Damos las gracias a William T. Eckert para narrar el video. Desarrollo del protocolo de lana de vidrio era parte del agua de Wisconsin y de prueba de Riesgos de Salud (entéricos WAHTER Estudio), financiado por la EPA de EE.UU. Star Grant R831630. Alaska muestras fueron recogidas por A. Reeves, Ramey A. y B. Meixell con el apoyo financiero de la USGS. Cualquier uso de nombres comerciales, productos, o de una empresa es para propósitos descriptivos solamente y no implica aprobación por parte del Gobierno de los EE.UU..

1. Preparación de la lana de vidrio <ol><li> Antes y después de hacer cada lote de filtros, esterilizar el área de trabajo con una solución de lejía al 10%. </li><li> Póngase los guantes y la bata. Esterilizar un cubo en autoclave a 121 ° C y 15 psi durante al menos 20 minutos. Colocar la lana de vidrio en la cubeta estéril. </li><li> Saturar la lana de vidrio con agua de ósmosis inversa y deje en remojo durante 15 minutos. </li><li> Vacíe el agua de ósmosis inversa del cubo. </li><li> Saturar la lana de vidrio con HCl 1 M y deje en remojo durante 15 minutos. </li><li> Escurrir el HCl 1 M de la cubeta. </li><li> Lavar la lana de vidrio con agua de ósmosis inversa. </li><li> Mezclar bien. </li><li> Comprobar el pH con papel pH y repetir el agua de ósmosis inversa enjuague hasta un pH neutro se consigue. </li><li> Vierta el agua de enjuague. </li><li> Saturar la lana de vidrio con 1 M NaOH y deje en remojo durante 15 minutos. </li><li> Escurrir el NaOH 1 M de la cubeta. </li><li> Repita la ósmosis inversa enjuague hasta un pH neutro se consigue. </li><li> Vierta el agua de enjuague. </li><li> En lugar de un cubo, una arandela de lana de vidrio puede ser construida, que es similar en diseño a una lavadora de vidrio pipeta (Figura 1). </li><li> Cubrir la lana de vidrio completamente con fosfato estéril salino tamponado (PBS) se ajustó a pH 6,8. </li><li> El uso de lana de vidrio preparada inmediatamente o se almacena a 4 º C. Se puede almacenar por hasta dos semanas. Antes de utilizarlo, asegúrese de que el pH es neutro, ya que se incrementará con el tiempo. Si el pH no es neutral, vuelva a enjuagar con solución salina estéril tamponada de fosfato (pH 6,8). </li></ol> 2. Montaje del filtro de lana de vidrio <ol><li> Haga un agujero 11/16 pulgadas en las tapas de PVC y los hilos grifo por lo que los accesorios de adaptador macho de nylon pueden ser atornillados en las tapas. Este paso es necesario sólo para la primera asamblea. Posteriormente, las tapas pueden ser utilizados para años. Aplique teflónla cinta en las roscas de los tornillos y herrajes de nylon a las tapas. </li><li> Paquete de la tubería de PVC con pequeños trozos de lana de vidrio. Use un émbolo metálico, como una válvula de motor de un coche, para empacar apretado. Embalaje no requiere mucha fuerza. Pack suficientemente ajustado para que la lana de vidrio se mantiene en su lugar y no forman canales. Sin embargo, no se empaquetan tan fuertemente el agua no puede fluir a través del filtro. Cuando embalado apropiadamente, con un caudal de 4 a 5 litros por minuto debe ser alcanzable cuando el filtro está unido a los grifos de agua con presiones entre 40-60 psi. Por el tamaño de tubo de PVC se especifica en este protocolo, aproximadamente 85 gramos de lana lavada y lleno de vidrio se utiliza por tubo. Tara el vacío tubería de PVC en un equilibrio de carga superior y un paquete de lavado de lana de vidrio hasta que la masa aumenta tubo de 85 gramos. </li><li> Inserte la malla de polipropileno en las tapas de PVC con accesorios masculinos del adaptador de nylon atadas. </li><li> Aplique cinta de teflón a las partes roscadas de la tubería de PVC. </li><li> Tapas de rosca de PVC a la página de PVCIPE y la etiqueta de un extremo del filtro del flujo de entrada y el otro extremo de la salida. Esto es importante para la posterior etapa de elución. Lo cual se denomina entrada no importa. </li><li> Empuje 60 ml de solución salina estéril tamponada de fosfato (pH = 6,8) en el filtro utilizando un catéter con punta de jeringa. El exceso va a salir el extremo opuesto. </li><li> Envuelva bien termina con Parafilm para evitar fugas. Los filtros pueden ser almacenados hasta por 30 días a 4 ° C. </li></ol> 3. Muestreo <ol><li> Esterilizar tubería utilizada para el muestreo por recirculación o sumergiendo los elementos durante 30 minutos en% NaClO 0,525 (es decir, una solución al 10% de cloro de uso doméstico estándar). Sigue este drenando la solución de hipoclorito y neutralizar con tiosulfato de sodio al 0,05%, fabricado por la adición de 25 ml de un stock de 2% anhidro solución de tiosulfato de sodio a un litro de agua por ósmosis inversa. </li><li> Para la muestra de agua con un pH superior a 7,5, ajustar el pH entre 6,5 y 7,0 con HCl. Concentración de HCl puede variar desde 0,25M a 1 M. Cuatro litros HCl 0,5 M es generalmente suficiente para dos muestras de 800 litros, dependiendo del pH del