Cette recherche a été soutenue par le programme Small Business Innovation Research des National Institutes of Health (NIAID, 1R43AI082698-01) et l&#39;USDA Institut national de l&#39;alimentation et l&#39;agriculture (NIFA, 2009-33610-20028).

1. Y. l&#39;inoculation plaque pestis <ol><li> Streak Y. pestis A1122 (BeiResources # NR15) les cultures de stock sur Luria-Bertani (LB) agar (Miller). Utiliser une technique stérile et effectuer toutes Y. manipulations pestis dans un type de classe II A enceinte de sécurité biologique. Y. pestis A1122 est un confinement de niveau (BSL) 2 souche exclus agent de sélection. Il manque à la fois les 75 paires ko faible teneur en calcium de réponse (LCR) plasmide de virulence, et le locus PGM qui sont nécessaires pour la virulence. Cultivez Y. pestis à 28 ° C pendant 48 h dans une température d&#39;air statique contrôlé incubateur. Le taux de croissance de Y. pestis est lente avec un temps de génération de 1.25 h 9. </li></ol> 2. Y. pestis milieux liquides d&#39;inoculation <ol><li> Transfert d&#39;un seul Y. pestis dans une colonie de stériles 17 x 100 mm tube de culture contenant 2 ml de bouillon LB utilisant une boucle métallique stérile inoculation. Sélectionnez une colonie qui est uniforme dans la taille, est révélateur des autres colonies, et il est clairement séparé du reste des colonies sur la plaque. Vortex brièvement et développer la culture à 28 ° C pendant environ 20 h dans un incubateur agitateur (225 rpm). </li></ol> 3. Y. excroissance pestis, outre phage journaliste, et la détection de bioluminescence <ol><li> Diluer le nuit Y. 01:20 pestis en bouillon de culture LB frais dans un tube Falcon de 50 ml (par exemple, 500 ul de cellules dans 9,5 ml de milieu) et de croître à 28 ° C avec agitation (225 rpm). Croître jusqu&#39;à une densité optique à 600 nm (600 A) de 0,2 est atteinte (environ 5 h). Cellules en croissance active sont préférables pour la détection puisque la capacité du phage pour transduire une réponse bioluminescents est corrélée à la viabilité et l&#39;aptitude de la bactérie hôte. Néanmoins, le système de détection a été montré pour être compatible avec les cellules récoltées à tous les stades du cycle de croissance de 7. </li><li> Générer le substrat nécessaire à la réaction de bioluminescence en préparant un 2% de n-décanale solution. Mélanger 200 uL de n-décanale avec 9,8 ml de bouillon LB. Vortex vigoureusement pendant 5 s. Premier la microplaque luminomètre injecteur avec 2 mL de 2% du n-décanale solution. Pré-régler le luminomètre d&#39;autoinject 67 ul de la solution décanale à chaque puits de microplaque puis lire immédiatement l&#39;échantillon pendant 10 s. </li><li> Distribuer des aliquotes de 1 ml de bouillon LB en 3 tubes de culture. Ajouter 20 ul de la solution stock journaliste phage (stock de 5 x 10 9 unités formant plaque [UFP] / ml) dans chaque tube, les phages et les médias seuls échantillons sert de contrôle négatif. En l&#39;absence de Y. cellules pestis, l&#39;ajout du phage journaliste ne devrait pas susciter une réaction de bioluminescence. </li><li> Distribuer des aliquotes de 1 mL de la Y. la culture pestis (de l&#39;étape 3.1) dans 6 tubes de culture. Ajouter 20 uL de la journaliste de stock de phages à 3 sur les