Os autores gostariam de agradecer o apoio relevante para este manuscrito da Comissão Europeia Sétimo Programa-Quadro (PIOF-GA-2009-255466) e do Conselho de Pesquisa irlandês Saúde (HRA_POR/2010/138). Lux-marcado E. coli foi um presente tipo de Dr. Cormac Gahan, Universidade College Cork.

1. Indução de tumores <ol><li> Para a indução do tumor de rotina, a dose tumorigénica mínima de células em suspensão em 200 uL de meio isento de soro da cultura foi injectado por via subcutânea (sc) no flanco de infecção livre 6-8 semanas de idade, ratinhos fêmea Balb / C ou atímicos MF1-nu/nu n = 6 (Harlan, Oxfordshire, Reino Unido) (1 x 10 6 células 4T1), utilizando uma agulha de seringa de calibre 21. A viabilidade de células utilizadas para inoculação foi superior a 95%, tal como determinado por contagem visual utilizando um hemocitómetro e Exclusão Trypan Blue Dye (Gibco). </li><li> Seguindo estabelecimento do tumor, os tumores foram deixados crescer e desenvolver e foram monitorados duas vezes por semana. O volume do tumor foi calculado de acordo com a fórmula V = (ab 2) Π / 6, em que a é o diâmetro maior do tumor e b é o maior diâmetro perpendicular ao diâmetro a. </li></ol> 2. Preparação bacteriana <ol><li> A estirpe bacteriana utilizada neste protocoEu era E. coli K-12 MG1655, um não-proteína toxina que expressam, estirpe portadora de um plasmídeo que codifica luxABCDE que permite que as bactérias a ser detectada pelo BLI. E. coli MG1655 contendo o luxABCDE integrado foi cultivada aerobicamente a 37 ° C em meio LB (Sigma-Aldrich, Irlanda) suplementado com 300 ug / ml de eritromicina (Em). O derivado de bioluminescente MG1655 foi criada usando o plasmídeo p16S lux que contém a P AJUDA constitutiva luxABCDE operon 1. </li><li> Para a preparação para a administração a ratinhos, as culturas foram incubadas em meio LB a 37 ° C num agitador a 200 rpm a mid-log de fase (densidade óptica a 600 nm). As bactérias foram colhidas por centrifugação (6000 xg durante 5 min), lavadas com PBS (Sigma), diluído em PBS e 1 x 10 7 unidades formadoras de colónias (ufc) / ml para administração IV, ou 1 x 10 10 por meio de sonda. </li></ol> 3. Administração bacterianação <ol><li> Os ratos foram aleatoriamente divididos em grupos experimentais quando os tumores atingiram cerca de 100 mm 3 em volume. Para administração intravenosa, os ratos receberam cada conteve 10 6 células em 100 uL, injectado directamente para dentro da veia lateral da cauda utilizando uma agulha de seringa de 28G. A contagem de viáveis ​​de cada inoculo foi determinada por plaqueamento retrospectiva. </li><li> Para os estudos de colonização GIT, 10 9 células bacterianas foram administradas oralmente em 100 ul por rato por gavagem, em três dias consecutivos. Pré-existentes comensais níveis bacterianos foram reduzidos, antes da alimentação, por adição de 5 mg de estreptomicina / ml, em água potável rato durante 7 dias antes do início de uma sonda oral. </li></ol> 4. Imagem de bioluminescência <ol><li> 2D in vivo BLI imagem foi realizado usando o IVIS100 (Caliper). No tempo definido após a administração pontos bacteriana, os ratos foram anestesiados usando XGI-8 Caliper Anestesia GasSistema com Isofluorano 3%, e em todo o corpo a análise de imagens foi realizada nos 100 IVIS sistema para 2-5 min com uma sensibilidade elevada. </li><li> Para imagens em 3D, os ratos anestesiados foram colocados em um ônibus de imagem do rato dentro do sistema de imagem óptico para imagens dorsal (IVIS Spectrum, Caliper). Para a aquisição de imagens do sinal de luciferase bacteriana para a reconstrução 3D óptica, comprimentos de