ambiente del agua y la capacidad de almacenamiento en búfer. Inyectar HCl durante el muestreo utilizando una bomba peristáltica o venturi (Figura 2). Ajustar la velocidad de la bomba o abertura Venturi para lograr el pH deseado. Medir el pH a la salida de línea de la muestra utilizando un medidor de pH campo. </li><li> Utilice un filtro previo si se bloquea la lana de vidrio de filtro de agua con alta turbidez. (Las especificaciones para el prefiltro y su alojamiento están en la lista de materiales en línea.) Debido a que los patógenos se puede unir a las partículas atrapadas por el prefiltro debe eluyó junto con el filtro de lana de vidrio (véase más adelante). </li><li> Ajustar el flujo a entre 2 y 4 litros por minuto. Generalmente, los volúmenes de la muestra son entre 200 y 1.500 litros. </li><li> Desconecte el filtro de lana de vidrio y guardar en una bolsa de plástico estéril a 4 ° C durante un máximo de 48 horas. </li></ol> 4. Elución <ol><li> Coloque el filtro de lana de vidrio para un anilloreposar con el extremo afluencia muestra apuntando hacia abajo en una botella de polipropileno. Eluir opuesto de la dirección de flujo de la muestra. </li><li> Empuje 80 ml de extracto de carne de vaca estéril 3% en 0,05 M de glicina con un pH de 9,5 en el filtro. </li><li> Espere 15 minutos. </li><li> Empujar otro 80 ml de extracto de carne de vaca estéril 3% en 0,05 M de glicina con un pH de 9,5 en el filtro. </li><li> Empujar aire a fondo el filtro hasta que la espuma que sale de la entrada del filtro. </li><li> Ajustar el pH a eluido entre 7,0 y 7,5 con HCl 1 M. </li><li> Tienda eluato a 4 ° C durante hasta 24 horas o menos -20 ° C durante períodos más largos de tiempo. </li><li> Eluir el prefiltro si se usa uno. Desatornille la parte superior del cartucho de la vivienda y se vierte toda el agua residual en el interior de la vivienda mientras se mantiene el pre-filtro en su lugar con las manos enguantadas estériles. </li><li> Quite el filtro previo del cartucho de la vivienda y el deslizamiento en una de 15 &quot;x 6&quot; bolsa zip-lock. </li><li> Verter 200 ml de extracto de carne de vaca estéril 3% en 0,05 M glicina con un pH de 9,5 en la bolsa y sellar con seguridad con el cierre de cremallera. </li><li> Invertir la bolsa prefiltro varias veces para asegurar toda la superficie entra en contacto con el extracto de carne. </li><li> Espere 15 minutos, invirtiendo en ocasiones y el masaje del prefiltro en sacos. </li><li> Abra el zip-lock. Adherencia de la bolsa alrededor del prefiltro herméticamente para exprimir como extracto de carne tanto como sea posible y tire el prefiltro con estériles manos enguantadas,. </li><li> Verter el eluato extracto de carne en una botella de polipropileno. </li><li> Ajustar el pH a eluido entre 7,0 y 7,5 con HCl 1M. </li><li> Tienda eluato a 4 ° C durante hasta 24 horas o menos -20 ° C durante períodos más largos de tiempo. El eluato prefiltro puede ser analizado por separado para los agentes patógenos o combinado con el filtro de vidrio eluato lana. </li></ol> 5. Los resultados representativos <table border="1"><tbody><tr><td> Patógeno </td><td> Nivel de agua de turbidez (NTU) una </td><td> Cantidad Sembrado / L b, c, d </td><td> Recuperación% ± 1 </td><td> Número de ensayos independientes </td></tr><tr><td> Enterovirus-poliovirus Sabin III </td><td> 0.5 </td><td> 500 </td><td> 81% ± 11 </td><td> 7 </td></tr><tr><td> Enterovirus poliovirus Sabin III </td><td> 0.5 </td><td> 5000 </td><td> 67% ± 12 </td><td> 8 </td></tr><tr><td> Enterovirus-poliovirus Sabin III </td><td> 215 </td><td> 500 </td><td> 59% ± 32 </td><td> 7 </td></tr><tr><td> Enterovirus-poliovirus Sabin III </td><td> 215 </td><td> 5000 </td><td> 38% ± 22 </td><td> 6 </td></tr><tr><td> Enterovirus-poliovirus Sabin III </td><td> 447 </td><td> 500 </td><td> 56% ± 18 </td><td> 8 </td></tr><tr><td> Enterovirus-poliovirus Sabin III </td><td> 447 </td><td> 5000 </td><td> 63% ± 37 </td><td> 8 </td></tr><tr><td> Cryptosporidium parvum </td><td> 0.5 </td><td> 5 </td><td> 38% ± 14 </td><td> 7 </td></tr><tr><td> Cryptosporidium parvum </td><td> 0.5 </td><td> 50 </td><td> 53% ± 19 </td><td> 8 </td></tr><tr><td> Llorarptosporidium parvum </td><td> 215 </td><td> 5 </td><td> 40% ± 16 </td><td> 7 </td></tr><tr><td> Cryptosporidium parvum </td><td> 215 </td><td> 50 </td><td> 30% ± 6 </td><td> 6 </td></tr><tr><td> Cryptosporidium parvum </td><td> 447 </td><td> 5 </td><td> 33% ± 13 </td><td> 8 </td></tr><tr><td> Cryptosporidium parvum </td><td> 447 </td><td> 50 </td><td> 28% ± 11 </td><td> 8 </td></tr><tr><td> Salmonella enterica </td><td> 0.5 </td><td> 5 </td><td> 29% ± 24 </td><td> 7 </td></tr><tr><td> Salmonella enterica </td><td> 0.5 </td><td> 500 </td><td> 56% ± 16 </td><td> 8 </td></tr><tr><td> Salmonella enterica </td><td> 215 </td><td> 5 </td><td> 32% ± 24 </td><td> 7 </td></tr><tr><td> Salmonella enterica </td&gt; <td> 215 </td><td> 500 </td><td> 34% ± 11 </td><td> 6 </td></tr><tr><td> Salmonella enterica </td><td> 447 </td><td> 5 </td><td> 34% ± 18 </td><td> 8 </td></tr><tr><td> Salmonella enterica </td><td> 447 </td><td> 500 </td><td> 31% ± 24 </td><td> 8 </td></tr></tbody></table><ol class="lalpha"><li> Unidades de Turbidez Nefelométrica </li><li> Los enterovirus se enumeran por qPCR como copias genómicas / L </li><li> C. parvum por inmunofluorescencia como se enumeran los ooquistes </li><li> S. enterica dividido por la cultura como formadoras de colonias de unidades </li></ol> Tabla 1. Vidrio concentración de lana con turbidez del agua y densidades diferentes patógenos. <table border="1"><tbody><tr><td> Patógeno </td><td> Localización de la Muestra de agua </td><td> Cantidad Granados / L a </td><td> Reco%<html> muy </td></tr><tr><td> La gripe aviar H5N2 </td><td> Sundi Lago, Municipio de Anchorage </td><td> 2500 </td><td> 42,9% </td></tr><tr><td> La gripe aviar H5N2 </td><td> Minto Pisos, Fairbanks North Star Borough </td><td> 2500 </td><td> 36,7% </td></tr><tr><td> La gripe aviar H5N2 </td><td> Portage Valley, condado de Anchorage </td><td> 2500 </td><td> 7,8% </td></tr><tr><td> La gripe aviar H5N2 </td><td> Potter Marsh, del condado de Anchorage </td><td> 2500 </td><td> 41,5% </td></tr><tr><td> La gripe aviar H5N2 </td><td> Willow Lake, Yukon-Koyukuk Municipio </td><td> 2500 </td><td> 15,5% </td></tr></tbody></table><ol class="lalpha"><li> Medido por qPCR como copias genómicas / L </li></ol> Tabla 2. La concentración de lana de vidrio de los virus de la gripe aviar, utilizando el agua de cinco sitios en Alaska. les/ftp_upload/3930/3930fig1.jpg &quot;alt =&quot; Figura 1 &quot;/&gt; Figura 1. Diagrama de la lavadora de lana de vidrio. Esto puede ser usado en lugar de un cubo, ahorrando tiempo de aclarado. El concepto es similar a una lavadora pipeta de vidrio. <img src="/files/ftp_upload/3930/3930fig2.jpg" alt="Figura 2" /> Figura 2. Vidrio filtración lana con inyección de ácido por la bomba peristáltica. Tenga en cuenta la &quot;T&quot; donde la tubería de la bomba introduce ácido en la línea de muestreo entre el grifo del agua y el filtro de lana de vidrio. el ajuste del pH es necesaria sólo si la muestra de agua tiene un pH&gt; 7,5. Filtros de lana de vidrio son eficaces en la concentración de agentes patógenos de agua con una amplia gama de niveles de turbidez y densidades de patógenos (Tabla 1). Para probar esta, 20 litros de agua del grifo sin cloro se mezcló con limo seco suelo franco (0, 1,27, o 2,75 g / l) para alcanzar el nivel deseado de turbidez y luego se siembra con densidades diferentes agentes patógenos. El aguase pasó a través de un filtro de lana de vidrio, los patógenos concentrados en el eluato se enumeraron, y este valor era el numerador en el cálculo de recuperación por ciento. La cantidad de agentes patógenos sembradas en el agua, es decir, el denominador del cálculo porcentaje de recuperación, se determinó por primera siembra los patógenos en un eluato negativo, entonces la enumeración de los patógenos. El eluato negativo se preparó haciendo pasar una cabeza de serie 20 litros de muestra a través de un filtro y eluyendo. La cuantificación de los patógenos sembradas en un eluato negativo evita diferencias en la enumeración de patógeno que podría derivarse de diferencias de la matriz creada por el filtro de lana de vidrio. La importancia de esta etapa cuando la cuantificación de los patógenos por qPCR se discute en Lambertini et al. 3. Una muestra de 20 litros de control negativo se concentró por filtración de lana de vidrio para determinar que no eran patógenos presentes nativo que podrían confundir el cálculo de porcentaje de recuperación. Un 10 micras de tamaño de poro nominal prefiltro wtal como se utiliza cuando el nivel de turbidez fue ≥ 215 NTU. Poliovirus se cuantificó por PCR cuantitativa en tiempo real utilizando la concentración de secundaria y los procedimientos de extracción de ácidos nucleicos y los cebadores y la sonda se describe en Lambertini et al. 3. Cryptosporidium parvum se cuantificó en el volumen final de la muestra concentrada (FCSV) creado por el procedimiento de concentración secundaria de los poliovirus . Los ooquistes fueron visualizados por inmunofluorescencia (Cryptosporidium y Giardia MERIFLUOR Kit de detección, Meridian Life Science, Inc., Cincinnati, OH). Salmonella enterica se cuantificó en el FCSV en placas en agar XLD (Remel, Lenexa, KS) y contando con la formación de colonias- unidades. Filtros de lana de vidrio son eficaces en la concentración de virus de influenza aviar (Tabla 2). Virus de baja patogenicidad de la influenza aviar (H5N2) se sembró en el agua de varias localidades de Alaska y el porcentaje de recuperación calculado como se describe Above. Concentración secundaria y procedimientos de ácidos nucleicos se realizaron como para el poliovirus, el virus se cuantificó por qPCR utilizando los cebadores y sondas descritas en Spackman et al 4.

