Nos gustaría dar las gracias a Elisabeth Guillemet, Buisson Christophe y Ludovic Bridoux por su excelente asistencia técnica. Estamos en deuda con Sylvie Salamitou y Fedhila Sinda para la configuración inicial del sistema.

1. La cría de insectos El ciclo completo de huevo a larvas de último estadio dura alrededor de 5 semanas a 25 ° C. Una o dos semanas adicionales son necesarios para obtener las mariposas adultas. <ol><li> Coloque por lo menos 100 pupas o adultos recién fusionada G. mariposas mellonella en un 5-litros de malla de alambre jaula. Mariposas macho medir 10 a 15 mm. La polilla macho adulto es de color beige con luz tenue y marcas oscuras. Mariposas medida Femenino alrededor de 20 mm. Las hembras son más oscuras que los machos con un color marrón / gris. </li><li> Suspender dos paquetes de cuatro capas de papel en la jaula para la puesta de huevos. Después de 2 días, la hembra adulta se ponen los huevos en el borde entre los papeles. </li><li> Dos veces por semana, coloque los paquetes de papel de huevo en las nuevas cajas de crianza de plástico con rejillas para que el aire circule, que contiene polen y cera de abeja. Los huevos eclosionan en unos 3 días y pequeñas larvas comienzan a desarrollarse alimentándose de cera y polen. Alternativamente, larvas se colocan sobre una dieta artificial consisting de una mezcla de miel líquida 500 g, 400 g de glicerina, 100 g de levadura de cerveza seca, harina de trigo 250 g, 200 g de leche en polvo descremada y 400 g polenta. Proporcionar proporción adecuada de alimento por larvas, regularmente separar las larvas en cajas nuevas y reponer la comida cada dos días. </li><li> Las larvas se crían durante las seis etapas del estadio de larva. Cada estadio se caracteriza por el deslizamiento de la cápsula de la cabeza de las larvas. Cuando las larvas alcanzan la última etapa antes de la pupación, dejan de alimentarse y empezar a construir un capullo de seda. </li><li> En sus capullos protectores, las larvas se apoyará y se transforman en pupas. Gusanos de cera permanecerá en la fase de pupa de una a dos semanas para emerger en polillas adultas. Las polillas adultas no beber ni comer. El apareamiento se produce y el ciclo de los gusanos de cera &quot;la vida comienza de nuevo. </li><li> Para estandarizar el ensayo de patología, tomar las larvas durante la etapa larval pasado. Durante esta etapa dejan de alimentarse, moverse a la tapa de la caja de la cría y empezar a producir seda. Este stage dura alrededor de 5 días. </li></ol> 2. Selección de insectos para la infección <ol><li> Seleccione último estadio larvario, que son de 2-3 cm de largo y 180-250 mg de peso, 24 horas antes de las infecciones y los puso en una caja vacía a morir de hambre. </li><li> Quitar el capullo de seda alrededor de la naciente larvas. </li></ol> 3. Preparación bacteriana <ol><li> Preparar suspensiones bacterianas de Bacillus para obtener concentraciones finales que varían desde 10 4 unidades formadoras de colonias (ufc) / ml a 10 8 ufc / ml para inyección intra haemocoelic y de 3x10 6 ufc / ml a 1x10 8 ufc / ml para la ingestión (el ufc para obtener un LD 50 depende de la especie bacteriana). En nuestro caso, B. cereus se cultiva en medio Luria-Bertani caldo (LB, 10 g / l de triptona, 5 g / l de extracto de levadura, 10 g / l de NaCl) a 37 ° C bajo agitación hasta que la entrada en fase estacionaria, correspondiente a la incurvación del crecimiento curva. Mida las OD 600 nm (entre 1 y 2 por B. cereus) y se utilizan para evaluar la concentración bacteriana, utilizando una curva de valoración previamente hecha. Cosecha bacterias por centrifugación a 5.000 xg durante 10 min a temperatura ambiente y se resuspende en tampón fosfato salino (PBS: 1 M KH 2 PO 4, 1M K 2 HPO 4, 5 M NaCl, pH 7,2). Cada larva recibirá 10 l de suspensión bacteriana en la concentración deseada. </li><li> Alternativamente, la suspensión de esporas se pueden preparar para la infección. Obtener esporas cultivando las bacterias en el medio de esporulación HCT (5 g / L de triptona, 2 g / L de hidrolizado de caseína, 12,5 mM K 2 HPO 4, 12,5 mM MgSO 4, 0,05 mM MnSO 4, 1,2 mM de ZnSO 4, 1,2 mM Fe 2 (SO 4) 3, 0,5% H 2 SO 4, 25 mM CaCl 2) 20 a 30 ° C durante 3 días. Esporas cosecha por centrifugación (10.000 xg, 15 min) y lavar dos veces en agua destilada estéril. Resuspender el pel esporasdejar en agua destilada estéril y calor durante 15 min a 78 ° C para eliminar el resto de las bacterias vegetativas. Numerate