Esta investigación fue financiada por el programa Small Business Innovation Research de los Institutos Nacionales de Salud (NIAID, 1R43AI082698-01) y el Departamento de Agricultura Instituto Nacional de la Agricultura y la Alimentación (NIFA, 2009-33610-20028).

1. Y. pestis inoculación placa <ol><li> Racha de Y. pestis A1122 (BeiResources # NR15) los cultivos de acciones en Luria-Bertani (LB) agar (Miller). Una técnica estéril y realizar todas las Y. pestis manipulaciones en un tipo de clase II Un gabinete de bioseguridad. Y. pestis A1122 es un nivel de bioseguridad (BSL) 2 cepa excluidos agente de selección. Se carece de los 75 kb par bajo contenido de calcio de respuesta (LCR) plásmido de virulencia, y el locus PGM que se requieren para la virulencia. Crecer Y. pestis a 28 ° C durante 48 horas a una temperatura controlada de aire estático incubadora. La tasa de crecimiento de Y. pestis es lenta, con un tiempo de generación de 1,25 h 9. </li></ol> 2. Y. pestis medios líquidos inoculación <ol><li> La transferencia de un solo Y. pestis colonia en una estéril 17 x 100 mm tubo de cultivo que contiene 2 ml de caldo LB con un asa metálica inoculación estéril. Seleccione una colonia que es de tamaño uniforme, es indicativo de las otras colonias, y está claramente separada del resto de las colonias en la placa. Tubo de vórtice brevemente y hacer crecer la cultura a 28 ° C durante aproximadamente 20 horas en una incubadora de agitación (225 rpm). </li></ol> 3. Y. consecuencia pestis, además de fago reportero, y la detección de bioluminiscencia <ol><li> Diluir la noche a la mañana Y. pestis 1:20 cultura en medio LB fresco en un tubo de 50 ml halcón (por ejemplo, 500 l de las células en 9,5 ml de medio) y crecer a 28 ° C con agitación (225 rpm). Crecer hasta una densidad óptica a 600 nm (A 600) de 0,2 que se alcanza (aproximadamente 5 h). Las células en crecimiento activo son preferibles para la detección ya que la capacidad de los fagos para la transducción de una respuesta bioluminiscente se correlaciona con la viabilidad y la adecuación de la bacteria huésped. Sin embargo, el sistema de detección se ha demostrado que es compatible con las células recolectadas en todas las etapas del ciclo de crecimiento del 7. </li><li> Generar el sustrato necesario para la reacción bioluminiscente en la preparación de un 2% de n-decanal solución. Mezcla de 200 l de n-decanal con 9,8 ml de caldo de LB. Agitar vigorosamente durante 5 s. El primer inyector de la microplaca luminómetro con 2 ml de 2% de la n-decanal solución. Pre-establecido para el luminómetro autoinject 67 l de la solución decanal a cada micro pozo e inmediatamente leer la muestra durante 10 s. </li><li> Dispensar alícuotas de 1 ml de caldo de LB en 3 tubos de cultivo. Añadir 20 l de la solución de fago reportero de acciones (stock de 5 x 10 9 unidades formadoras de placa [UFP] / mL) a cada tubo, el fago y las muestras de los medios de comunicación solo sirve como control negativo. En ausencia de Y. células pestis, la adición del fago reportero no debe provocar una respuesta bioluminiscente. </li><li> Dispensar alícuotas de 1 ml de la Y. pestis mediante cultivo (desde el paso 3.1) en 6 tubos de cultivo. Añadir 20 l de la acción del fago reportero a 3 de los cultivos (cultivos de ensayo). Los restantes tres culturas servir como &quot;células por sí solas&quot; control negativo (autobioluminescence de fondo). Mezcla de culturas con el vortex brevemente, y la cosecha de 200 l de cada muestra (por el momento 0 leer) y se distribuye en una placa de microtitulación de 96 pocillos de blanco. Incubar el cultivo restante a los 28 ° C con agitación (225 rpm). </li><li> Medir el tiempo de 0 muestras de bioluminiscencia (unidades relativas de luz, RLU) con el luminómetro microplaca. </li><li> Cosecha de 200 l de cada muestra después de 10, 20, 30, 40, 50 y 60 minutos después de la periodista además de fagos, y medir la muestra de la bioluminiscencia. Si Y. pestis células están presentes, el fago reportero se unirán específicamente a la célula, inyecta su ADN, y el uso de los anfitriones de maquinaria de transcripción y traducción de transcribir y traducir los genes reportero luxAB (Figura 1). </li><li> La fuerza de la señal y el tiempo de respuesta de la señal es proporcional al número de células presentes. En altas concentraciones de células (10 5 10 8 UFC / ml), un aumento significativo en RLU comparación con los controles deben ser evidentes dentro de los 20 min. En las concentraciones de células inferior, (10 2 -10 4 UFC / ml), un aumento significativo en RLU debe ser evidente en 40-60 min. </li><li> Por otra parte, para acelerar el proceso de detección sin necesidad de Y. consecuencia pestis, una colonia de una placa recién crecido puede ser mixto (mediante agitación) directamente en un tubo Eppendorf de 1,5 ml con 200 l de caldo de LB albergar el fago reportero. Prescindir de la mezcla de células / fago en un pozo de una placa de microtitulación. Incubar la placa de microtitulación de 28 ° C y leer la muestra de la bioluminiscencia después de 60 minutos (solo punto de tiempo solamente). </li></ol> 4. Los resultados representativos: Un experimento de tiempo por supuesto representante del fago reportero mediada la detección de Y. pestis se muestra en la Figura 2. Los controles negativos de 1) fago solo (sin células), o 2) células por sí solas (sin fago) proporcionar los niveles de referencia de la bioluminiscencia de aproximadamente 20 RLU toda la inc 60 minubation (niveles de referencia son específicos luminómetro). Por el contrario, un aumento de la bioluminiscencia de las muestras de ensayo (fago reportero y células) se hace evidente a los 15 minutos después de la adición del fago. La intensidad de la señal debe aumentar de manera constante durante 60 min. Incubaciones por períodos de tiempo prolongado (más de 80 min), se traducirá en una señal de que se reducirá desde el pico de la señal debido a la lisis mediada por fagos de las células huésped. Resultados similares se obtienen a temperaturas de incubación de 37 ° C aunque la temperatura óptima para la Y. crecimiento pestis es de 28 ° C 9. <img src="/files/ftp_upload/2740/2740fig2.jpg" alt="Figura 2"/> Figura 2. Fagos mediada por la detección de bioluminiscente de Y. pestis. En el momento 0, fago reportero de las células y se mezclan, se incuba a 28 ° C, y la bioluminiscencia (RLU) fue monitorizada a través del tiempo. Un aumento significativo en RLU (* Estudiantes t-test, p &lt;0,05) es evidente en los 15 minutos.