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Filtros de lana de vidrio han sido utilizados por diversos equipos de investigación 3,5,6 para concentrar virus entéricos humanos a partir de una variedad de fuentes de agua tales como agua potable terminado 7, el agua subterránea 8,9, el agua superficial 10, 11 de agua de mar, las aguas residuales 12, y escorrentía agrícola 13. Aquí mostramos los filtros también son eficaces en la concentración de virus de influenza aviar, así como los agentes p...

<table border="1"><tbody><tr><td> Nombre de reactivo o elemento </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Ácido clorhídrico </td><td> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific</a> </td><td> A144-500 </td></tr><tr><td height="24"> Hidróxido de sodio </td><td> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific</a> </td><td> BP359-212 </td></tr><tr><td> Tampón fosfato salino Cloruro de sodio Fosfato potásico dibásico- Fosfato potásico monobásico- </td><td> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific</a> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific</a> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific</a> </td><td><html> BP358-212 BP363-500 BP362-500 </td></tr><tr><td> El hipoclorito de sodio, es decir, cloro de uso doméstico </td><td> <a href="http://www.clorox.com" target="_blank">La Compañía Clorox</a> </td><td></td></tr><tr><td> El tiosulfato de sodio, anhidro </td><td> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific</a> </td><td> S 475-212 </td></tr><tr><td> Extracto de carne, desecado </td><td> <a href="http://www.bd.com" target="_blank">Becton, Dickinson and Company</a> </td><td> 211520 </td></tr><tr><td> Glicina </td><td> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific</a> </td><td> G46-500 </td></tr><tr><td> Lana de vidrio engrasado sodocalcic O R-11 de fibra de vidrio aislamiento no revestido </td><td> <a href="http://www.isover.com" target="_blank">Isover</a> <html> ref = &quot;http://www.jm.com&quot; target = &quot;_blank&quot;&gt; Johns Manville </td><td> Bourre 725 QN </td></tr><tr><td> Malla de polipropileno </td><td> <a href="http://www.industrialnetting.com" target="_blank">Redes Industriales</a> </td><td> xN4510 </td></tr><tr><td> 2 &quot;x 4&quot; PVC Sch 80 niple roscado </td><td> <a href="http://www.grainger.com" target="_blank">Grainger</a> </td><td> 6MW35 </td></tr><tr><td> 2 &quot;cédula 40 de PVC tapa </td><td> <a href="http://www.grainger.com" target="_blank">Grainger</a> </td><td> 5WDW3 </td></tr><tr><td> Adaptador macho de nylon (1/2 &quot;x1 / 2&quot;) </td><td> <a href="http://www.usplastic.com" target="_blank">EE.UU. Plastic Corp.</a> </td><td> 62178 </td></tr><tr><td> Recipientes de las muestras de efluente de 1 litro </td><td> <a href="http://www.fishersci.com" target="_blank">Fisher Scientific</a> </td><td> 03 a 313-4F </td></tr><tr><td> 60 ml jeringa </td><td>&quot;Http://www.fishersci.com&quot; target = &quot;_blank&quot;&gt; Fisher Scientific </td><td> NC9661991 </td></tr><tr><td> tiras de pH </td><td> <a href="http://www.whatman.com" target="_blank">Whatman</a> </td><td> 2614 991 </td></tr><tr><td> Prefiltro, polipropileno, 10 cartuchos pulgadas, 10 m </td><td> McMaster-Carr </td><td> 4411K75 </td></tr><tr><td> Prefiltro de vivienda </td><td> Cole-Parmer </td><td> S-29820-10 </td></tr></tbody></table>