la suspensión mediante siembra de diluciones seriadas en placas de agar LB. Cada larva recibirá 10 l de suspensión de esporas a la concentración deseada. </li></ol> 4. Preparación de toxina Cry <ol><li> Preparar la toxina Cry1C de la B. thuringiensis cepa 407 transformada con el plásmido 21 pHTF31C llevar el gen que codifica Cry1C. Cultivar la cepa en medio HCT 100 ml a 30 ° C durante 72 horas con antibióticos (eritromicina 10mg/ml) para permitir la esporulación completa, la producción de toxina de cristal y la liberación en el sobrenadante del cultivo. </li><li> Purificar cristales del sobrenadante en un gradiente de sacarosa 72% -79%. En primer lugar añadir 17 ml de 79% de sacarosa en un tubo de 40 ml. Cuidadosamente capa 17 ml de 72% de sacarosa en la parte superior de la sacarosa del 79%. Finalmente, la capa de 5-6 ml del cultivo esporulado concentrado 10 veces y cerrar el tubo. Se centrifuga el tubo durante 14 horas a 20,000 xg, 4 º C, para separar los cristales de las esporas. Recoger los cristales en la interfaz de gradiente. Resuspender el precipitado de cristales en 25 ml de agua estéril fría para lavar. Centrifugar a 8.000 xg durante 20 min. Este paso se repitió tres veces para eliminar todo sacarosa se pegue a los cristales. Resuspender los cristales en 5 ml de agua estéril y observar al microscopio para evaluar la pureza. </li><li> Evaluar la concentración de toxina de proteína por tinción clásica Bradford en solución de cristal presolubilized en 50 mM NaOH. Cristales de toxina puede ser almacenado a -20 ° C hasta su uso. </li></ol> 5. Inyector de Preparación <ol><li> Para controlar el volumen de inyección precisa, utilizar una bomba de jeringa automatizada (por ejemplo, KD Scientific KDS 100) con una velocidad de configuración a 1 l / seg (Figura 1A). </li><li> Llene una jeringa de 1 ml hipodérmica con 300 l de agua o la solución bacteriana (1 jeringa por condición) para la infección de larvas de 20-25. Eliminar las burbujas con cuidado tapping la jeringa, después coloque un 0,45 x 12 mm aguja a la jeringa. </li><li> Coloque la jeringa en el inyector. </li><li> Realizar una inyección en blanco de 10 l en un tubo de vacío para controlar el volumen inyectado. </li></ol> 6. Inyección Intrahaemocoelic insectos <ol><li> Coloque el insecto manualmente entre el pulgar y el índice. A continuación, insertar la aguja fijada en el inyector en la piel larva (cutícula) (Figura 1C). </li><li> Mantenga el insecto en su lugar mientras se inyecta la solución 10 l bacteriana (bacterias o esporas vegetativo). </li><li> Retire con cuidado el insecto de la aguja. </li><li> Coloque el insecto infectado en una pequeña placa de Petri (5 cm de diámetro), 5 larvas por plato. </li><li> Infect al menos 20 larvas para cada condición experimental. </li><li> Colocar las cápsulas a 37 º C en una incubadora durante 48 hr. </li></ol> 7. La alimentación forzada de insectos (Ingestión) <ol><li> En un tubo Eppendorf de 2 ml, mezclar 0,2 g / &amp; mu; l de la toxina Cry1C con cualquiera de suspensión de células o esporas vegetativo para obtener concentraciones finales que van desde 3x10 a 1x10 4 7 por 10 l. </li><li> Llene una jeringa de 1 ml con un 30 G, 25 mm aguja hipodérmica con 300 l de la solución de toxina bacteriana-Cry1C en la infección de larvas de 20-25. Eliminar las burbujas de la jeringa. </li><li> Coloque el insecto manualmente de modo que su boca entra en la aguja fija en el inyector (Figura 1D), y alimentar a la fuerza con 10 l de la solución bacteriana usando la bomba de jeringa automatizada. </li><li> Infect al menos 20 larvas para cada condición experimental. </li><li> Coloque el insecto infectado en un pequeño plato Petri de 5 cm (5 larvas por plato). Colocar las cápsulas a 37 º C en una incubadora durante 48 hr. </li></ol> 8. Grabación de la mortalidad de insectos <ol><li> Después de los experimentos de alimentación forzada o de inyección, mantener las larvas en la placa de Petri sin alimento a 25 ° C o 37 ° C. Compruebe larval mortality regularmente durante un período de 48 horas. Larvas muertas son inertes y por lo general vuelve negro (fig. 1B). </li><li> Los datos de mortalidad se pueden analizar utilizando el programa de log-probit 14. Este programa pone a prueba la linealidad de las curvas de mortalidad de dosis y proporciona dosis letales (LD 50). Alternativamente, otros programas, tales como Prism (Graph Pad), analizando la supervivencia o los datos de mortalidad se puede utilizar. </li></ol>