onda de emissão de filtro que variam 500-580 nm foram utilizados com caixa 16 tempos de aquisição de 3-4 min por filtro para maximizar a relação sinal-ruído. Como parte desta sequência de aquisição de imagem, uma imagem de luz estruturada foi obtida para definir um mapa de altura. Este mapa foi difuso de luz de entrada de imagem de tomografia (DLIT) reconstruções algoritmos que foram utilizados para formar uma imagem 3D óptico usando um não-negativo, pelo menos optimização quadrados 2. </li><li> Análise de Imagem: Regiões de interesse foram identificados e quantificados utilizando software imagem viva (Caliper). </li></ol> 5. RepresentanteResultados Neste estudo, as bactérias não patogénicas de E. coli comensal K-12 MG1655 expressando o operão luxABCDE IV foi administrada a ratos portadores de tumores de xenoenxerto sc 4T1. Bacterial lux sinal foi detectado especificamente em tumores de ratos pós-administração IV (Figura 2). Recuperação da cultura de bactérias a partir de ratinhos de amostra valida a existência de uma relação linear entre o número de bactérias viáveis ​​bem como a quantidade de luz detectada (Figura 3). In vivo imaging-administrados por via oral de bactérias comensais na GIT é também conseguida utilizando 3D BLI. <img src="/files/ftp_upload/4318/4318fig1.jpg" alt="Figura 1" fo:content-width="4in" fo:src="/files/ftp_upload/4318/4318fig1highres.jpg"/> Figura 1. Protocolo Timeline. Tumores subcutâneos são induzidas em ratos, e as bactérias administrado mediante o desenvolvimento de tumores (100 mm 3). Camundongos vivos são BLI imenvelhecido a administração pós vários pontos temporais bacteriana (setas exibir vezes típicas). <img src="/files/ftp_upload/4318/4318fig2.jpg" alt="Figura 2" /> Figura 2. A administração de E. luxABCDE coli MG1655 a ratinhos portadores de tumor. tumores subcutâneos 4T1 foram induzidos em MF1 nu / nu e E. luxABCDE coli MG1655 administrado mediante o desenvolvimento de tumores. Cada animal recebeu 10 6 células injectadas directamente na veia lateral da cauda. Ratos foram fotografadas em quatro momentos durante o estudo (pontos pretos eixo z e imagens) com subsequente recuperação de células viáveis ​​(cfu) de tumores de ratinhos sacrificados amostra (gráfico de barras). Aumento no número de bactérias e expressão gênica plasmídeo especificamente nos tumores foi observada ao longo do tempo (mouse representante ilustrado por ponto de tempo). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/4318/4318fig2large.jpg" target="_blank">Clique aqui para ver maior figura</a> .<img src="/files/ftp_upload/4318/4318fig3.jpg" alt="Figura 3" /> Figura 3. Relação entre intratumoral Números bacteriana e Bioluminescência. Bactérias viáveis ​​em tumores foram enumerados pelo ex vivo cultura bacteriana de tumores após a BLI em vários pontos temporais pós administração IV. Os valores de log de ​​números bacterianas (cfu) em relação a unidades vivo bioluminesce estão representados graficamente. Uma correlação estreita entre as contagens bacterianas e sinais de bioluminescência é observada R 2 = 0,9717 1. <img src="/files/ftp_upload/4318/4318fig4.jpg" alt="Figura 4" /> Figura 4. Imagem 3D IVIS do trato gastrointestinal Murino colonizada por E. coli MG1655. O GIT de ratinhos foi colonizada por administração oral de 10 9 CFU de E. coli durante três dias consecutivos. A imagem de amostra isolada de tomografia 3D do mouse colonizado é mostrado.Imagens em 3D mostram um atlas do mouse digital do esqueleto para fornecer registro anatômico. E. bioluminescência coli MG1655 é visível em verde na inferior, e roxo em níveis mais elevados.

