Financiado en parte por subvenciones de NIH RO1AI015503. Ratones infectados de schistosome fueron proporcionados por del NIAID esquistosomiasis Resource Center en el Instituto de investigaciones biomédicas (Rockville, MD) a través de NIH NIAID contrato HHSN272201000005I para la distribución a través de recursos del BEI.

Todos los procedimientos experimentales y cuidado de los animales fueron aprobados por el Comité de uso de la Universidad de Wisconsin-Madison bajo protocolo y atención institucional del Animal no. V005717. El siguiente protocolo consiste en trabajar en un centro de bioseguridad nivel 2 (BSL2) para patógenos humanos, aunque ninguna de las etapas de esquistosoma, representadas en este protocolo son infecciosas para los seres humanos o de otros mamíferos. Seguir las políticas institucionales para el manejo de grupo de riesgo 2 (RG2) patógenos humanos.
Nota: Utilizamos mujer ratones Swiss-Webster (6 - semanas de edad) debido a su mayor susceptibilidad a la infección10, produciendo así mayores cargas de huevo. Tucker et al. , describen los protocolos de infección del ratón y caracol utilizados en este modelo de sistema11. La Tabla de materiales enumera todos los equipos específicos, materiales, reactivos y fuentes animales para llevar a cabo este protocolo experimental.
1. proceso de hígados infectados
<ol><li>Eutanasia a ratones, post-infección 6-7 semanas, por asfixia de CO2 (tasa de 10-30% del volumen de la cámara por minuto). Ratones de monitor para el cese de la respiración y del latido del corazón. Nota: Por lo general, a 20 ratones pueden procesarse en un momento dado.</li>
	<li>Después de la transferencia sacrificaron ratones a un gabinete de bioseguridad, poner ratones en sus espaldas y rocíe las superficies ventrales con etanol al 70%. Remojar durante 1 minuto.</li>
	<li>Apriete y tire hacia arriba la piel del abdomen inferior y en toda la base de la piel que mantiene pellizcada más cercana al abdomen con tijeras quirúrgicas estériles. Tire la piel corte anterior (es decir, hacia la cabeza) para revelar el hígado. Tenga en cuenta que el hígado agrandado y manchado debido a una respuesta granulomatosa inducida por huevo (figura 2A).</li>
	<li>Retirar el hígado y colocarlo en un vaso que contiene 200 mL de estéril solución de NaCl del 1,2% que contiene pluma/strep.</li>
	<li>Repita los pasos 1.3 y 1.4 con los ratones restantes. Nota: Veinte ratones generalmente se procesan en una sola cosecha.</li>
	<li>Coloque los hígados en una placa Petri estéril y retirar tejidos de hígado sin grasa/conectivos con pinzas estériles.</li>
	<li>Transferencia ajustado/limpiar hígado a una botella de centrífuga estériles de 250 mL que contiene ~ 200 mL de solución estéril de NaCl de 1.2% con pluma/strep.</li>
	<li>Enérgicamente sacudir la botella que contiene el hígado y, con una pipeta desechable estéril, retire la superficie excesiva espuma/flotación ruina. Poco a poco retirar la solución salina restante en un recipiente o fregadero que contiene 1% cloro (desinfectante).</li>
	<li>Añadir ~ 200 mL de solución salina fresca a los hígados y repita el paso 1.8 tres veces más.</li>
</ol>2. hígado extracción y miracidial aislamiento
<ol><li>Coloque los hígados en una taza pequeña batidora de acero inoxidable estéril y añadir 2 mL de solución de NaCl al 1.2% (figura 2B).</li>
	<li>Cubra la taza con papel de aluminio y una placa de Petri para evitar derrames y mezclar durante 1 minuto alternando baja y alta velocidad (20 s baja velocidad/10 s velocidad; repetición) (figura 2).</li>
	<li>Distribuir la suspensión de hígado mezclada en botellas de centrífuga estériles de 250 mL (para 20 hígados usan 4 botellas).</li>
	<li>Añadir solución salina estéril con antibióticos a ~ 200 mL botella de volumen total. Pesa/balanza botellas antes de centrifugar.</li>
	<li>Centrifugar el hígado mezclado a 290 x g durante 15 min a 4 oC. suavemente retirar el sobrenadante en un recipiente con lejía antes de desecharlos.</li>