<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>

Este método demuestra la capacidad del fago reportero para detectar rápidamente Y. pestis desde el fago reportero puede transducir una señal de respuesta bioluminiscente de cultivo Y. pestis células dentro de los 20 minutos después de la adición del fago. El fago reportero también es capaz de detectar directamente Y. pestis en las matrices de clínica, sin el requisito de aislamiento y posterior cultivo 7. En comparación con los ensayos de lisis del fago estándar que genera...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th></tr><tr><td> Difco agar LB, Miller </td><td> VWR </td><td> 90003-346 </td></tr><tr><td> LB caldo Difco, Miller </td><td> VWR </td><td> 90003-350 </td></tr><tr><td> 17 x 100 mm tubos de la cultura </td><td> EE.UU. Científico </td><td> 1485-0810 </td></tr><tr><td> n-Decanal </td><td> Sigma </td><td> D7384 </td></tr><tr><td> Microplaca Veritas luminómetro </td><td> Turner Biosystems </td><td> 9100-001 </td></tr><tr><td> Microlite microtitulación de 96 pocillos </td><td> VWR </td><td> 62402-984 </td></tr></tbody></table>

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

Yersinia pestis y Bacillus anthracis son de categoría A patógenos bacterianos que son los agentes causales de la peste y el ántrax, respectivamente 1. A pesar de la presencia natural de ambas enfermedades es ahora relativamente rara, la posibilidad de que grupos terroristas que utilizan estos agentes patógenos como un arma biológica es real. Debido a la comunicabilidad inherente de la enfermedad, curso clínico rápido y alta tasa de mortalidad, es fundamental que un brote se detectó rápidamente. Por lo tanto, las metodologías que proporcionan una rápida detección y el diagnóstico son esenciales para garantizar la aplicación inmediata de medidas de salud pública y la activación de la gestión de crisis. Recombinante fago reportero puede ofrecer un enfoque rápido y específico para la detección de Y. pestis y B. anthracis. Los Centros para el Control y Prevención de Enfermedades en la actualidad el uso de los ensayos de fagos lisis clásico para la identificación confirmado de estas bacterias patógenas 2-4. Estos ensayos de aprovechar de forma natural fagos que son específicos y lítico para sus huéspedes bacterianos. Después de un crecimiento durante la noche de la bacteria cultivada en la presencia de los fagos específicos, la formación de placas (lisis bacteriana) proporciona una identificación positiva de la diana bacteriana. A pesar de estas pruebas son sólidas, sufren de tres problemas: 1) son de laboratorio basado, 2) que requieren aislamiento bacteriano y el cultivo de la muestra sospechosa, y 3) tomar 24-36 horas para completar. Para abordar estas cuestiones, un recombinante de &quot;luz-con etiqueta&quot; fago reportero fueron genéticamente manipulados mediante la integración de los Vibrio harveyi luxAB genes en el genoma de la Y. pestis y B. anthracis específicos fago 5-8. El fago reportero luxAB resultantes fueron capaces de detectar su objetivo específico de acuerdo rápidamente (en minutos) y la sensibilidad que confiere un fenotipo bioluminiscente a las células receptoras. Es importante destacar que la detección se obtuvo, ya sea con las células receptoras cultivadas o con modelo de infectados muestras clínicas 7. Para fines de demostración, aquí se describe el método para la detección de fagos mediada de un conocido Y. pestis aislar utilizando un fago reportero luxAB construido a partir de los CDC plaga de diagnóstico fago ΦA1122 6,7 (Figura 1). Un método similar, con ligeras modificaciones (por ejemplo, cambio en la temperatura de crecimiento y los medios de comunicación), se puede utilizar para la detección de B. anthracis aislamientos de la B. fago reportero anthracis Wβ:: luxAB 8. Este método describe la transducción mediada por fagos de un fenotipo de biolumescent cultivada Y. pestis células que posteriormente se midió utilizando un luminómetro de microplacas. Las principales ventajas de este método en los ensayos de lisis del fago tradicionales es la facilidad de uso, los resultados rápidos, y la capacidad para analizar muestras de forma simultánea en un formato de microtitulación de 96 pocillos. <img src="/files/ftp_upload/2740/2740fig1.jpg" alt="Figura 1"/> Figura 1. Esquema de detección. El fago se mezclan con la muestra, el fago infecta a la célula, luxAB expresadas, y el bioluminesces celular. Procesamiento de las muestras no es necesario, el fago y las células se mezclan y posteriormente se valoran por la luz.

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 método simple para la identificación de los patógenos bacterianos prioridad es el uso de fagos reportero de la ingeniería genética. Estos fagos reportero, que son específicos de su especie huésped particular, son capaces de transducir una respuesta rápida de la señal bioluminiscente a las células huésped. En este documento, se describe el uso de fagos reportero para la detección de<em&gt; Yersinia pestis</em&gt;.

&quot;Bioluminiscente &#39;reportero fagos para la detección de patógenos bacterianos de la categoría 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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Research

JoVE Journal

Immunology and Infection

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

13.3K vistas.  Guild Associates, Inc.. Un método simple para la identificación de los patógenos bacterianos prioridad es el uso de fagos reportero de la ingeniería genética. Estos fagos reportero, que son específicos de su especie huésped particular, son capaces de transducir una respuesta rápida de la señal bioluminiscente a las células huésped. En este documento, se describe el uso de fagos reportero para la detección de 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.

Utilizando imágenes bioluminiscentes encargado de investigar la sinergia entre Streptococcus pneumoniae Y el Virus de la Influenza en ratones lactantes

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

Investigación

Inmunología e Infección

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.

La introducción de la tensión de cizallamiento en el Estudio de la adhesión bacteriana

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.

La infección de embriones de pez cebra con patógenos bacterianos intracelulares

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.

Imágenes bacteriana Bioluminiscente<em&gt; En 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.

Transformación biolística de un fluorescente Tagged gen en el oportunista por hongos patógenos<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.

La detección de alto rendimiento de los patógenos respiratorios de especímenes animales por PCR nanoescala

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.

Desarrollo de un sistema reportero virus de la hepatitis B para supervisar las primeras etapas del ciclo de replicación

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 plataforma de alto rendimiento para la detección 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.

Opsonofagocítico asesinato ensayo para evaluar las respuestas inmunológicas contra patógenos bacterianos

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.

Prueba de inmunohistoquímica para la detección de antígenos de lisavirus en tejidos fijos de formalina

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