Este trabalho foi suportado pelo USDA Grants 1999-35212-8680, 2005-01378, e 2010-04288-01, Fundos USDA Formula eo Estado de Veterinária da Geórgia Grant Pesquisa Agrícola Medicina. O protocolo descrito no presente publicação foi elaborada para uso por alunos de graduação como parte de cursos de pesquisa dirigida: MIBO 4900L, GENE 4960H, BCMB 4960L e 4960H Biol da Universidade da Geórgia e utilizados pelos alunos em vários projetos de pesquisa em epidemiologia molecular na Maurer laboratório. Queremos agradecer os muitos estudantes de graduação que realizaram pesquisas em laboratórios Maurer e Lee, e nosso chefe de departamento, John Glisson para apoiar os nossos esforços para integrar ensino de graduação com a pesquisa.

<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>

Não há conflitos de interesse declarados.

Comercialmente disponíveis os testes de PCR para a detecção de genes alvos Salmonella exclusivo para o gênero (ex. inva). No entanto, nem todos são iguais Salmonella em sua capacidade de causar doenças em seres humanos ou prevalência em populações animais. O reconhecimento precoce das diferenças antigênicas em Salmonella LPS O antígeno e flagellinH1 e antígenos H2 levou à diferenciação de Salmonella em mais de 2.500 sorotipos com base nessas diferenças antigênicas. Há 46 diferentes antígenos O e 86 distintas flagelina (H) antígenos identificados no gênero Salmonella. Salmonella pode possuir um (H1) ou dois diferentes (H1 e H2) flagellins. A combinação diferente de O, H1, H2 e conta antígenos para os sorovares Salmonella 2500 reconheceu hoje. A sorovar é atribuída com base na fórmula antigênica derivada da identificação do Ó individuais e antígenos H. A fórmula antigênica é escrito como O: H1: H2, e onde &quot;-&quot;, refere-se à ausência de H1 ou H2 flagelina e &quot;NT&quot; (não-tipáveis) para Salmonella negativo para o antígeno O-. Por exemplo, o sorovar Typhimurium é atribuído a um S. enterica isolar com a fórmula antigênica B: i: 1,2, enquanto Enteritidis é dado a um isolado com o D1 fórmula: g, m: -. Essa variação antigênica na população Salmonella é a manifestação exterior de diferenças no nível de nucleotídeos que podem, portanto, ser facilmente explorada por PCR. Nós identificamos as diferenças genéticas associadas com O alelos específicos e H para desenvolver um PCR para a identificação de S. allelotyping enterica sorotipos: Enteritidis, Hadar, Heidelberg, e Typhimurium (3). A atribuição correta e comunicação de Salmonella sorovar requer testes para todos os alelos associados com O: H1: fórmulas antigênicas H2 para Enteritidis (D1: g, m: -), Hadar (C2: z10: e, n, x), Heidelberg (B : r: 1,2), e Typhimurium (B: i: 1,2). Sem o conhecimento de O alelo ou antígeno, a conclusão do g H1, m alelo por si só não significa necessariamente identificar a Salmonella Enteritidis como isolar como outros sorovares de Salmonella: Agona, Califórnia, e Montevidéu, também possuem este mesmo alelo. Enquanto a PCR allelotyping foi originalmente concebido para detectar a tona mencionado sorovares de Salmonella, este teste pode identificar um adicional de 19 sorovares (Tabela 3). Nosso allelotyping PCR foi originalmente concebido para detectar alelos S e H. Na prática, tornou-se mais prático e de custo eficaz para identificar a O antígeno pelo teste de aglutinação, utilizando anti-soros específicos O-, ao invés de executar o allelotyping O PCR quando se trabalha com culturas de Salmonella isoladas. No entanto, nós recomendamos o uso do allelotyping O PCR se o seu laboratório utiliza esta PCR como uma tela (2) ou para a digitação Salmonella isolados submetidos em FTA cards (6), e incluem uma PCR Salmonella específicas com a primeira tela de amostras (4 ). Limitar os PCRs allelotyping apenas aquelas amostras que são identificados como Salmonella pela PCR ou testes bioquímicos / antigênica. O protocolo descrito neste artigo foi otimizado para uso com a tecnologia Idaho Rapidcycler, um termociclador de ar quente que permite que um de reduzir volumes de reação e as condições de reação corre em menos tempo do que muitos termocicladores bloco de aquecimento. Para adaptar este protocolo para uso com um termociclador bloco de aquecimento pode exigir otimização das condições de reação empiricamente através da redução / elevando a temperatura do recozimento; ajustar as concentrações de primer; ou ajustar MgCl 2 concentrações. Por exemplo, tivemos