glass wool filters for concentrating waterborne viruses and agricultural zoonotic pathogens

El primer paso clave en la evaluación de los niveles de patógenos en el agua contaminada se sospecha es la concentración. Los métodos de concentración tienden a ser específicas para un grupo determinado patógeno, por ejemplo Protección Ambiental de EE.UU. Agencia Método 1623 para Giardia y Cryptosporidium 1, lo que significa que múltiples métodos son necesarios si el programa de muestreo se dirige a más de un grupo de patógenos. Otro inconveniente de los métodos actuales es el equipo puede ser complicado y caro, por ejemplo el método VIRADEL con el filtro de cartucho 1MDS para concentrar los virus 2. En este artículo se describe cómo construir filtros de lana de vidrio para la concentración de patógenos en el agua. Después de la elución de filtro, el concentrado es susceptible de una segunda etapa de concentración, tales como centrifugación, seguido por la detección de patógenos y la enumeración de los métodos de cultivo o molecular. Los filtros tienen varias ventajas. La construcción es sencilla y los filtros se pueden construir a unny el tamaño para satisfacer las necesidades específicas de muestreo. Las partes del filtro son baratos, por lo que es posible recoger un gran número de muestras sin afectar gravemente un presupuesto del proyecto. Los grandes volúmenes de muestra (100s a 1.000 s L) se puede concentrar en función de la tasa de obstrucción de turbidez de la muestra. Los filtros son fáciles de transportar y con un equipo mínimo, como una bomba y un medidor de flujo, se pueden implementar en el campo para el muestreo de agua potable final, las aguas superficiales, aguas subterráneas, y la escorrentía agrícola. Por último, la filtración de lana de vidrio es eficaz para concentrar una variedad de tipos de patógenos tan sólo un método es necesario. Aquí nos informe sobre la eficacia del filtro en la concentración de enterovirus humanos transmitidas por el agua, S ALMONELLA enterica, Cryptosporidium parvum, y el virus de la gripe aviar.

