Los autores desean agradecer el apoyo pertinente a este manuscrito de la Comisión Europea del Séptimo Programa Marco (PIOF-GA-2009-255.466) y el Consejo de Investigación en Salud de Irlanda (HRA_POR/2010/138). Lux-etiquetados E. coli fue una especie de regalo del doctor Cormac Gahan, University College Cork.

1. La inducción de tumores <ol><li> Para la inducción de tumores de rutina, la dosis tumorígena mínima de células suspendidas en 200 l de medio de cultivo libre de suero se inyecta por vía subcutánea (sc) en el flanco de la infección libre de 6-8 semanas de edad hembras Balb / C o ratones atímicos MF1-nu/nu n = 6 (Harlan, Oxfordshire, Reino Unido) (1 x 10 6 células 4T1) utilizando una aguja de jeringa de calibre 21-. La viabilidad de las células utilizadas para la inoculación fue mayor que 95% según se determinó por recuento visual usando un hemocitómetro y la exclusión del colorante azul de tripano (Gibco). </li><li> Después del establecimiento del tumor, los tumores se dejaron crecer y desarrollar y se monitorizó dos veces semanalmente. El volumen del tumor se calculó según la fórmula V = (ab 2) Π / 6, donde a es el diámetro más largo del tumor y b es el diámetro más largo perpendicular a un diámetro. </li></ol> 2. Preparación bacteriana <ol><li> La cepa bacteriana utilizada en este PROTOCOLO DEl fue E. coli K-12 MG1655, una proteína no-toxina que expresa cepa, que alberga un plásmido luxABCDE-codificación que permite a las bacterias para ser detectada por BLI. E. coli MG1655 que contienen el luxABCDE integrado se cultivó aeróbicamente a 37 ° C en medio LB (Sigma-Aldrich, Irlanda) suplementado con 300 mg / ml de eritromicina (Em). El derivado de MG1655 bioluminiscente se ha creado con el plásmido p16S lux que contiene la AYUDA P constitutiva luxABCDE operón 1. </li><li> Para la preparación para administración a ratones, los cultivos se incubaron en medio LB a 37 ° C en un agitador a 200 rpm hasta la fase logarítmica media-(densidad óptica a 600 nm). Las bacterias fueron cosechadas por centrifugación (6.000 xg durante 5 min), se lavaron con PBS (Sigma), y se diluyó en PBS 1 × 10 7 unidades formadoras de colonias (ufc) / ml para la administración IV, o 1 x 10 10 de alimentación forzada. </li></ol> 3. Bacteriana Administraciónción <ol><li> Los ratones se dividieron aleatoriamente en grupos experimentales cuando los tumores alcanzaron aproximadamente 100 mm 3 en volumen. Para la administración intravenosa, los ratones recibieron cada uno restringidas 10 6 células en 100 l, se inyecta directamente en la vena lateral de la cola usando una aguja de jeringa 28G. El recuento de viables de cada inóculo se determinó mediante siembra retrospectiva. </li><li> Para los estudios de colonización GIT, 10 9 células bacterianas se administra por vía oral en 100 l por ratón por sonda, en tres días consecutivos. Preexistentes niveles de bacterias comensales se redujeron antes de la alimentación mediante la adición de 5 mg / ml de estreptomicina en el agua potable del ratón durante 7 días antes del comienzo de sonda oral 1. </li></ol> 4. Bioluminiscencia de imágenes <ol><li> 2D in vivo BLI imágenes se realizó usando el IVIS100 (pinza). En la administración de tiempo definido después de los puntos bacteriana, los ratones fueron anestesiados utilizando XGI-8 Caliper Anestesia GasSistema con 3% isofluorano, y todo el cuerpo de análisis de imágenes se realizó en el sistema IVIS 100 para 2-5 min a una sensibilidad alta. </li><li> Para imágenes en 3D, los ratones anestesiados se colocaron en un servicio de transporte de imágenes del ratón dentro del sistema de formación de imágenes ópticas para obtener imágenes de dorsal (IVIS Spectrum, Caliper). Para adquirir imágenes