de adaptar o protocolo para a Pesquisa MJ PTC-200 termociclador como se segue. Trabalhando com a mesma reação volumes descritos na Tabela 1, o estoque de trabalho concentrationof o conjunto de primers i foi diluído a 2,5 mM, a temperatura de recozimento foi reduzida de 55 ° C a 45 ° C, tempos e incubação na desnaturação, anelamento e extensão passos foi alongado para 20 s para o i / g, m allelotyping PCR. Tendo os controles apropriados positivos e negativos são essenciais para o arranque inicial para cima e otimização para qualquer PCR, incluindo protocolos publicados. Recomendamos a aquisição de sorovares de Salmonella conhecidos, especificamente Anatum (E1: e, h: 1,6), Enteritidis (D1: g, m: -), Hadar (C2: z10: e, n, x), Heidelberg (B: r : 1,2), Montevidéu (C1: g, m, s: 1,2,7) e Typhimurium (B: i: 1,2), e um laboratório de Escherichia coli K12 tensão (DH5α, LE393, JM109, etc ) como controles positivos e negativos, respectivamente, para os testes de PCR allelotyping descrito neste artigo. Vários desses sorovares Salmonella servirá como controle positivo ou negativo, dependendo de qual alelo é testada. Certas precauções e diretrizes precisam ser seguidas durante a realização deste e de qualquer outro meio de diagnóstico PCR. Primeiro, a separação físicamodelo de preparação, PCR set-up, e eletroforese em gel é fundamental na prevenção da PCR possibilidade de reporte de contaminação e geração de relatórios errôneos de falso-positivos. Além disso, a inclusão de controles adequados é necessário em qualquer PCR de rotina como esses controles identificar problemas que possam surgir e ajudar na interpretação dos resultados (5). Mais importante ainda, a consistência é essencial, especialmente no que diz respeito às condições eletroforéticas para amplicon andestimation detecção (PCR produto) de tamanho amplicon. Para ilustrar este ponto, nós propositadamente suspenso eletroforese anteriormente que o previsto na Figura 1A. Os padrões de peso molecular (faixas 1 e 13) e controle positivo (pista 2) não são suficientemente separadas para estimar com precisão tamanhos ou observar a separação de fragmentos de DNA onde existem pequenas diferenças (~ 50 pb) em tamanhos. Separação adequada dos fragmentos de DNA, especialmente os padrões de peso molecular, é essencial como relatórios positivos é dependente da estimativa precisa do tamanho do amplicon e distinguindo &quot;verdadeiro positivo&quot; de não-específica amplicons que às vezes são observadas no cotidiano de qualquer PCR (5 ). Por exemplo, há um amplicon não-específicas na Figura 1B, pista 7 (~ 700 pb de tamanho). Eletroforese tinha sido interrompido cedo demais, quando a frente de corante foi somente no ponto a meio caminho do gel de agarose, haveria pouca diferenciação entre 500 pb e 700 pb fragmentos de DNA. Portanto, a amostra na pista 7 teria sido erroneamente reportados como positivos para ambos os alelos i e g, m. Finalmente, é preciso reconhecer as limitações associadas a qualquer teste. Vários dos allelotyping H1/H2 PCR não tem o poder discriminatório para discernir sutis diferenças genéticas na alelos pertencentes à H2 1,2; 1,5; 1,6; 1,7 complexo antígeno; e, n, x / e , n, z15 complexo antígeno ea H1 complexo antígeno-G, que inclui a g, m alelo. Uma ou duas mudanças de aminoácidos para a conta de epítopos presentes em diferentes esses antígenos flagelar. Foi, portanto, difícil para nós, para projetar primers que poderiam explorar essas diferenças de base, por vezes único par na seqüência do gene da flagelina (3). Por exemplo, Salmonella sorovares Dublin (D1: g, p: -) e Berta (D1: f, g, t: -) são também PCR positivos com o nosso g, mallelotyping primers. Os primers H2 allelotyping não pode 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), ou Remo (B: r: 1,7), ou Hadar (C2: z10: e, n, x) de Glostrup (C2: z10: e, n, z15). Enquanto a probabilidade de encontrar alguns desses sorovares: Lagos, Bradford, Winneba, Remo ou Glustrup é remota, é uma possibilidade e requer o fornecimento de aviso sobre a limitação dos testes ou outro teste confirmatório. Em conclusão, este sorotipagem baseados em PCR identifica rapidamente S. enterica sorotipos Enteritidis, Hadar, Heidelberg e Typhimurium, e O, H1, H2 e alelos associados com 19 sorotipos adicionais. Este ensaio é uma resposta rápida, alternativa de baixo custo para abordagem microbiológica tradicional para Salmonella sorotipagem.