cultures (cultures d&#39;essai). Les 3 autres cultures servir de «cellules seules« contrôle négatif (autobioluminescence fond). Mélanger les cultures au vortex brièvement, et la récolte 200 ul de chaque échantillon (pour l&#39;instant 0 en lecture) et répartir dans une plaque de microtitration de 96 puits blanc. Incuber la culture reste à 28 ° C avec agitation (225 rpm). </li><li> Mesurer le temps 0 échantillons de la bioluminescence (unités relatives de lumière, RLU) en utilisant le luminomètre pour microplaques. </li><li> Récolte 200 ul de chaque échantillon après 10, 20, 30, 40, 50 et 60 min après ajout de phages-reporter, et de mesurer l&#39;échantillon de la bioluminescence. Si Y. cellules pestis sont présents, le phage rapporteur sera lient spécifiquement à la cellule, injecter son ADN, et d&#39;utiliser les machines hôtes transcription et de traduction de transcrire et de traduire les gènes rapporteurs luxAB (figure 1). </li><li> La force du signal et le temps de réponse du signal est proportionnelle au nombre de cellules présentes. À des concentrations élevées de cellules (10 5 -10 8 UFC / ml), une augmentation significative en RLU par rapport aux contrôles devrait être évident dans les 20 min. A des concentrations inférieures cellulaire, (10 2 -10 4 UFC / ml), une augmentation significative des AVC devrait être évident dans les 40-60 min. </li><li> Sinon, pour accélérer le processus de détection sans avoir besoin de Y. excroissance pestis, une colonie d&#39;une plaque fraîchement cultivées peuvent être mixtes (au vortex) directement dans un tube Eppendorf de 1,5 ml avec 200 pi de bouillon LB abritant le phage journaliste. Répartir le mélange de cellules / phages dans un puits d&#39;une microplaque. Incuber la microplaque à 28 ° C et de lire l&#39;échantillon de la bioluminescence après 60 min (seul moment seulement). </li></ol> 4. Les résultats représentatifs: Une expérience représentative du temps bien sûr du phage journaliste de médiation de détection de Y. pestis est représenté dans la figure 2. Les contrôles négatifs de 1) phage seul (sans cellules), ou 2) des cellules seules (pas le phage) de fournir des niveaux de référence de la bioluminescence d&#39;environ 20 RLU dans le inc 60 minubation (niveaux de référence sont spécifiques luminomètre). En revanche, une augmentation de la bioluminescence pour les échantillons d&#39;essai (phage journaliste et cellules) est évidente à 15 minutes après l&#39;addition de phages. La force du signal devrait augmenter régulièrement plus de 60 min. Les incubations pour des périodes prolongées (plus de 80 min), se traduira par un signal qui va diminuer à partir du signal de pointe en raison de phage lyse des cellules hôtes. Des résultats similaires sont obtenus à des températures d&#39;incubation de 37 ° C même si la température optimale pour Y. la croissance pestis est de 28 ° C 9. <img src="/files/ftp_upload/2740/2740fig2.jpg" alt="Figure 2"/> Figure 2. Phage médiée par la détection de bioluminescence de Y. pestis. Au temps 0, phage journaliste et les cellules ont été mélangés, incubées à 28 ° C, et la bioluminescence (AVC) a été suivi dans le temps. Une augmentation significative des AVC (* Etudiants t-test, p &lt;0,05) est évidente dans les 15 min.

