Este trabajo ha sido subvencionado por el USDA 1999-35212-8680, 2005-01378, y 2010-04288-01, Fondos USDA Fórmula y el Estado de Beca de Investigación Veterinaria de Georgia Medicina Agrícola. El protocolo descrito en esta publicación se ha desarrollado para su uso por los estudiantes como parte de los cursos de investigación dirigido: MIBO 4900L, 4960H GENE, BCMB 4960L, y BIOL 4960H en la Universidad de Georgia y utilizados por los estudiantes en varios proyectos de investigación de epidemiología molecular en el Maurer laboratorio. Queremos agradecer a los muchos estudiantes de pregrado que se han realizado investigaciones en los laboratorios de Maurer y Lee, y nuestro jefe de departamento, John Glisson por apoyar nuestros esfuerzos para integrar la enseñanza de pregrado con la investigación.

<ol>
	<li>Dauga, C., Zabrovskaia, A., Grimont, P. A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restriction+fragment+length+polymorphism+analysis+of+some+flagellin+genes+of+Salmonella+enterica.">Restriction fragment length polymorphism analysis of some flagellin genes of Salmonella enterica.</a> J. Clin. Microbiol. 36, 2835-2843 .</li><li>Hong, Y., Liu, T., Lee, M. D., Hofacre, C. L., Maier, M., White, D. G., Ayers, S., Wang, L., Berghaus, R., Maurer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+screen+of+broth+enrichments+for+Salmonella+enterica+serovars+Enteritidis,+Hadar,+Heidelberg,+and+Typhimurium+using+an+allelotyping+multiplex+PCR+that+targets+O+and+H+antigen+alleles.">A rapid screen of broth enrichments for Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium using an allelotyping multiplex PCR that targets O and H antigen alleles.</a> J. Food Prot. 72, 2198-2201 (2009).</li><li>Hong, Y., Liu, T., Lee, H. o. f. a. c. r. e., Maier, C. L., White, M., Ayers, D. G., Wang, S., Berghaus, L., R, M. a. u. r. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=J.Rapid+screening+of+Salmonella+enterica+serovars+Enteritidis,+Hadar,+Heidelberg+and+Typhimurium+using+a+serologically-correlative+allelotyping+PCR+targeting+the+O+and+H+antigen+alleles.">J.Rapid screening of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg and Typhimurium using a serologically-correlative allelotyping PCR targeting the O and H antigen alleles.</a> BMC Microbiol. , (2008).</li><li>Liu, T., Liljebjelke, K., Bartlett, E., Hofacre, C. L., Sanchez, S., Maurer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+nested+PCR+to+detection+of+Salmonella+in+poultry+environments.">Application of nested PCR to detection of Salmonella in poultry environments.</a> J. Food Prot. 65, 1227-1232 (2002).</li><li>Maurer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+and+limitation+of+molecular+techniques.">Rapid detection and limitation of molecular techniques.</a> Ann. Rev. Food Sci. Tech. 2, 259-279 (2011).</li><li>Moscoso, H., Alvarado, I., Hofacre, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+infectious+bursal+disease+virus+from+bursal+tissues+collected+on+FTA+filter+paper.">Molecular analysis of infectious bursal disease virus from bursal tissues collected on FTA filter paper.</a> Avian Dis. 50, 391-396 (2006).</li><li>Sambrook, J., Fritsch, E. F., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory. , (1989).</li><li>Wittwer, C. T., Fillmore, G. C., Hillyard, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+polymerase+chain+reaction+capillary+tubes+with+hot+air.">Automated polymerase chain reaction capillary tubes with hot air.</a> Nucleic Acids Res. 17, 4353-4357 (1989).</li></ol>

No hay conflictos de interés declarado.

