Questa ricerca è stata sostenuta dal programma di ricerca Piccole Imprese per l&#39;Innovazione del National Institutes of Health (NIAID, 1R43AI082698-01) e l&#39;USDA Istituto Nazionale di Alimentazione e l&#39;Agricoltura (NIFA, 2009-33610-20028).

1. Y. pestis piastra inoculazione <ol><li> Streak Y. pestis A1122 (BeiResources # NR15) colture su Luria-Bertani (LB) agar (Miller). Utilizzare una tecnica sterile ed eseguire tutte le Y. manipolazioni pestis in un tipo di classe II A cabinet. biosicurezza Y. pestis A1122 è un livello di biosicurezza (BSL) 2 ceppo esclusi agente selezionare. Manca sia la coppia 75 kb basso contenuto di calcio di risposta (LCR) plasmide di virulenza, e il luogo pgm che sono necessari per la virulenza. Crescere Y. pestis a 28 ° C per 48 ore a temperatura statica aria incubatrice. Il tasso di crescita di Y. pestis è lenta con un tempo di generazione di 1,25 h 9. </li></ol> 2. Y. pestis liquido mezzi inoculazione <ol><li> Trasferire un singolo Y. colonia pestis in una sterile 17 x 100 mm Tubo di coltura contenente 2 ml di brodo LB sterile utilizzando un anello di metallo inoculazione. Seleziona una colonia che è uniforme nelle dimensioni, è indicativo delle altre colonie, ed è chiaramente separato dal resto delle colonie sulla piastra. Tubo vorticoso brevemente e far crescere la cultura a 28 ° C per circa 20 ore in una agitazione incubatore (225 giri). </li></ol> 3. Y. pestis escrescenza, fago Inoltre giornalista, e il rilevamento bioluminescenti <ol><li> Diluire la notte Y. pestis 1:20 cultura in brodo LB fresco in un tubo da 50 ml falco (ad esempio, 500 ml di cellule in 9,5 ml di terreno) e crescono a 28 ° C con agitazione (225 giri). Crescere fino a una densità ottica a 600 nm (A 600) di 0,2 si raggiunge (circa 5 h). Cellule in attiva crescita sono da preferire per il rilevamento in quanto la capacità del fago per trasdurre una risposta bioluminescente è correlato alla vitalità e benessere dei batteri ospitante. Tuttavia, il sistema di rilevazione ha dimostrato di essere compatibile con le cellule raccolte in tutte le fasi del ciclo di crescita 7. </li><li> Generare il substrato necessario per la reazione bioluminescente preparando un 2% di n-decanale soluzione. Mescolare 200 ml di n-decanale con 9,8 ml di brodo LB. Energicamente per 5 s. Primo la micropiastra luminometro iniettore con 2 mL di n-2% decanale soluzione. Pre-impostare il luminometro al autoinject 67 microlitri della soluzione del decanale ad ogni micropiastra bene e poi subito a leggere il campione per 10 s. </li><li> Dispensare 1 ml di brodo LB in 3 provette di coltura. Aggiungere 20 ml di soluzione madre giornalista fago (stock di 5 x 10 9 unità placca formando [UFP] / mL) a ciascuna provetta, il fago e dei media di campioni da solo funge da controllo negativo. In assenza di Y. cellule pestis, l&#39;aggiunta del fago giornalista non dovrebbe suscitare una reazione bioluminescente. </li><li> Dispensare 1 ml di Y. pestis cultura (dal punto 3.1) in 6 tubi cultura. Aggiungere 20 ml di stock fago giornalista a 3 delle culture (culture