<ol>
	<li>Darling, R. G., Catlett, C. L., Huebner, K. D., Jarrett, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Threats+in+bioterrorism.+I:+CDC+category+A+agents.">Threats in bioterrorism. I: CDC category A agents.</a> Emerg Med Clin North Am. 20, 273-309 (2002).</li><li>Chu, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory manual of plague diagnostic tests.. , (2000).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (1999).</li><li>Inglesby, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anthrax+as+a+biological+weapon,+2002:+updated+recommendations+for+management.">Anthrax as a biological weapon, 2002: updated recommendations for management.</a> JAMA. 287, 2236-2252 (2002).</li><li>Schuch, R., Fischetti, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detailed+genomic+analysis+of+the+Wbeta+and+gamma+phages+infecting+Bacillus+anthracis:+implications+for+evolution+of+environmental+fitness+and+antibiotic+resistance.">Detailed genomic analysis of the Wbeta and gamma phages infecting Bacillus anthracis: implications for evolution of environmental fitness and antibiotic resistance.</a> J Bacteriol. 188, 3037-3051 (2006).</li><li>Garcia, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genome+sequence+of+Yersinia+pestis+bacteriophage+phiA1122+reveals+an+intimate+history+with+the+coliphage+T3+and+T7+genomes.">The genome sequence of Yersinia pestis bacteriophage phiA1122 reveals an intimate history with the coliphage T3 and T7 genomes.</a> J Bacteriol. 185, 5248-5262 (2003).</li><li>Schofield, D. A., Molineux, I. J., Westwater, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+bioluminescent+phage+for+detection+of+Yersinia+pestis.">Diagnostic bioluminescent phage for detection of Yersinia pestis.</a> Journal of Clinical Microbiology. 47, 3887-3894 (2009).</li><li>Schofield, D. A., Westwater, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage-mediated+bioluminescent+detection+of+Bacillus+anthracis.">Phage-mediated bioluminescent detection of Bacillus anthracis.</a> Journal of Applied Microbiology. 107, 468-478 (2009).</li><li>Chu, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CDC: Basic laboratory protocols for the presumptive identification of Yersinia pestis. , 1-19 (2001).</li><li>Gunnison, J. B., Larson, A., Lazarus, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+differentiation+between+Pasteurella+pestis+and+Pasteurella+pseudotuberculosis+by+action+of+bacteriophage.">Rapid differentiation between Pasteurella pestis and Pasteurella pseudotuberculosis by action of bacteriophage.</a> J Infect Dis. 88, 254-255 (1951).</li><li>Lazarus, A. S., Gunnison, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Action+of+Pasteurella+pestis+Bacteriophage+on+Strains+of+Pasteurella,+Salmonella,+and+Shigella.">The Action of Pasteurella pestis Bacteriophage on Strains of Pasteurella, Salmonella, and Shigella.</a> J Bacteriol. 53, 705-714 (1947).</li><li>Sergueev, K. V., He, Y., Borschel, R. H., Nikolich, M. P., Filippov, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+sensitive+detection+of+Yersinia+pestis+using+amplification+of+plague+diagnostic+bacteriophages+monitored+by+real-time+PCR.">Rapid and sensitive detection of Yersinia pestis using amplification of plague diagnostic bacteriophages monitored by real-time PCR.</a> PLoS One. 5, e11337-e11337 (2010).</li></ol>