La mayoría de los disponibles en el mercado pruebas PCR para la detección de los objetivos de Salmonella único en el género (por ejemplo, invA) genes. Sin embargo, no todos los serotipos son iguales en su capacidad para causar enfermedades en humanos o la prevalencia en las poblaciones animales. El reconocimiento temprano de las diferencias antigénicas de Salmonella LPS O-antígeno y antígenos H2 flagellinH1 y ha dado lugar a la diferenciación de Salmonella en más de 2.500 serotipos sobre la base de estas diferencias antigénicas. Hay 46 diferentes antígenos O y 86 antígenos distintos flagelina (H) identificados en el género Salmonella. Salmonella puede poseer uno (H1) o de dos diferentes (H1 y H2) flagellins. La combinación diferente de O, H1, H2 y cuenta antígenos de los serotipos de Salmonella 2.500 reconocido hoy en día. Un serotipo es asignado sobre la base de la fórmula antigénica derivada de la identificación de la persona antígenos O y H. La fórmula antigénica se escribe como O: H1: H2, y donde &quot;-&quot;, se refiere a la ausencia de flagelina H1 o H2 y &quot;NT&quot; (no tipificable) para Salmonella negativo para el antígeno-O. Por ejemplo, el serovar Typhimurium se asigna a una S. enterica aislada con la fórmula antigénica B: i: 1,2, mientras que Enteritidis se le da a un aislamiento con la fórmula D1: g, m: -. Esta variación antigénica en la población de Salmonella es la manifestación externa de las diferencias a nivel de nucleótidos que por lo tanto pueden ser fácilmente explotados por PCR. Hemos identificado las diferencias genéticas específicas asociadas con los alelos S y H para desarrollar un PCR para la identificación de S. allelotyping enterica serovar: Enteritidis, Hadar, Heidelberg, y Typhimurium (3). La correcta asignación y presentación de informes de Salmonella serovar requiere una prueba para todos los alelos asociados con O: H1: H2 fórmulas antigénicas para Enteritidis (D1: g, m: -), Hadar (C2: z10: e, n, x), Heidelberg (B : r: 1,2) y Typhimurium (B: i: 1,2). Sin el conocimiento del alelo S o antígeno, el hallazgo de la g H1, el alelo m por sí sola no se identifica necesariamente la Salmonella Enteritidis en aislar como otros serotipos de Salmonella: Agona, California, y Montevideo, también poseen este mismo alelo. Mientras que la PCR allelotyping fue originalmente diseñado para detectar un primer plano mencionado serotipos de Salmonella, esta prueba puede identificar otros 19 serotipos (tabla 3). Nuestra allelotyping PCR fue diseñada originalmente para detectar alelos S y H. En la práctica, se ha vuelto más práctico y rentable para que podamos identificar el antígeno-O por la prueba de aglutinación, utilizando antisueros específicos O, en lugar de realizar el PCR allelotyping O cuando se trabaja con cultivos de Salmonella aisladas. Sin embargo, se recomienda utilizar el O allelotyping PCR si el laboratorio utiliza el PCR como una pantalla (2) o para la clasificación presentada en los aislamientos de Salmonella tarjetas FTA (6), e incluyen una PCR específica de Salmonella con la primera pantalla de las muestras (4 ). Limitar la PCR allelotyping a sólo aquellas muestras que se identifican como Salmonella por PCR o pruebas bioquímicas / antigénica. El protocolo se describe en este artículo ha sido optimizado para su uso con el Rapidcycler Idaho Technology, un termociclador de aire caliente que permite a reducir los volúmenes de reacción y las condiciones de reacción se ejecuta en menos tiempo de lo que muchos termocicladores calefacción bloque. Para adaptar este protocolo para su uso con un termociclador un calentador pueden requerir la optimización de las condiciones de reacción empíricamente mediante la reducción / aumento de las temperaturas de recocido, el ajuste de concentración de los cebadores, o el ajuste de concentraciones de MgCl 2. Por ejemplo, hemos tenido que adaptar el protocolo para el MJ Research PTC-200 termociclador de la siguiente manera. Trabajar con los volúmenes misma reacción en la tabla 1, las acciones de trabajo concentrationof se diluyó el primer set i de 2,5 M, la temperatura de hibridación se redujo de 55 ° C a 45 ° C, y los tiempos de incubación en la desnaturalización, hibridación y extensión de las medidas se alargó a 20 s para el i / g, m allelotyping PCR. Tener el control, positivas y negativas son esenciales en la puesta en marcha inicial y la optimización de los PCR, incluyendo los protocolos publicados. Recomendamos adquirir conocido serotipos de Salmonella, especialmente Anatum (E1: e, h: 1,6), Enteritidis (D1: g, m: -), Hadar (C2: z10: e, n, x), Heidelberg (B: r : 1,2), Montevideo (C1: g, m, s: 1,2,7) y Typhimurium (B: i: 1,2), y un laboratorio de Escherichia coli K12 (DH5a, LE393, JM109, etc ) como controles positivos y negativos, respectivamente, para las pruebas de PCR allelotyping descrito en este artículo. Varios de estos serotipos de Salmonella servirá como control positivo o negativo, dependiendo de qué alelo se pone a prueba. Ciertas precauciones y pautas se deben seguir al realizar esta y cualquier otro diagnóstico de PCR. En primer lugar, la separación físicade la preparación de la plantilla, PCR puesta en marcha, y la electroforesis en gel es fundamental en la prevención de posibles PCR traspaso de la contaminación y la información errónea de los falsos positivos. Además, la inclusión de los controles adecuados es necesario en cualquier rutina de PCR ya que estos controles identificar los problemas que puedan surgir y ayudar en la interpretación de los resultados (5). Lo más importante, la consistencia es esencial, especialmente en lo que respecta a las condiciones de electroforesis para la detección andestimation amplificación (PCR producto) de tamaño de amplificación. Para ilustrar este punto, a propósito de electroforesis suspendido antes de lo esperado en la figura 1A. Los patrones de peso molecular (calles 1 y 13) y el control positivo (carril 2), no están lo suficientemente separados para estimar con precisión los tamaños y observar la separación de fragmentos de ADN, donde hay pequeñas diferencias (~ 50 pb) en los tamaños. La adecuada separación de los fragmentos de ADN, especialmente los patrones de peso molecular, es fundamental la presentación de informes positivos depende de la estimación precisa del tamaño de amplificación y la distinción &quot;verdaderos positivos&quot; de no amplicones específicos que a veces se observan en el funcionamiento diario de cualquier PCR (5 ). Por ejemplo, hay una amplificación no específicos en la Figura 1B, carril 7 (~ 700 pb de tamaño). Electroforesis había sido descontinuado demasiado pronto, cuando el frente de colorante sólo en el punto a mitad de camino en el gel de agarosa, habría poca diferencia entre 500 pb y 700 pb fragmentos de ADN. Por lo tanto, muestra en la calle 7 que han sido erróneamente reportado como positivo para ambos i y g, m alelos. Finalmente, es necesario reconocer las limitaciones asociadas a cualquier prueba. Varios de los PCR H1/H2 allelotyping no tiene el poder discriminatorio para distinguir sutiles diferencias genéticas en los alelos que pertenecen a la H2 1,2, 1,5, 1,6, 1,7 complejo antígeno, e, n, x / e , n, Z15 complejo antígeno y el complejo antígeno H1 G, que incluye la g, m alelo. Uno o dos cambios de aminoácidos en cuenta los diferentes epítopos presentes en estos antígenos flagelares. Por lo tanto, difícil para nosotros el diseño de cebadores que podría explotar estas diferencias de base en ocasiones de un solo par en la secuencia del gen de flagelina (3). Por ejemplo, Salmonella serovares Dublin (D1: g, p: -) y Berta (D1: f, g, t: -) también son positivas en la PCR con los g, mallelotyping primers. Los cebadores H2 allelotyping no puede distinguir Typhimurium (B: i: 1,2) de Lagos (B: i: 1,5), Heidelberg (B: r: 1,2) de Bradford (B: r: 1,5), Winneba (B: r: 1,6), o Remo (B: r: 1,7), o Hadar (C2: z10: e, n, x) de Glostrup (C2: z10: e, n, Z15). Mientras que la probabilidad de encontrar algunos de estos serotipos: Lagos, Bradford, Winneba, Remo o Glustrup es remota, es una posibilidad y requiere ya sea proporcionando aviso legal sobre la limitación de las pruebas o de otra prueba confirmatoria. En conclusión, este serotipo basado en la PCR identifica rápidamente S. enterica serovar Enteritidis, Hadar, Heidelberg y Typhimurium, y O, H1, H2 y los alelos asociados con 19 serotipos adicionales. Este ensayo es una rápida y rentable alternativa al enfoque tradicional microbiológicos de Salmonella serotipo.