di prova). I restanti 3 culture servire come &#39;cellule solo&#39; di controllo negativo (autobioluminescence sfondo). Mescolare culture vortexando brevemente, e la raccolta 200 ul di ciascun campione (per leggere il tempo 0) ed erogare in un piatto bianco microtitolo a 96 pozzetti. Incubare la cultura rimanendo a 28 ° C con agitazione (225 giri). </li><li> Misurare il tempo 0 campioni per bioluminescenza (unità di luce relativa, RLU) utilizzando il luminometro per micropiastre. </li><li> Harvest 200 ul di ciascun campione dopo 10, 20, 30, 40, 50 e 60 minuti post-giornalista Inoltre fago, e misurare il campione per bioluminescenza. Se Y. pestis cellule sono presenti, il fago giornalista si legano specificamente alla cellula, inietta il suo DNA, e utilizzare i padroni di casa macchinario trascrizionale e traslazionale di trascrivere e tradurre i geni reporter luxAB (Figura 1). </li><li> La forza del segnale e il tempo di risposta del segnale è proporzionale al numero di cellule presenti. Ad alte concentrazioni di cellule (10 5 -10 8 UFC / ml), un significativo aumento RLU rispetto ai controlli dovrebbe essere evidente entro 20 min. A concentrazioni cella inferiore, (10 2 -10 4 UFC / ml), un significativo aumento RLU dovrebbe essere evidente entro 40-60 min. </li><li> In alternativa, per accelerare il processo di rilevamento senza la necessità di Y. escrescenza pestis, una colonia da un piatto fresco cresciuto possono essere mescolati (nel vortex) direttamente in una provetta Eppendorf 1,5 ml con 200 ml di brodo LB ospitare il fago reporter. Dispensare la cellula / fago miscela in un pozzo della micropiastra. Incubare la micropiastra a 28 ° C e leggere il campione per bioluminescenza dopo 60 min (punto di volta sola). </li></ol> 4. Rappresentante dei risultati: Un rappresentante esperimento durata di fago giornalista mediata rilevamento di Y. pestis è illustrato nella figura 2. I controlli negativi di 1) fago da solo (senza cellule), o 2) le cellule da solo (senza fago) forniscono livelli basali di bioluminescenza di circa 20 RLU tutto il min 60 incubation (livelli basali sono specifici luminometro). Al contrario, un aumento della bioluminescenza per i campioni di prova (fagi giornalista e cellule) è evidente a 15 minuti dopo l&#39;aggiunta dei fagi. La potenza del segnale dovrebbe aumentare costantemente più di 60 min. Incubazioni per periodi di tempo prolungati (oltre 80 min), si tradurrà in un segnale che scenderà dal segnale di picco a causa di fagi lisi mediata delle cellule ospiti. Risultati simili si ottengono a temperature di incubazione di 37 ° C anche se la temperatura ottimale per Y. crescita pestis è di 28 ° C 9. <img src="/files/ftp_upload/2740/2740fig2.jpg" alt="Figura 2"/> Figura 2. Fago-mediata rilevazione bioluminescenti di Y. pestis. Al tempo 0, fagi giornalista e le cellule sono state mescolate, incubate a 28 ° C, e la bioluminescenza (RLU) è stata monitorata nel tempo. Un aumento significativo RLU (* Studenti t-test, p &lt;0,05) è evidente entro 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>