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El uso de insectos y especialmente la etapa larval, como modelos de infección para los varios patógenos, se está convirtiendo frecuente. Un modelo de elección para algunos aspectos es Drosophila (mosca del modelo) que se utiliza como adultos y 1,2 etapa larval. El insecto lepidóptero G. mellonella también se ha utilizado principalmente para ensayar la virulencia bacteriana por inyección. La ventaja de tolerar temperaturas más altas (por encima de 37 ° C) que en Drosophila (...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> Cera y polen </td><td> La Ruche Roanaise </td><td> 303000 </td><td> Los productores de miel </td></tr><tr><td> Bomba de jeringa automatizada </td><td> KD Scientific </td><td> KDS 100 </td><td></td></tr><tr><td> Jeringa 1 ml </td><td> Terumo </td><td> BS 01T </td><td></td></tr><tr><td> Aguja de 0,45 x 12 mm </td><td> Terumo </td><td> NN 2613R </td><td></td></tr><tr><td> Placa de Petri de 5 cm </td><td> VWR </td><td> 89000-300 </td><td></td></tr><tr><td> Aguja 30G, 25 mm hipodérmica </td><td> Burkard Mfg Co. Ltd. </td><td> PDE0005 </td><td></td></tr></tbody></table>

the insect galleria mellonella as a powerful infection model to investigate bacterial pathogenesis

El estudio de la virulencia bacteriana a menudo requiere un modelo animal adecuado. Modelos de mamíferos de infección son costosos y pueden plantear cuestiones éticas. El uso de insectos como modelos de infección proporciona una alternativa valiosa. En comparación con otros anfitriones modelo no vertebrados tales como los nematodos, insectos tienen un sistema relativamente avanzado de defensas antimicrobianas y por tanto son más propensos a producir información relevante para el proceso de infección de mamífero. Al igual que los mamíferos, los insectos poseen un complejo sistema inmune innato 1. Las células en la hemolinfa son capaces de fagocitar o encapsular los invasores microbianos, y las respuestas humorales incluyen la producción inducible de los péptidos antibacterianos de lisozima y 2,3 pequeño. Además, las analogías se encuentran entre las células epiteliales del intestino medio de larvas de insectos y células intestinales de los sistemas digestivos de mamíferos. Finalmente, varios componentes básicos esenciales para el proceso de infección bacteriana, como la adhesión celular, la resistencia apéptidos antimicrobianos, la degradación del tejido y la adaptación al estrés oxidativo es probable que sean importantes tanto en los insectos y mamíferos 1. Así, los insectos son herramientas polivalentes para la identificación y caracterización de factores de virulencia microbianos implicados en las infecciones de mamíferos. Las larvas de la polilla mayor de la cera Galleria mellonella se ha demostrado que proporcionan una información útil sobre la patogénesis de una amplia gama de infecciones microbianas, incluyendo hongos de mamífero (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) y los patógenos bacterianos, tales como Staphylococcus aureus, Proteus vulgaris , Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes o Enterococcus faecalis 4-7. Independientemente de las especies bacterianas, los resultados obtenidos con larvas de Galleria infectados por inyección directa a través de la cutícula se correlaciona necesariamente con los de Similar estudios de mamíferos: las cepas bacterianas que son atenuados en modelos de mamíferos demostrar menor virulencia en Galleria, y las cepas que causan graves infecciones humanas son también altamente virulento en el modelo Galleria 8-11. Infección oral de la Galleria es mucho menos utilizado y otros compuestos, como toxinas específicas, son necesarios para llegar a la mortalidad. G. mellonella larvas presentes varias ventajas técnicas: son relativamente grandes (larvas de último estadio antes de la pupación son aproximadamente 2 cm de largo y mg de peso 250), lo que permite la inyección de dosis definidas de bacterias, ya que pueden ser criados a diversas temperaturas (20 ° estudios C a 30 ° C) y la infección puede llevarse a cabo entre 15 ° C a 37 ° C por encima de 12,13, lo que permite experimentos que imitan un entorno de mamífero. Además, la cría de insectos es fácil y relativamente barato. La infección de las larvas permite monitorizar la virulencia bacteriana por varios medios, including cálculo de LD 50 14, la medición de la supervivencia bacteriana 15,16 y el examen del proceso de infección 17. Aquí se describe la cría de los insectos, que cubre todas las etapas de vida de G. mellonella. Proporcionamos un protocolo detallado de la infección por dos vías de inoculación: haemocoelic intravaginal. El modelo bacteriano utilizado en este protocolo es Bacillus cereus, un patógeno Gram positivo implicado en gastrointestinal, así como en otras infecciones oportunistas graves local o sistémica 18,19.