<table border="1"><tbody><tr><th> Nombre del Material </th><th> Tipo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentario </th></tr><tr><td> Luria Bertani </td><td></td><td> Pescador </td><td></td><td> (O cualquier otra fuente similar) </td></tr><tr><td> Microfuga tubos </td><td> 1,5 ml de capacidad </td><td> Pescador </td><td></td><td> (O cualquier otra fuente similar) </td></tr><tr><td> Etanol </td><td> Prueba 200 </td><td> Pescador </td><td></td><td> (O cualquier otra fuente similar) </td></tr><tr><td> dH 2 O </td><td> Grado de Biología Molecular </td><td> Sigma </td><td></td><td> (O cualquier otra fuente similar, sólo PCR) </td></tr><tr><td> 10 x PCR Buffer: </td><td> 20 mM MgCl 2 </td><td> Idaho Tecnología </td><td></td><td> (Tampón viene con BSA, Ficoll y tartrazina) </td></tr><tr><td></td><td> 30 mM MgCl 2 </td><td></td><td></td><td></td></tr><tr><td></td><td> 40 mM MgCl 2 </td><td></td><td></td><td></td></tr><tr><td> PCR primers </td><td></td><td></td><td></td><td></td></tr><tr><td> DMSO </td><td></td><td></td><td></td><td></td></tr><tr><td> dNTP </td><td></td><td> Roche </td><td></td><td> (O cualquier otra fuente similar) </td></tr><tr><td> Taq ADN polimerasa </td><td></td><td> Denville </td><td></td><td> (O cualquier otra fuente similar) </td></tr><tr><td> Pipetas capilares </td><td> Un volumen de 10 l </td><td> Idaho Tecnología </td><td></td><td></td></tr><tr><td> Rápida termociclador </td><td></td><td> Idaho Tecnología </td><td></td><td></td></tr><tr><td> Agarosa </td><td> Baja EEO; Grado de Biología Molecular </td><td> BioRad </td><td></td><td> (O cualquier otra fuente similar) </td></tr><tr><td> 50 X TAE buffer o tampón TBE 5X </td><td></td><td> Pescador </td><td></td><td> (O cualquier otra fuente similar) </td></tr><tr><td> El bromuro de etidio </td><td></td><td> BioRad </td><td></td><td> (O cualquier otra fuente similar) </td></tr><tr><td> Horizontal, sumergible cámara de electroforesis en gel con molde de gel </td><td></td><td> BioRad </td><td></td><td> (O cualquier otra fuente similar) </td></tr><tr><td> Fuente de alimentación </td><td></td><td> BioRad </td><td></td><td> (O cualquier otra fuente similar) </td></tr><tr><td> Molecular Imager sistema Gel Doc </td><td></td><td> BioRad </td><td></td><td> (O cualquier otra fuente similar) </td></tr></tbody></table> Tabla 4. Materiales