	<li>Repita los pasos del 2.4-2.5 una vez. Asegúrese de resuspender completamente el hígado sedimento antes de centrifugar.</li>
	<li>Después de descartar la última solución salina lavar (paso 2.6), agregue 200 mL de agua estéril estanque con pluma/estreptococos al tejido hepático alimentos Peletizado, agitar para Resuspender el sedimento y transferir la suspensión a un matraz aforado estéril de 1 L que ha sido completamente cubierto con aluminio papel de aluminio, excepto en la parte superior 3 cm del cuello (Figura 2D). Nota: Use dos botellas (= 10 hígados) cada frasco de 1 L. Agua de estanque simula el entorno natural de agua dulce necesario para estimular miracidial eclosión de los huevos.</li>
	<li>Usar agua estéril, llenar el frasco a unos 3 cm por encima de la lámina que cubre el cuello. Entonces, sin demora (como miracidia pronto comienzan a eclosionar), quitar la espuma residual y tejido hepático flotando a la superficie mediante pipeteo rápidamente hasta ~ 12 mL de agua del estanque que contenía desechos y descartarlo en un separado deseche el tubo. Vuelva a colocar con agua estéril fresca.</li>
	<li>Repetir limpieza paso 2.8, tres veces, comprobando la presencia de miracidia en el tubo de descarte. Dejar de repetición de la limpieza paso si miracidia aparece en la solución de lavado.</li>
	<li>Después del último lavado, agregue lentamente ~ 12 mL de agua tibia charca estéril (previamente calentado a 30 oC) para crear un gradiente de temperatura con agua caliente limpia y estéril en la parte superior. Nota: Esto se logra mejor descargando lentamente a lo largo de la parte del cuello del frasco para evitar la mezcla.</li>
	<li>Coloque una cubierta de caja de Petri pequeña sobre la apertura del frasco y brillar una luz brillante en la parte superior del matraz por encima de la cubierta de aluminio para iluminar la superficie superior del agua. Nota: Normalmente, dentro de 5-10 minutos, nadando miracidia, atraído por la luz, se concentrará en gran número dentro de los primeros 2-3 cm de agua de los estanques (Figura 3A).</li>
</ol>3. Miracidial cosecha y cultivo
<ol><li>Cuando miracidia han acumulado en la superficie después de 10 min, utilizando una pipeta de transferencia estéril, retire ~ 8 mL de agua del estanque y transfiéralo a un tubo de centrífuga de 15 mL estéril. Coloque el tubo que contienen miracidia en hielo.</li>
	<li>Añadir back 8 mL de agua estéril tibia por el interior del matraz volumétrico y esperar otros 10 minutos.</li>
	<li>Utilizando tubos de nuevo cada vez, repita los pasos 3.1 y 3.2 tres veces más. Después de la colecciónth 4, mantenga el tubo final en hielo durante 10 minutos permitir que el miracidia resolver. Este conjunto de tubos consta de la primera cosecha.</li>
	<li>Tubos de centrífuga que contienen miracidia 290 x g durante 1 min a 4 oC en una centrífuga refrigerada previamente enfriada.</li>
	<li>Retire inmediatamente la mayor parte del sobrenadante (~ 7 mL) teniendo cuidado de no perturbar el sedimento del parásito (figura 3B).</li>
	<li>Piscina todas la miracidia de esta primera cosecha en un tubo, luego lavar todos los tubos originales con agua estéril ~ 1 mL y añada al tubo "agrupado".</li>
	<li>Permite parásitos a reposar en hielo ~ 5 min y otra vez centrifugar los parásitos a 290 x g durante 1 min a 4 oC.</li>
	<li>Cuidadosamente Quite el sobrenadante y añadir que 6-8 mL estéril Chernin equilibrada solución de sal (CBSS +; véase tabla de materiales) que contienen pluma/strep.</li>
	<li>Suspender suavemente las miracidia y alícuota en una placa de cultivo de 24 pozos (1 mL por pozo), seguido por enjuague el tubo en 6-8 mL CBSS + y añadir 1 mL de enjuague a cada pocillo. Incubar los parásitos a 26 oC normoxic condiciones. Concentraciones de miracidia aislado varían, pero generalmente promediarán ~ 7000/mL.</li>