Watch this Scientific Journal Video about Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens at JoVE.com

Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens

Filtros de lana de vidrio se han utilizado para concentrar los virus transmitidos por el agua por un número de grupos de investigación en todo el mundo. Aquí se muestra un método sencillo para la construcción de filtros de lana de vidrio y demostrar los filtros también son eficaces en la concentración de patógenos transmitidos por el agua virus, bacterias y protozoos.

Los filtros de lana de vidrio para la concentración de virus transmitidas por el agua y la agricultura patógenos zoonóticos

The key first step in evaluating pathogen levels in suspected contaminated water is concentration.  Concentration methods tend to be specific for a ...

glass-wool-filters-for-concentrating-waterborne-viruses-agricultural

Research

JoVE Journal

Immunology and Infection

17.9K vistas.  United States Geological Survey. Filtros de lana de vidrio se han utilizado para concentrar los virus transmitidos por el agua por un número de grupos de investigación en todo el mundo. Aquí se muestra un método sencillo para la construcción de filtros de lana de vidrio y demostrar los filtros también son eficaces en la concentración de patógenos transmitidos por el agua virus, bacterias y protozoos.

Video: Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens

Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1. A tick-borne murine model 2 has been developed to study Lyme disease in the laboratory. While na&iacute;ve ticks can be infected with B. burgdorferi by feeding them on infected mice, the molting process takes several weeks to months to complete. Therefore, development of more rapid and efficient tick infection techniques, such as a microinjection-based procedure, is an important tool for the study of Lyme disease 3,4. The procedure requires only hours to generate infected ticks and allows control over the delivery of equal quantities of spirochetes in a cohort of ticks. This is particularly important as the generation of B. burgdorferi infected ticks by the natural feeding process using mice fails to ensure 100% infection rate and potentially results in variation of pathogen burden amongst fed ticks. Furthermore, microinjection can be used to infect ticks with B. burgdorferi isolates in cases where an attenuated strain is unable to establish infection in mice and thus can not be naturally acquired by ticks 5. This technique can also be used to deliver a variety of other biological materials into ticks, for example, specific antibodies or double stranded RNA 6. In this article, we will demonstrate the microinjection of nymphal ticks with in vitro-grown B. burgdorferi. We will also describe a method for localization of Lyme disease pathogens in the tick gut using confocal immunofluorescence microscopy.

Lyme disease research studies often require generation of ticks infected with the pathogen Borrelia burgdorferi, a process that typically takes several weeks. Here we demonstrate a microinjection-based tick infection procedure that can be accomplished within hours. We also demonstrate an immunofluorescence method for in situ localization of B. burgdorferi within ticks.

Métodos para la transferencia rápida y localización de la enfermedad de Lyme patógenos en el intestino garrapatas

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1.  ...

Investigación

Inmunología e Infección

'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.

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

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. ...

Avian Influenza Surveillance with FTA  Cards: Field Methods, Biosafety, and Transportation Issues Solved