an allelotyping pcr for identifying salmonella enterica serovars enteritidis, hadar, heidelberg, and typhimurium

Las pruebas actuales de terminación de proyecto comercial para la identificación de genes de Salmonella objetivo único de este género. Sin embargo, hay dos especies, subespecies y seis, y más de 2.500 serotipos de Salmonella diferentes, y no todos son iguales en su importancia para la salud pública. Por ejemplo, la búsqueda de S. enterica subespecie IIIa Arizona en una granja ponedora de huevos de mesa es insignificante en comparación con el aislamiento de S. enterica serovar Enteritidis subespecie I, la principal causa de salmonelosis asociados al consumo de huevos de mesa. Serotipos se identifican con base en las diferencias antigénicas de lipopolisacárido (LPS) (antígeno O) y flagelina (H1 y H2 antígenos). Estas diferencias antigénicas son la apariencia externa de la diversidad de los genes y alelos de genes asociados con este fenotipo. Hemos desarrollado un allelotyping, PCR multiplex que las claves en las diferencias genéticas entre los cuatro principales S. enterica serovar subespecie que se encuentra en las aves de corral y se asocia con la enfermedad humana significativa en los EE.UU.. Las parejas de cebadores de PCR fueron dirigidos a genes o secuencias clave única de un serovar de Salmonella específico y diseñado para producir una amplificación con un tamaño específico para que el gen o alelo. Salmonella serovar se asigna a un aislamiento basado en la combinación de resultados de las pruebas de PCR para LPS específicos y flagelina alelos del gen. La PCR multiplex descrito en este artículo son específicos para la detección de S. enterica serovar Enteritidis subespecie I, Hadar, Heidelberg, y Typhimurium. Aquí se demuestra cómo utilizar el multiplex PCR para identificar los serotipos de Salmonella para aislar.

Watch this Scientific Journal Video about An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium at JoVE.com

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Se describe una técnica de PCR múltiple para la detección rápida de Salmonella enterica serovar Enteritidis, Hadar, Heidelberg, y Typhimurium. Serotipos de Salmonella específicos se pueden identificar por objetivo una PCR múltiple para los genes y las secuencias únicas para el grupo O-antígeno de la biosíntesis y la flagelina de un serotipo determinado. Serovar se le asignó a un aislamiento de Salmonella basado en la apariencia de los amplicones específicos de tamaño, (producto de PCR), correspondiente al alelo objetivo.