Nessun conflitto di interessi dichiarati.

Questo metodo dimostra la capacità del fago giornalista per rilevare rapidamente Y. pestis dal fago giornalista può trasdurre una risposta bioluminescente segnale colto Y. pestis cellule entro 20 minuti dopo l&#39;aggiunta dei fagi. Il fago giornalista è anche in grado di rilevare direttamente Y. pestis in matrici clinica, senza il presupposto di isolamento e 7 successiva coltivazione. Rispetto ai test standard di lisi fagica che in genere richiedono 48 ore per il completamento, ...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> Difco agar LB, Miller </td><td> VWR </td><td> 90003-346 </td></tr><tr><td> Difco LB brodo, Miller </td><td> VWR </td><td> 90003-350 </td></tr><tr><td> 17 x 100 mm cultura tubi </td><td> USA scientifico </td><td> 1485-0810 </td></tr><tr><td> n-decanale </td><td> Sigma </td><td> D7384 </td></tr><tr><td> Veritas luminometro per micropiastre </td><td> Turner Biosystems </td><td> 9100-001 </td></tr><tr><td> Microlite micropiastra a 96 pozzetti </td><td> VWR </td><td> 62402-984 </td></tr></tbody></table>

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

Yersinia pestis e Bacillus anthracis sono di categoria A batteri patogeni che sono gli agenti causali della peste e antrace, rispettivamente 1. Anche se la presenza naturale di entrambe le malattie &#39;è ora relativamente rara, la possibilità di gruppi terroristici con questi patogeni come arma biologica è reale. A causa della intrinseca comunicabilità della malattia, rapido decorso clinico, e alto tasso di mortalità, è fondamentale che un focolaio essere rilevati rapidamente. Pertanto le metodologie che consentono il rilevamento rapido e la diagnosi sono essenziali per garantire l&#39;attuazione immediata di misure di sanità pubblica e l&#39;attivazione della gestione delle crisi. Fago ricombinante giornalista può fornire un approccio rapido e specifico per l&#39;individuazione di Y. pestis e B. anthracis. I Centri per il Controllo delle Malattie e la Prevenzione attualmente utilizzano il classico test lisi dei fagi per l&#39;identificazione ha confermato di questi batteri patogeni 2-4. Questi test sfruttare naturale fago che sono specifici e litico per i loro ospiti batterici. Dopo una crescita durante la notte del batterio coltivato in presenza del fago specifico, la formazione di placche (lisi batterica) fornisce una identificazione positiva del target batterico. Anche se questi test sono robusti, soffrono da tre difetti: 1) sono di laboratorio, 2) che richiedono l&#39;isolamento e la coltura batterica dal campione sospetto, e 3) prendono 24-36 ore per essere completato. Per affrontare questi problemi, ricombinante &quot;light-tag&quot; fago giornalista sono stati geneticamente modificati, integrando le Vibrio harveyi luxAB geni nel genoma di Y. pestis e B. anthracis fago specifico 5-8. La risultante dei fagi giornalista luxAB sono stati in grado di rilevare i loro target specifico, mediante una rapida (pochi minuti) e sensibile che conferisce un fenotipo bioluminescente di cellule riceventi. È importante sottolineare che il rilevamento è stato ottenuto sia con cellule riceventi coltivate o con campioni clinici mock-infettate 7. A scopo dimostrativo, qui si descrive il metodo per il fago-mediata rilevamento di un noto Y. pestis isolare con un fago luxAB giornalista costruito dalla peste CDC diagnostica fago ΦA1122 6,7 (Figura 1). Un metodo simile, con piccole modifiche (ad esempio cambiamento della temperatura di crescita e media), possono essere utilizzati per la rilevazione di B. anthracis isola con il B. anthracis fago Wβ reporter:: luxAB 8. Il metodo descrive il fago-mediata trasduzione di un fenotipo biolumescent ad essere coltivato Y. pestis cellule che vengono successivamente misurata con un luminometro per micropiastre. I principali vantaggi di questo metodo il test tradizionale lisi dei fagi è la facilità di utilizzo, i risultati rapidi e la possibilità di testare campioni multipli simultaneamente in un formato a 96 pozzetti di micropiastre. <img src="/files/ftp_upload/2740/2740fig1.jpg" alt="Figura 1"/> Figura 1. Schema di rilevamento. I fagi sono mescolati con il campione, il fago infetta il cellulare, luxAB sono espressi, e il bioluminesces cellula. Trattamento dei campioni non è necessaria; il fago e le cellule sono mescolate e successivamente valutate per la luce.

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

Un metodo semplice per l&#39;identificazione di batteri patogeni priorità è quella di utilizzare fago giornalista geneticamente modificato. Queste fago reporter, che sono specifici per la loro specie ospite particolare, sono in grado di trasduzione rapidamente una risposta bioluminescente segnale alle cellule ospite. Qui, descriviamo l&#39;uso del fago giornalista per la rilevazione di<em&gt; Yersinia pestis</em&gt;.

Phage &#39;Bioluminescent&#39; Reporter per il rilevamento di batteri patogeni di categoria 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 Views.  Guild Associates, Inc.. Un metodo semplice per l'identificazione di batteri patogeni priorità è quella di utilizzare fago giornalista geneticamente modificato. Queste fago reporter, che sono specifici per la loro specie ospite particolare, sono in grado di trasduzione rapidamente una risposta bioluminescente segnale alle cellule ospite. Qui, descriviamo l'uso del fago giornalista per la rilevazione di Yersinia pestis.

Video: '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.

Utilizzando Imaging Bioluminescent per Indagare il sinergismo tra Streptococcus pneumoniae E Influenza A virus in topi neonati

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

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Immunologia e infezioni

Introducing Shear Stress in the Study of Bacterial Adhesion

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

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

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

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

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;infezione di embrioni di zebrafish con batteri patogeni intracellulari

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 Imaging batterica<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.

Trasformazione Biolistic di una fluorescente Tagged Gene nel Opportunistic Fungal patogeni<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.

Detection High-throughput respiratorie patogeni nei campioni animali da 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.

Sviluppo di un virus System Reporter dell&#39;epatite B per monitorare la fasi iniziali del ciclo di replicazione

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.

Una piattaforma di High-throughput per lo Screening della 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.

Anticorpli uccisione test per valutare le risposte immunologiche contro batteri patogeni

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

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