de la señal de luciferasa bacteriana para la reconstrucción óptica 3D, longitudes de onda de emisión de filtro que van desde 500 hasta 580 nm se utilizaron con tiempos de adquisición de bin 16 min de 3-4 por filtro para maximizar la relación señal a ruido. Como parte de esta secuencia de adquisición de imágenes, una imagen de luz estructurada se obtuvo para definir un mapa de altura. Este mapa fue difusa luz de entrada de imágenes de tomografía (dlit) reconstrucciones algoritmos que se utilizan para formar una imagen en 3D óptica usando un no negativo mínimo optimización cuadrados 2. </li><li> Análisis de imágenes: Las regiones de interés se identificaron y cuantificaron utilizando el software Image estar (pinza). </li></ol> 5. RepresentanteResultados En este estudio, las bacterias no patógenas comensales E. coli K-12 MG1655 que expresa el operón luxABCDE fue IV administrada a ratones portadores de xenoinjertos de tumores sc 4T1. Señal bacteriana lux se detectó específicamente en los tumores de los ratones después de administración IV-(Figura 2). Recuperación cultivo de bacterias a partir de ratones de muestra valida la existencia de una relación lineal entre el número de bacterias viables y la cantidad de luz detectada (Figura 3). Imágenes in vivo de administración oral las bacterias comensales en el GIT se consigue también el uso de 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 de línea de tiempo. Tumores subcutáneos son inducidos en ratones, y bacterias administrado en el desarrollo del tumor (100 mm 3). Ratones vivos son BLI imaños en la administración de diferentes puntos de tiempo posteriores bacteriana (flechas muestran los tiempos típicos). <img src="/files/ftp_upload/4318/4318fig2.jpg" alt="Figura 2" /> Figura 2. Administración de E. coli MG1655 luxABCDE a ratones portadores de tumor. subcutáneos tumores 4T1 fueron inducidos en MF1 ratones nu / nu y E. coli MG1655 luxABCDE administrado en el desarrollo del tumor. Cada animal recibió 10 6 células inyectadas directamente en la vena lateral de la cola. Los ratones fueron imágenes a cuatro puntos de tiempo durante el estudio (puntos negros eje z e imágenes) con la subsiguiente recuperación de bacterias viables (cfu) de tumores de los ratones sacrificados de muestra (gráfico de barras). Aumento en el número de bacterias y la expresión génica en tumores específicamente plásmido se observó en el tiempo (ratón representante ilustrado por punto de tiempo). <a href="http://www.jove.com/files/ftp_upload/4318/4318fig2large.jpg" target="_blank">Haga clic aquí para ampliar la cifra</a> .<img src="/files/ftp_upload/4318/4318fig3.jpg" alt="Figura 3" /> Figura 3. Relación entre el número de bacterias intratumorales y bioluminiscencia. Bacterias viables en los tumores fueron enumerados por ex cultivo bacteriano vivo de tumores posteriores a BLI en varios puntos de tiempo después de la administración IV. Los valores de log del número de bacterias (ufc) en relación con las unidades in vivo bioluminesce se grafican. Se observó una correlación robusta entre la carga bacteriana y señales bacterianas bioluminiscencia se observa R 2 = 0,9717 1. <img src="/files/ftp_upload/4318/4318fig4.jpg" alt="Figura 4" /> Figura 4. 3D Image IVIS del tracto gastrointestinal murino colonizados por E. coli MG1655. El tracto gastrointestinal de los ratones fue colonizada por la administración oral de 10 9 ufc de E. coli por tres días consecutivos. Una imagen muestra aislada de tomografía 3D del ratón colonizados se muestra.Imágenes en 3D muestran un atlas ratón digital del esqueleto para proporcionar un registro anatómica. E. coli MG1655 bioluminiscencia es visible en verde en la inferior, y púrpura a niveles más altos.