Cette méthode démontre la capacité du phage journaliste de détecter rapidement Y. pestis depuis le phage journaliste peut transduire un signal de réponse à la culture bioluminescents Y. cellules pestis dans les 20 minutes après l&#39;addition de phages. Le phage journaliste est également capable de détecter directement Y. pestis dans des matrices cliniques, sans le préalable de l&#39;isolement et la culture 7 ultérieures. Par rapport à la lyse de phages tests st...

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th></tr><tr><td> LB agar Difco, Miller </td><td> VWR </td><td> 90003-346 </td></tr><tr><td> Difco LB bouillon, Miller </td><td> VWR </td><td> 90003-350 </td></tr><tr><td> 17 x 100 mm tubes de culture </td><td> USA Scientific </td><td> 1485-0810 </td></tr><tr><td> n-décanale </td><td> Sigma </td><td> D7384 </td></tr><tr><td> Veritas microplaques luminomètre </td><td> Turner Biosystems </td><td> 9100-001 </td></tr><tr><td> Microlite microtitration 96 puits </td><td> VWR </td><td> 62402-984 </td></tr></tbody></table>

'bioluminescent' reporter phage for the detection of category a bacterial pathogens

Yersinia pestis et Bacillus anthracis sont de catégorie A des bactéries pathogènes qui sont les agents responsables de la peste et l&#39;anthrax, respectivement 1. Bien que la présence naturelle de ces deux maladies est maintenant relativement rare, la possibilité à des groupes terroristes d&#39;utiliser ces agents pathogènes comme une arme biologique est bien réelle. En raison de la communicabilité inhérente de la maladie, l&#39;évolution clinique rapide, et le taux de mortalité élevé, il est essentiel qu&#39;une flambée être détectés rapidement. Par conséquent les méthodes qui permettent la détection et le diagnostic rapides sont essentiels pour assurer la mise en œuvre immédiate de mesures de santé publique et l&#39;activation de la gestion de crise. Recombinante de phage journaliste peut fournir une approche rapide et spécifique pour la détection de Y. pestis et B. anthracis. Les Centers for Disease Control and Prevention utilisent actuellement les tests classiques lyse de phages pour l&#39;identification confirmée de ces bactéries pathogènes 2-4. Ces dosages profiter des phages naturels qui sont spécifiques et lytique pour leurs hôtes bactériens. Après une croissance de nuit de la bactérie cultivée en présence du phage spécifique, la formation de plaques (lyse bactérienne) fournit une identification positive de la cible bactérienne. Bien que ces tests sont robustes, ils souffrent de trois défauts: 1) ils sont en laboratoire, 2) ils ont besoin d&#39;isolement bactérien et la culture de l&#39;échantillon suspect, et 3) ils prennent 24 à 36 h pour terminer. Pour répondre à ces questions, recombinante de phage &quot;lumière taggés« journaliste ont été génétiquement modifiées en intégrant les Vibrio harveyi gènes luxAB dans le génome de Y. pestis et B. anthracis phage spécifique 5-8. Le phage résultant luxAB journaliste ont été capables de détecter leurs cibles spécifiques par rapidement (quelques minutes) et sensible conférant un phénotype bioluminescent dans des cellules receveuses. Surtout, la détection a été obtenue soit avec des cellules cultivées bénéficiaires ou avec maquette infectés échantillons cliniques 7. Pour fins de démonstration, ici nous décrire la méthode pour la détection de phages à médiation d&#39;un appelé Y. pestis isoler en utilisant un phage journaliste luxAB construit à partir du phage CDC peste diagnostic ΦA1122 6,7 (figure 1). Une méthode similaire, avec des modifications mineures (par exemple changement de la température de la croissance et les médias), peuvent être utilisés pour la détection de B. anthracis isole à l&#39;aide de la B. Wβ anthracis phage journaliste:: luxAB 8. La méthode décrit la transduction de phages à médiation d&#39;un phénotype biolumescent d&#39;cultivées Y. pestis cellules qui sont ensuite mesurés à l&#39;aide d&#39;un luminomètre pour microplaques. Les principaux avantages de cette méthode sur les tests traditionnels lyse de phages est la facilité d&#39;utilisation, les résultats rapides, et la possibilité de tester plusieurs échantillons en même temps dans un format de 96 puits de plaques de microtitration. <img src="/files/ftp_upload/2740/2740fig1.jpg" alt="Figure 1"/> Figure 1. Schéma de détection. Les phages sont mélangées avec l&#39;échantillon, les phages infecte la cellule, luxAB sont exprimés, et le bioluminesces cellule. Le traitement des échantillons n&#39;est pas nécessaire; le phage et les cellules sont mélangés et ensuite évalués pour la lumière.

Watch this Scientific Journal Video about 'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens at JoVE.com

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

Une méthode simple pour l&#39;identification de pathogènes bactériens priorité est d&#39;utiliser le phage journaliste génétiquement modifiés. Ces phages journaliste, qui sont spécifiques à leur espèce hôte particulier, sont capables de transduire rapidement une réponse de signal bioluminescent aux cellules hôtes. Nous décrivons ici l&#39;utilisation de phages journaliste pour la détection de<em&gt; Yersinia pestis</em&gt;.

Phage »Bioluminescent&quot; Reporter pour la détection des bactéries pathogènes de la catégorie A

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

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JoVE Journal

Immunology and Infection

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

13.3K vues.  Guild Associates, Inc.. Une méthode simple pour l'identification de pathogènes bactériens priorité est d'utiliser le phage journaliste génétiquement modifiés. Ces phages journaliste, qui sont spécifiques à leur espèce hôte particulier, sont capables de transduire rapidement une réponse de signal bioluminescent aux cellules hôtes. Nous décrivons ici l'utilisation de phages journaliste pour la détection de Yersinia pestis.

Vidéo: 'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

Utiliser l&#39;imagerie Bioluminescent chargé d&#39;enquêter sur une synergie entre Streptococcus pneumoniae Et virus Influenza A de souriceaux

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Recherche

Immunologie et infection

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

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

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

L&#39;infection des embryons de poisson zèbre avec bactéries pathogènes intracellulaires

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

Bioluminescent imagerie bactérienne<em&gt; In Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

Transformation biolistique d&#39;un fluorescent Tagged Gene dans la opportuniste pathogène fongique<em&gt; Cryptococcus neoformans</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

À haut débit de détection de pathogènes dans respiratoires Spécimens animaux par Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Développement d&#39;un système de virus de l&#39;hépatite B Reporter pour surveiller les premiers stades du cycle de réplication

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Une plate-forme haut débit pour la détection de Salmonella spp /Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Opsonophagocytic meurtre dosage pour évaluer les réponses immunologiques contre les bactéries pathogènes

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

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. ...