	<li>Si los parásitos se concentran aún en la fuente de luz, repita los pasos 3.1 a 3.9 a continuar cosechando tarde eclosión miracidia pero aumentar el intervalo de tiempo entre colecciones de 15-20 minutos. Nota: Este nuevo conjunto de tubos representa la segunda cosecha. Sin embargo, porque miracidial números por lo general son bajos, las larvas de todos los tubos generalmente son agrupadas en una sola cultura, bien que contiene 2 mL CBSS +.</li>
	<li>En cultivo y poscosecha de 24 h, resuspender suavemente esporoquistes en sus pozos por pipeteo.</li>
	<li>Espere unos minutos para permitir que los parásitos resolver en el fondo de los pozos. Una vez que han colocado, retire con cuidado el sobrenadante (~1.5 mL), que contendrá la mayoría de las placas epidérmicas ciliadas derramada por miracidia durante transformación larvaria. Esta "transformación" sobrenadante puede desechar o incorporada a estudios de seguimiento de productos de secreción de larvas.</li>
	<li>Añadir 1,5 mL de CBSS fresco + a cada bien y suavemente la pipeta para resuspender esporoquistes. Repita el paso 3.12 si se desea más lavar o cambiar a un medio diferente.</li>
</ol>

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Autores no tienen nada que revelar.

Miracidia de S. mansoni, aislados y manipulados como se describe en este documento, puede infectar solamente el anfitrión intermedio del caracol y por lo tanto no representan un riesgo biológico humano durante esta fase de desarrollo larvario. Sin embargo, para evitar la exposición accidental, infección de los caracoles, debe tenerse cuidado para realizar aislamientos miracidial en una ubicación diferente de áreas donde especies de caracoles Biomphalaria susceptibles pueden ser presente o mantenid...

El ser humano sangre platijas Schistosoma spp., son los agentes causativos de la esquistosomiasis, una enfermedad tropical desatendida que afecta a un estimado de 230 millones de personas en todo el mundo1. La especie más difundida, Schistosoma mansoni, se distribuye geográficamente en América del sur, el archipiélago Caribe, Oriente Medio y África subsahariana2. El ciclo de vida de S. mansoniy otros esquistosomas, es complejo, que implica un mamífero hospedador definitivo y los caracoles de agua dulce del género Biomphalaria que sirven como hospederos intermediarios obligados.
S. mansoni macho y hembra adulto del gusano pares viven en las venas mesentéricas posteriores de la reproducción del huésped mamífero, lo que resulta en la liberación de huevos embrionados (ova) que se metieran en las pequeñas vénulas de la mucosa intestinal. Huevos se rompe a través de las paredes del recipiente, migran a través de tejido mucoso y finalmente entrar en la luz intestinal donde son anuladas con las heces. Cuando óvulos entran las larvas nadan libremente y de agua dulce (miracidia) Portilla, deben, en poco tiempo, encontrar un caracol Biomphalaria adecuado para infectar a fin de continuar su ciclo de vida. Este proceso de infección consiste en miracidial accesorio a la superficie de cuerpo exterior de caracol seguida por la penetración activo y la entrada de la larva en el host. Pronto después de la entrada, el miracidium comienza a arrojar sus placas epidérmicas ciliadas externas como forma un sincitio se convertirá en la nueva superficie exterior (tegumento) de la siguiente etapa larval, el sporocyst primario o madre. A través de reproducción asexual, cada sporocyst primario produce y libera una segunda generación de esporoquistes, denominados secundarios o esporoquistes de hija, que a su vez, generaran y liberan grandes cantidades de la fase final de la intramolluscan, la cercaria3 . A escapar de la sede de caracol, cercariae nado libre es capaces de conectar a y penetrar directamente en la piel de un humano o de otro huésped mamífero adecuado. Al entrar en su nuevo host, cercariae transforma a la etapa de parásito esquistosómula y invade el sistema vascular. A su vez, migran a los pulmones y luego a la vena hepática donde maduran a gusanos adultos, forman pares masculinos y femeninos y viajar a las venas mesentéricas para completar su ciclo de vida.