<table border="1"><tbody><tr><th> Nome do material </th><th> Tipo </th><th> Companhia </th><th> Número Catálogo </th><th> Comentário </th></tr><tr><td> Luria Bertani </td><td></td><td> Pescador </td><td></td><td> (Ou qualquer outra fonte comparável) </td></tr><tr><td> Tubos de microcentrífuga </td><td> 1,5 ml de capacidade </td><td> Pescador </td><td></td><td> (Ou qualquer outra fonte comparável) </td></tr><tr><td> Etanol </td><td> 200 prova </td><td> Pescador </td><td></td><td> (Ou qualquer outra fonte comparável) </td></tr><tr><td> dH 2 O </td><td> Grau Biologia Molecular </td><td> Sigma </td><td></td><td> (Ou qualquer outra fonte comparáveis; PCR apenas) </td></tr><tr><td> 10 x PCR Buffer: </td><td> 20 mM MgCl 2 </td><td> Idaho Tecnologia </td><td></td><td> (Buffer vem com BSA, Ficoll e 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 </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> (Ou qualquer outra fonte comparável) </td></tr><tr><td> Taq DNA polimerase </td><td></td><td> Denville </td><td></td><td> (Ou qualquer outra fonte comparável) </td></tr><tr><td> Pipetas capilares </td><td> 10 mL de volume </td><td> Idaho Tecnologia </td><td></td><td></td></tr><tr><td> Rápida Cycler </td><td></td><td> Idaho Tecnologia </td><td></td><td></td></tr><tr><td> Agarose </td><td> EEO baixa; Grau Biologia Molecular </td><td> BioRad </td><td></td><td> (Ou qualquer outra fonte comparável) </td></tr><tr><td> 50 X tampão TAE ou TBE 5X </td><td></td><td> Pescador </td><td></td><td> (Ou qualquer outra fonte comparável) </td></tr><tr><td> Brometo de etídio </td><td></td><td> BioRad </td><td></td><td> (Ou qualquer outra fonte comparável) </td></tr><tr><td> Horizontal, câmara submersível de eletroforese em gel com o molde de gel </td><td></td><td> BioRad </td><td></td><td> (Ou qualquer outra fonte comparável) </td></tr><tr><td> Fonte de alimentação </td><td></td><td> BioRad </td><td></td><td> (Ou qualquer outra fonte comparável) </td></tr><tr><td> Molecular Imager Gel Doc Sistema </td><td></td><td> BioRad </td><td></td><td> (Ou qualquer outra fonte comparável) </td></tr></tbody></table> Tabela 4. Materiais

