 0.14% in the external bait matrix. We collected a serum sample prior to bait offering and then weekly for up to eight weeks post offering. We extracted Iophenoxic acids from sera into acetonitrile and quantified using liquid chromatography/mass spectrometry. We analyzed sera for et-IPA or me-IPA by liquid chromatography-mass spectrometry. We found adequate marking ability for at least eight and four weeks for et- and me-IPA, respectively. Both IPA derivatives could be suitable for field evaluation of ORV bait uptake in mongooses. Due to the longevity of the marker in mongoose sera, care must be taken to not confound results by using the same IPA derivative during consecutive evaluations.

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose Herpestes Auropunctatus Sera for Use as an Oral Rabies Vaccination Biological Marker

We offered captive mongooses placebo oral rabies vaccine baits with ethyl or methyl iophenoxic acid as a biomarker and verified bait uptake using a novel liquid chromatography with tandem mass spectrometry (LC-MS/MS) method.

Analyse des analogues de l'acide iophénoxique chez la petite mangouste indienne (Herpestes Auropunctatus) Sera pour utilisation comme marqueur biologique de vaccination contre la rage buccale

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

Infection récurrente des voies urinaires à Escherichia coli déclenchée par Gardnerella vaginalis Exposition à la vessie chez la souris

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.
B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4+ T cells decreases while that of CD8+ T cells expands dramatically during acute EBV infection, and the persistence of CD8+ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.
The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4+ T cells, CD8+ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

Séparation des sous-populations de cellules immunitaires dans les échantillons de sang périphérique des enfants atteints de mononucléose infectieuse

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein&#8211;Barr virus (EBV) infection. The main clinical symptoms ...

Standardization of Transfer across Labs between Flow Cytometers for Detection of Lymphocytes in Japanese Encephalitis Vaccinated Children

An increasing number of laboratories need to collect data from multiple flow cytometers, especially for research projects performed across multiple centers. The challenges of using two flow cytometers in different labs include the lack of standardized materials, software compatibility issues, inconsistencies in instrument setup, and the use of different configurations for different flow cytometers. To establish a standardized flow cytometry experiment to achieve the consistency and comparability of experimental results across multiple centers, a rapid and feasible standardization method was established to transfer parameters across different flow cytometers. 
The methods developed in this study allowed the transfer of experimental settings and analysis templates between two flow cytometers in different laboratories for the detection of lymphocytes in Japanese encephalitis (JE)-vaccinated children. A consistent fluorescence intensity was obtained between the two cytometers using fluorescence standard beads to establish the cytometer settings. Comparable results were obtained in two laboratories with different types of instruments. Using this method, we can standardize analysis for evaluating the immune function of JE-vaccinated children in different laboratories with different instruments, diminish the differences in data and results among flow cytometers in multiple centers, and provide a feasible approach for the mutual accreditation of laboratory results. The standardization method of flow cytometer experiments will ensure the effective performance of research projects across multiple centers.

In this study, a method was developed to facilitate the transfer of experimental settings and analysis templates between two flow cytometers in two laboratories for the detection of lymphocytes in Japanese encephalitis-vaccinated children. The standardization method for the flow cytometer experiments will allow research projects to be effectively conducted in multiple centers.

Normalisation du transfert entre laboratoires entre cytomètres en flux pour la détection des lymphocytes chez les enfants vaccinés contre l’encéphalite japonaise

An increasing number of laboratories need to collect data from multiple flow cytometers, especially for research projects performed across multiple ...