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No contexto da terapia genética, o uso de agentes biológicos para a entrega de genes terapêuticos para pacientes mostrou uma grande promessa 3-5. Como vírus, as propriedades biológicas de bactérias inatas permitir a entrega eficiente do ADN a células ou tecidos, em particular no contexto do cancro. Tem sido demonstrado que as bactérias são naturalmente capazes de homing para tumores quando administrados sistemicamente, resultando em níveis elevados de replicação localmente, quer externas (para não-invasivos espécie) ou no interior das células tumorais (patógenos). Câncer específico de replicação bacteriana foi inicialmente atribuído à natureza hipóxica dos tumores sólidos (baixos níveis de O 2), com a natureza anaeróbia de regiões hipóxicas / necrose nos tumores que promovem o crescimento de bactérias anaeróbicas e anaeróbios facultativos. Mais recentemente, os factores, tais como o fornecimento de sangue irregular, permeável e a supressão imunitária local nos tumores têm sido propostas para desempenhar um papel. A maioria dos estudos pré-clínicos utilizaram xenograft modelos de tumor para investigar a colonização bacteriana tumor vector. Modelos murinos de tumores que surgem espontaneamente mais se assemelham a realidade clínica em termos de vasculatura (um potencial variável de colonização bacteriana de tumores), e as bactérias têm também demonstrado que tais modelos colonizar 6. Vários estudos pré-clínicos e clínicos têm demonstrado a capacidade de diferentes estirpes de bactérias para o transporte e amplificar os genes que codificam factores, tais como pró-fármaco de conversão de enzimas, toxinas, inibidores de angiogénese, e de citoquinas, especificamente em tumores 4,7. Embora a colonização bacteriana tumor tem sido mostrado para ser independente da estirpe bacteriana e do tipo de tumor 6, a escolha da estirpe óptima para um determinado modelo pode variar - por exemplo, anaeróbios estritos podem ser mais adequados para grandes tumores necróticos. Temos concebido um certo número de estirpes para expressar o cassete luxABCDE 1,8-11. O protocolo apresentado no vídeoanimação usa lux-marcado E. coli como um exemplo. E. coli faz parte da flora do GIT humana. Vários estudos têm descrito a segurança de administração IV de não-patogênico E. coli para ratos, e sua capacidade de crescer dentro de tumores especificamente 4,12. E. coli MG1655 (tal como utilizado no presente estudo) também coloniza o GIT rato para níveis elevados 13. O estudo de bactérias em modelos animais de pequeno porte é de grande importância para uma série de campos de pesquisa médica, incluindo doenças infecciosas, saúde intestinal e terapia gênica. Por exemplo, adaptações deste protocolo pode ser aplicado a estudos de acompanhamento de infecção bacteriana, de formação de biofilme, etc Outros genes baseados em sistemas de repórter também foram examinadas, incluindo Topografia de Emissão de Positrões (PET) de digitalização, em combinação com a expressão bacteriana de timidina-quinase (tk ), seja a expressão endógena de E. coli ou S. Typhimurium engineered para expressar o gene tk do vírus Herpes Simplex (HSVtk) 14. Ambos fluorescente (Green Fluorescent Protein e suas variantes) e luminescentes (lux) genes estão disponíveis para as bactérias. Uma vantagem de utilizar a luciferase bacteriana é a de que a cassete de lux codifica as enzimas necessárias para a biosíntese do substrato, resultando em um agente directamente imagable 15. BLI é baseado na detecção de luz bioluminescente do sujeito através da utilização de um dispositivo de acoplamento por carga refrigerado câmara (CCD). A instrumentação relativamente simples e à falta de necessidade de radioactividade coloca a tecnologia bem dentro do alcance do laboratório média. BLI apresenta muitas vantagens quando comparado com o outro em modalidades in vivo. É fácil de utilizar, de baixo custo, rápida e facilita imagiologia de animais simultaneamente, produzindo pouco fundo com alta sensibilidade.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Linha celular 4T1 </td><td> ATCC </td><td> CRL-2539 </td><td> Modelo de cancro da mama singeneico derivado de um tumor espontâneo resultante BALB / c mamária </td></tr><tr><td> DMEM </td><td> Sigma-Aldrich </td><td> D6429 </td><td> Meio de Dulbecco Modificado de Eagle </td></tr><tr><td> PBS </td><td> Sigma-Aldrich </td><td> D8537 </td><td> Solução salina tamponada com fosfato </td></tr><tr><td> Xenogen IVIS </td><td> Caliper Life Sciences </td><td></td><td> IVIS 100 para 2D imagem; IVIS Spectrum para 3D. </td></tr><tr><td> Luria Broth Miller (LB) </td><td> Sigma-Aldrich </td><td> L2542 </td><td> O meio de crescimento para E. coli </td></tr&gt; <tr><td> Eritromicina </td><td> Sigma-Aldrich </td><td> E5389 </td><td> Antibiótico </td></tr><tr><td> Estreptomicina </td><td> Sigma-Aldrich </td><td> S9137 </td><td> Antibiótico </td></tr><tr><td> Camundongos MF1nu/nu </td><td> Harlan (UK) </td><td> 069 (nu) / 070 (nu / +) </td><td> Hsd: atímicos nu-Foxn1nu </td></tr><tr><td> Camundongos Balb / c </td><td> Harlan (UK) </td><td> 066 </td><td> Haplotipo H-2 d </td></tr><tr><td> Agulha de gavagem </td><td> Vet-Tech Solutions (UK) </td><td> DE009 </td><td> 22G x 38mm agulha reta gavagem </td></tr><tr><td> Seringa para injeção IV </td><td> BD BioSciences </td><td> 309309-1 ml </td><td> Seringa de insulina com 28 x ½ polegadas G micro-agulha fina IV. </td></tr><tr><td> Seringa para inoculação do tumor </td><td> Braun </td><td> 9161376V </td><td> Omnifix 26 G x &amp;frac12; agulha polegadas </td></tr></tbody></table>