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No hay conflictos de interés declarado.

En el contexto de la terapia génica, la utilización de agentes biológicos para la entrega de genes terapéuticos a los pacientes ha demostrado una gran promesa 3-5. Al igual que los virus, las propiedades biológicas innatas de bacterias permitir el suministro eficiente de ADN a células o tejidos, en particular en el contexto del cáncer. Se ha demostrado que las bacterias son naturalmente capaces de homing a los tumores por administración sistémica que resulta en niveles altos de replicación a nivel l...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Línea celular 4T1 </td><td> ATCC </td><td> CRL-2539 </td><td> Mama singénico modelo de cáncer derivado de un surja espontáneamente BALB / c tumor mamario </td></tr><tr><td> DMEM </td><td> Sigma-Aldrich </td><td> D6429 </td><td> Dulbecco modificada de Eagle </td></tr><tr><td> PBS </td><td> Sigma-Aldrich </td><td> D8537 </td><td> Phosphate Buffered Saline </td></tr><tr><td> Xenogen IVIS </td><td> Caliper Life Sciences </td><td></td><td> IVIS 100 para imágenes 2D; Spectrum IVIS para 3D. </td></tr><tr><td> Caldo Luria Miller (LB) </td><td> Sigma-Aldrich </td><td> L2542 </td><td> Crecimiento medio 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> MF1nu/nu ratones </td><td> Harlan (UK) </td><td> 069 (nu) / 070 (nu / +) </td><td> HSD: atímicos Nude-Foxn1nu </td></tr><tr><td> Ratones Balb / c </td><td> Harlan (UK) </td><td> 066 </td><td> Haplotipo: H-2 d </td></tr><tr><td> Aguja Gavage </td><td> Vet tecnología Solutions (Reino Unido) </td><td> DE009 </td><td> 22G x 38 mm aguja sonda recta </td></tr><tr><td> Jeringa para inyección IV </td><td> BD Biosciences </td><td> 309309 a 1 ml </td><td> La insulina jeringa con 28 G x ½ pulgada micro-fina aguja intravenosa. </td></tr><tr><td> Jeringa para la inoculación del tumor </td><td> Braun </td><td> 9161376V </td><td> Omnifix 26 G x &amp;frac12; aguja pulgadas </td></tr></tbody></table>

bioluminescent bacterial imaging in vivo

Este video describe el uso de todo el cuerpo bioluminesce formación de imágenes (BLI) para el estudio del tráfico bacteriana en ratones vivos, con énfasis en el uso de bacterias en terapia génica y celular para el cáncer. Las bacterias presentes una clase de vector atractivo para la terapia del cáncer, que posee una capacidad natural para crecer preferentemente dentro de los tumores después de la administración sistémica. Las bacterias genéticamente para expresar el gen lux casete permiso BLI detección de las bacterias y al mismo tiempo sitios de tumor. La ubicación y los niveles de las bacterias dentro de los tumores con el tiempo puede ser fácilmente examinado, visualizado en dos o tres dimensiones. El método es aplicable a una amplia gama de especies bacterianas y tipos de tumores de xenoinjertos. En este artículo se describe el protocolo para el análisis de bacterias bioluminiscentes en ratones portadores de tumores subcutáneos. La visualización de las bacterias comensales en el tracto gastrointestinal (GIT) por BLI también se describe. Este potente y barato, imágenes en tiempo real representación estrategiats un método ideal para el estudio de las bacterias in vivo en el contexto de la investigación del cáncer, en terapia génica particular, y las enfermedades infecciosas. Este video muestra el procedimiento para el estudio de lux-etiquetados E. coli en ratones vivos, lo que demuestra la lectura espacial y temporal alcanzable BLI utilizar con el sistema IVIS.

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

Bioluminescent Bacterial Imaging In Vivo

En este artículo se describe la administración de<em&gt; Lux-etiquetado</em&gt; Bacterias a ratones y posteriores<em&gt; In vivo</em&gt; Análisis utilizando imágenes IVIS bioluminiscencia.

Imágenes bacteriana Bioluminiscente<em&gt; En Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Research

JoVE Journal

Immunology and Infection

Bioluminescent Bacterial Imaging In Vivo

15.1K vistas.  University College Cork. En este artículo se describe la administración de Lux-etiquetado Bacterias a ratones y posteriores In vivo Análisis utilizando imágenes IVIS bioluminiscencia.

Video: 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.

Imágenes in vivo de los cultivos transgénicos parásitos Leishmania en un host en vivo

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

Investigación

Inmunología e Infección

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.

De imagen no invasivas de Homing leucocitos y Migración 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.

Evaluación de la respuesta estomática de las células bacterianas en vivo utilizando imágenes de Hoja Entera

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.

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

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

'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;Bioluminiscente &#39;reportero fagos para la detección de patógenos bacterianos de la categoría A

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

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.

La saliva, la glándula salival, y la Colección hemolinfa de las garrapatas 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.

En Vivo Imaging Systems (IVIS) La detección de un virus invasor Neuro-encefálico

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.

In vivo método de imagen para distinguir la inflamación aguda y crónica

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.

Generación y multi-fenotípicos cribado de alto contenido de<em&gt; Coxiella burnetii</em&gt; transposón mutantes

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 estudiar la adhesión bacteriana a la pared del recipiente en condiciones de flujo

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 ...