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral subtypes2. Human pandemics of flu originally stem from AIVs3. Many fatal human cases during the H5N1 outbreaks in recent years were reported. Lately, a new AIV related strain swept through the human population, causing the 'swine flu epidemic'4. Although human trading and transportation activity seems to be responsible for the spread of highly pathogenic strains5, dispersal can also partly be attributed to wild birds6, 7. However, the actual reservoir of all AIV strains is wild birds.
In reaction to this and in face of severe commercial losses in the poultry industry, large surveillance programs have been implemented globally to collect information on the ecology of AIVs, and to install early warning systems to detect certain highly pathogenic strains8-12. Traditional virological methods require viruses to be intact and cultivated before analysis. This necessitates strict cold chains with deep freezers and heavy biosafety procedures to be in place during transport. Long-term surveillance is therefore usually restricted to a few field stations close to well equipped laboratories. Remote areas cannot be sampled unless logistically cumbersome procedures are implemented. These problems have been recognised13, 14 and the use of alternative storage and transport strategies investigated (alcohols or guanidine)15-17. Recently, Kraus et al.18 introduced a method to collect, store and transport AIV samples, based on a special filter paper. FTA cards19 preserve RNA on a dry storage basis20 and render pathogens inactive upon contact21. This study showed that FTA cards can be used to detect AIV RNA in reverse-transcription PCR and that the resulting cDNA could be sequenced and virus genes and determined.
In the study of Kraus et al.18 a laboratory isolate of AIV was used, and samples were handled individually. In the extension presented here, faecal samples from wild birds from the duck trap at the Ottenby Bird Observatory (SE Sweden) were tested directly to illustrate the usefulness of the methods under field conditions. Catching of ducks and sample collection by cloacal swabs is demonstrated. The current protocol includes up-scaling of the work flow from single tube handling to a 96-well design. Although less sensitive than the traditional methods, the method of FTA cards provides an excellent supplement to large surveillance schemes. It allows collection and analysis of samples from anywhere in the world, without the need to maintaining a cool chain or safety regulations with respect to shipping of hazardous reagents, such as alcohol or guanidine.

A method to preserve, detect and sequence RNA from Avian Influenza Viruses was validated and extended using natural faecal samples from birds. This technique removes the necessity of maintaining a cool chain and handling of infectious viruses and can be applied in a 96-well high-throughput setup.

Vigilancia de la Gripe Aviar, con tarjetas FTA: Métodos de Campo, la bioseguridad y los problemas de transporte resueltos

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral ...

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Detección del virus infeccioso de campo recogidos por los mosquitos del ensayo cultivo celular Vero

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

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 ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

El establecimiento de un cultivo líquido cubierto de las vías respiratorias humanas epiteliales polarizadas Calu-3 células para estudiar la respuesta del huésped celular a patógenos respiratorios<em&gt; In vitro</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

Screening de alto rendimiento para los de amplio espectro químico Inhibidores de Virus ARN

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Administración intranasal de vacunas contra la influenza recombinante en modelos de ratón quimérico para el Estudio de la mucosa Inmunidad

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging techniques require propagation of light through glass, but on glass macrophage fusion is a rare event. This protocol presents the fabrication of several optical-quality glass surfaces where adsorption of compounds containing long-chain hydrocarbons transforms glass into a fusogenic surface. First, preparation of clean glass surfaces as starting material for surface modification is described. Second, a method is provided for the adsorption of compounds containing long-chain hydrocarbons to convert non-fusogenic glass into a fusogenic substrate. Third, this protocol describes fabrication of surface micropatterns that promote a high degree of spatiotemporal control over MGC formation. Finally, fabricating glass bottom dishes is described. Examples of use of this in vitro cell system as a model to study macrophage fusion and MGC formation are shown.

This protocol describes the fabrication of optical-quality glass surfaces adsorbed with compounds containing long-chain hydrocarbons that can be used to monitor macrophage fusion of living specimens and enables super-resolution microscopy of fixed specimens.

Fabricación de superficies de cristal de calidad óptica para estudiar la fusión de macrófagos

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging ...

Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. While the direct fluorescent antibody (DFA) test or the direct rapid immunohistochemical test (DRIT) are most commonly utilized for the antigen detection, both tests require fresh and/or frozen tissues for impressions on slides prior to the antigen detection using antibodies. If samples are collected and fixed in formalin, neither test is optimal for the antigen detection, however, testing can be performed by conventional immunohistochemistry (IHC) after embedding in paraffin blocks and sectioning. With this IHC method, tissues are stained with anti-rabies antibodies, sections are deparaffinized, antigen retrieved by partial proteolysis or other methods, and incubated with primary and secondary antibodies. Antigens are stained using horseradish peroxidase / amino ethyl carbazole and counterstained with hematoxylin for the visualization using a light microscope. In addition to the specific antigen detection, formalin fixation offers other advantages like the determination of histological changes, relaxed conditions for specimen storage and transport (under ambient temperatures), ability to test retrospective cases and improved biological safety through the inactivation of infectious agents.

Here, we present an immunohistochemistry test protocol for the detection of rabies virus antigen as an alternative diagnostic test for formalin-fixed tissues.

Prueba de inmunohistoquímica para la detección de antígenos de lisavirus en tejidos fijos de formalina

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. ...