bioluminescent bacterial imaging in vivo

Este vídeo descreve o uso de todo o corpo bioluminesce imaging (BLI) para o estudo de tráfico bacteriana em ratos vivos, com ênfase no uso de bactérias na terapia génica e celular para o cancro. As bactérias presentes uma classe atraente de vetor para terapia de câncer, possuindo uma capacidade natural de crescer preferencialmente dentro de tumores após a administração sistêmica. As bactérias manipuladas para expressar o gene lux cassete autorização BLI detecção das bactérias e simultaneamente locais tumorais. A localização e níveis de bactérias ao longo do tempo dentro de tumores pode ser facilmente examinado, visualizado em duas ou três dimensões. O método é aplicável a uma ampla variedade de espécies bacterianas e tipos de xenoenxertos de tumor. Este artigo descreve o protocolo para a análise de bactérias bioluminescentes dentro ratinhos portadores de tumores subcutâneos. A visualização das bactérias comensais no tracto gastrointestinal (TGI) por BLI é também descrito. Este poderoso e barato, em tempo real representação estratégia de imagemts um método ideal para o estudo da bactéria in vivo no contexto da pesquisa de cancro, em particular, a terapia génica, e doenças infecciosas. Este vídeo descreve o procedimento para estudar lux-marcado E. coli em murganhos vivos, demonstrando a leitura espacial e temporal BLI alcançável utilizando o sistema IVIS.

Watch this Scientific Journal Video about Bioluminescent Bacterial Imaging In Vivo at JoVE.com

Bioluminescent Bacterial Imaging In Vivo

Este artigo descreve a administração de<em&gt; Lux-marcado</em&gt; Bactérias a ratos e subseqüentes<em&gt; In vivo</em&gt; Análise usando IVIS imagem de bioluminescência.

Imagem bacteriana Bioluminescent<em&gt; Em 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 ...

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Bioluminescent Bacterial Imaging In Vivo

15.1K visualizações.  University College Cork. Este artigo descreve a administração de Lux-marcado Bactérias a ratos e subseqüentes In vivo Análise usando IVIS imagem de bioluminescência.

Vídeo: Bioluminescent Bacterial Imaging In Vivo

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

In vivo de imagens de parasitas Leishmania transgênicas em um host ao vivo

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

Imunologia e Infecção

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Não-invasiva por imagem do Homing leucócitos e Migração In vivo</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Avaliar a resposta estomática ao vivo células bacterianas usando imagens Folha Inteira

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

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.

Usando imagens Bioluminescent para Investigar sinergismo entre Streptococcus pneumoniae E Vírus da Influenza A em Ratos infantil

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

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

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

&quot;Bioluminescent &#39;Phage Reporter para a detecção de patógenos bacterianos Categoria A

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

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

A saliva, glândula salivar, e Coleta de hemolinfa de carrapatos Ixodes scapularis

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

In Vivo Imaging Systems Detecção (IVIS) de um vírus de Neuro-invasiva encefalítico

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

No método de imagem in vivo Distinguir inflamação aguda e crônica

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Geração e Multi-fenotípica rastreio de alto teor de<em&gt; Coxiella burnetii</em&gt; mutantes por transposões

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In Vitro e In Vivo modelo para estudar a adesão bacteriana para o Vaso de parede em condições de escoamento

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...