Inyección Intra haemocoelic de bacterias en G. mellonella ha demostrado ser muy útil para la identificación de factores de virulencia que se ocupan de los daños y la resistencia a los factores inmunes innatas de varios patógenos humanos. Como ejemplo, la figura 2A representa mortalidad de los insectos después de la inyección de varias dosis de B. bacterias cereus (tipo salvaje y cepas mutantes) 22. Figura 2B representa la supervivencia bacter...

Watch this Scientific Journal Video about The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis at JoVE.com

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

Haemocolic infección intra oral y de las larvas de la polilla mayor de la cera<em&gt; Galleria mellonella</em&gt; Se describe. Este insecto puede ser usado para estudiar los factores de virulencia de entomopatógeno así como mamíferos bacterias oportunistas. La cría de los insectos, los métodos de infección y ejemplos de<em&gt; In vivo</em&gt; Análisis se describen.

El insecto<em&gt; Galleria mellonella</em&gt; Como un modelo de infección poderosa para investigar la patogénesis bacteriana

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

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Research

JoVE Journal

Immunology and Infection

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

31.8K vistas.  INRA, Micalis UMR1319, France. Haemocolic infección intra oral y de las larvas de la polilla mayor de la cera Galleria mellonella Se describe. Este insecto puede ser usado para estudiar los factores de virulencia de entomopatógeno así como mamíferos bacterias oportunistas. La cría de los insectos, los métodos de infección y ejemplos de In vivo Análisis se describen.

Video: The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

En la formación de biofilm in vitro en una diapositiva Cámara de 8 pocillos

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Investigación

Inmunología e Infección

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Luciferasa utilizando a las infecciones bacterianas de la imagen en ratones

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

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

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

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 ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

El uso de<em&gt; Galleria mellonella</em&gt; Como organismo modelo para estudiar<em&gt; Legionella pneumophila</em&gt; Infección

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

Imaging InlC secreción de investigar la infección celular por el patógeno bacteriano<em&gt; Listeria monocytogenes</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac&#160;edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.

Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

El pez cebra como modelo para evaluar el potencial teratogénico de nitrito

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can be utilized as an alternative animal model for the study of virulence, host-pathogen interaction, innate immune response, and drug efficacy. Details for the rearing, care and breeding of the hissing cockroach are provided. We also illustrate how it can be infected with bacteria such as the intracellular pathogens Burkholderia mallei, B. pseudomallei, and B. thailandensis. Use of the hissing cockroach is inexpensive and overcomes regulatory issues dealing with the use of mammals in research. In addition, results found using the hissing cockroach model are reproducible and similar to those obtained using mammalian models. Thus, the Madagascar hissing cockroach represents an attractive surrogate host that should be explored when conducting animal studies.

We present a protocol to utilize the Madagascar hissing cockroach as an alternative non-mammalian animal model to conduct bacterial virulence, pathogenesis, drug toxicity, drug efficacy, and innate immune response studies.

La cucaracha que silba como un modelo alternativo de animales no mamíferos para investigar la eficacia de la droga, virulencia y patogenia de Madagascar

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Un modelo de cultivo primario de células Bursal de pollo Ex Vivo para estudiar la patogenesia de la enfermedad Bursal infecciosa Virus

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease&#39;s pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients.

Establecimiento del modelo de ratón TK-NOG humanizado dual para la patogénesis hepática asociada al VIH

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...

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