Una PCR para la identificación de Allelotyping Salmonella enterica Serotipos Enteritidis, Hadar, Heidelberg, y Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus.  However, there are two species, six subspecies, and over ...

an-allelotyping-pcr-for-identifying-salmonella-enterica-serovars

Research

JoVE Journal

Immunology and Infection

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

21.6K vistas.  University of Georgia. Se describe una técnica de PCR múltiple para la detección rápida de Salmonella enterica serovar Enteritidis, Hadar, Heidelberg, y Typhimurium. Serotipos de Salmonella específicos se pueden identificar por objetivo una PCR múltiple para los genes y las secuencias únicas para el grupo O-antígeno de la biosíntesis y la flagelina de un serotipo determinado. Serovar se le asignó a un aislamiento de Salmonella basado en la apariencia de los amplicones específicos de ...

Video: An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of pleural effusion lymphomas (PEL) and some forms of multicentric Castleman's disease.3-5 Only 1-5% of cells in KS lesions actively support lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent associated with KS, and it is clear that cellular factors must interact with viral factors in the process of oncogenesis and tumor progression.6,7 Identifying novel host-factor determinants which contribute to KS pathology is essential for developing prognostic markers for tumor progression and metastasis as well as for developing novel therapeutics for the treatment of KS.8 The accompanying video details the methods we use to identify host cell gene expression programs altered in dermal microvascular endothelial cells (DMVEC) after KSHV infection and in KS tumor tissue.9 Once dysregulated genes are identified by microarray analysis, changes in protein expression are confirmed by immunoblot and dual labeled immunofluorescence. Changes in transcriptional expression of dysregulated genes are confirmed in vitro by quantitative real-time polymerase chain reaction (qRT-PCR). Validation of in vitro findings using archival KS tumor tissue is also performed by dual labeled immunochemistry and tissue microarrays.8,10 Our approach to identifying dysregulated genes in the KS tumor tissue microenvironment will allow the development of in vitro and subsequently in vivo model systems for discovery and evaluation of potential novel therapeutic for the treatment of KS.

Identifying Dysregulated Genes Induced by Kaposi&#039;s Sarcoma-associated Herpesvirus KSHV

Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.

La identificación de genes inducida por la desregulación de Sarcoma de Kaposi asociado al virus del herpes (HVSK)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

Investigación

Inmunología e Infección

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Un<em&gt; In vitro</em&gt; Modelo para estudiar la heterogeneidad de diferenciación de los macrófagos humanos y polarización

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. 
We generated model xenograft tumors in mice by subcutaneous injection of a human ovarian cancer cell line, OVCAR-8 (NCI DCTD Tumor Repository, Frederick, MD). 
We transformed attenuated strains of S. typhimurium bacteria (ELH430:SL1344 phoPQ- 1) with a constitutively expressed luciferase (luxCDABE) plasmid for visualization2. These strains specifically colonize tumors while remaining essentially non-virulent to the mouse1. 
Once measurable tumors were established, bacteria were injected intravenously via the tail vein with varying dosage. Tumor-localized, bacterial gene expression was monitored in real time over the course of 60 hours using an in vivo imaging system (IVIS). At each time point, tumors were excised, homogenized, and plated to quantitate bacterial colonies for correlation with gene expression data. 
Together, this data yields a quantitative measure of the in vivo growth and gene expression dynamics of bacteria growing inside tumors.

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. This video covers tumor cell preparation and implantation, bacteria preparation and injection, whole-animal luminescence imaging, tumor excision, and bacterial colony counting.

Growth La medición y de la Expresión Génica Dinámica de Tumor-Targeted<em&gt; S. Typhimurium</em&gt; Las bacterias

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

Procedimientos para la Identificación de priones infecciosos después de pasar por el sistema digestivo de una especie de aves

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans has emerged as a new genetic model to study host-pathogen interactions. Here we describe a protocol to infect C. elegans with Salmonella typhimurium coupled with the double-strand RNAi interference technique to examine the role of host genes in defense against Salmonella infection.

Un protocolo para infectar<em&gt; Caenorhabditis elegans</em&gt; Con<em&gt; Salmonella typhimurium</em

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

Ensayo de alto rendimiento para Fenotipo<em&gt; Salmonella enterica</em&gt; Asociación Typhimurium, Invasión y replicación en macrófagos

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Establecimiento de un programa de instalación de alto rendimiento para la detección de pequeñas moléculas que modulan la c-di-GMP Señalización en el<em&gt; Pseudomonas aeruginosa</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

genotipado de<em&gt; Staphylococcus aureus</em&gt; por Ribosómica espaciador de PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

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

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

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

Aislamiento de Salmonella typhimurium-que contienen fagosomas de los macrófagos

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

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 ...