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

Comercial atual testes PCR para identificação de genes-alvo Salmonella exclusivo para este gênero. No entanto, existem duas espécies, seis subespécies, e mais de 2.500 sorotipos de Salmonella diferentes, e nem todos são iguais em sua importância para a saúde pública. Por exemplo, encontrar S. enterica subespécie IIIa Arizona em uma fazenda camada tabela de ovos é insignificante comparado com o isolamento de S. enterica subespécie I serovar Enteritidis, a principal causa de salmonelose relacionados ao consumo de ovos de mesa. Sorovares são identificados com base nas diferenças antigênicas de lipopolissacarídeo (LPS) (antígeno O) e flagelina (H1 e H2 antígenos). Essas diferenças antigênicas são a aparência externa da diversidade de genes e alelos do gene associado a este fenótipo. Nós desenvolvemos um allelotyping, multiplex PCR que as chaves sobre as diferenças genéticas entre os quatro grandes S. sorovares enterica subespécie I encontrados em aves domésticas e associados com doenças humanas significativas em os EUA. Os pares de primers de PCR foram direcionados para os principais genes ou seqüências únicas para um sorotipo Salmonella específicas e destinadas a produzir um amplicon com tamanho específico para o gene ou alelo. Salmonella sorovar é atribuída a um isolado com base na combinação dos resultados dos testes de PCR para LPS específicas e alelos do gene flagelina. Os PCRs multiplex descritos neste artigo são específicos para a detecção de S. enterica subespécie I sorovares Enteritidis, Hadar, Heidelberg, e Typhimurium. Aqui demonstramos como usar o PCRs multiplex para identificar serovar Salmonella para uma isolar.

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

Nós descrevemos um multiplex PCR para a detecção rápida de Salmonella enterica sorotipos Enteritidis, Hadar, Heidelberg, e Typhimurium. Sorovares de Salmonella específicos podem ser identificados por alvo um multiplex PCR para os genes e as seqüências exclusivas para o cluster biossíntese O antígeno-flagelina e de um sorovar dado. Sorovar é atribuída então a uma Salmonella isolar baseado na aparência de amplicons específicos, o tamanho (produto de PCR) correspondente ao alelo alvo.

Uma PCR Allelotyping para Identificar Salmonella enterica Sorovares Enteritidis, Hadar, Heidelberg, e Typhimurium

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

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Research

JoVE Journal

Immunology and Infection

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

21.6K visualizações.  University of Georgia. Nós descrevemos um multiplex PCR para a detecção rápida de Salmonella enterica sorotipos Enteritidis, Hadar, Heidelberg, e Typhimurium. Sorovares de Salmonella específicos podem ser identificados por alvo um multiplex PCR para os genes e as seqüências exclusivas para o cluster biossíntese O antígeno-flagelina e de um sorovar dado. Sorovar é atribuída então a uma Salmonella isolar baseado na aparência de amplicons específicos, o tamanho (produto de PCR) corre...

Vídeo: 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.

Identificação de genes desregulados Induzida pelo Herpesvirus sarcoma de Kaposi-associado (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 ...

Imunologia e Infecção

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.

Um<em&gt; In vitro</em&gt; Modelo para estudar a heterogeneidade de diferenciação de macrófagos humanos e Polarização

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.

Medindo o Crescimento e Expressão Gênica Dynamics do Tumor-Targeted<em&gt; S. Typhimurium</em&gt; Bactérias

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.

Procedimentos para identificar Infecciosas príons após a passagem através do sistema digestivo de uma espécie 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.

Um protocolo para infectar<em&gt; Caenorhabditis elegans</em&gt; Com<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.

De alta capacidade: Ensaio para Fenótipo<em&gt; Salmonella enterica</em&gt; Typhimurium Association, invasão e replicação em 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.

Estabelecimento de um programa de configuração de alta capacidade para a triagem de pequenas moléculas que modulam c-di-GMP Sinalização<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.

genotipagem de<em&gt; Staphylococcus aureus</em&gt; por Ribossômico 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, ...

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.

Isolamento de Salmonella typhimurium-contendo Phagosomes de 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.

Uma plataforma de alto rendimento para o rastreio 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 ...