Exitoso intramolluscan o el desarrollo de la "fase Caracol" de esquistosomas larvales es esencial para la continuación del ciclo de transmisión humana de host a host. De importancia crítica es la entrada siguiente períoda del miracidium nadan libremente en el host de caracol y su transformación temprana a la etapa del sporocyst primario. Dependiendo de la compatibilidad fisiológica e inmunológica entre huésped y parásito, es durante esta fase de desarrollo larval inicial éxito o el fracaso para establecer infecciones es determinado4,5,6 . Desarrollo posterior de esporocisto primaria capaz de producir asexualmente esporoquistes secundarios, que luego generan cercariae infeccioso en humanos, además requieren un entorno de host fisiológica permisiva que ofrece para todos los del crecimiento del parásito y necesidades de reproducción.
Hasta la fecha, poco se sabe sobre el subyacente fisiológico, bioquímico y moleculares procesos controlar las interacciones de schistosome caracol, especialmente las moléculas y vías implicados en regulación de la reproducción y desarrollo larval, o modulación de respuestas inmunitarias del huésped. Acceso a un gran número de estos estadios larvarios en condiciones en vitro cultura que permiten manipulación experimental facilitaría enormemente el descubrimiento de vías de desarrollo y los mecanismos subyacentes del crecimiento celular, diferenciación y la reproducción. Crítico parásito vías o mecanismos, identificados a través de la experimentación en vitro , podrían utilizarse para interrumpir el crecimiento/reproducción larvaria en el host de caracol, o caracol huésped inmune mecanismos implicados en el reconocimiento y eliminación de miracidial o sporocyst etapas.
En este artículo y el vídeo de presentación, ofrecemos una descripción detallada de un método para aislar gran cantidad de nado libre S. mansoni miracidia para la introducción en cultivo en vitro que luego puede ser sometida a seguimiento experimental manipulación (ver figura 1 para una descripción esquemática del flujo de trabajo de protocolo). Aunque se han descrito procedimientos similares previamente7,8,9, sentimos que un protocolo detallado sería útil a los investigadores que deseen emplear este modelo para abordar preguntas todavía sin respuesta relacionadas con intramolluscan desarrollo larvario, proliferación celular e interacciones inmunes schistosome caracol. Además, este enfoque se podría adaptar fácilmente para el aislamiento de huevos de otros trematodos médicamente importantes tales como vejiga-vivienda S. hematobium, hígado trematodos o tremátodos del pulmón.

<table><tbody><tr><td>&lt;strong&gt;Chernin&#039;s balanced salt solution (CBSS+)&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;For 1L of solution&lt;/strong&gt;</td></tr><tr><td>2.8 g sodium chloride</td><td>Fisher Scientific</td><td>S271-3</td><td>Dissolve salts, except calcium chloride, and&amp;nbsp;</td></tr><tr><td>0.15 g of potassium chloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P5405</td><td>sugars in 800 mL ddH2O</td></tr><tr><td>0.07 g sodium phosphate, dibasic anhydrous</td><td>Fisher Scientific</td><td>S374-500</td><td>Dissolve calcium chloride separately in 200&amp;nbsp;</td></tr><tr><td>0.45 g magnesium sulfate heptahydrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M1880</td><td>mL ddH2O</td></tr><tr><td>0.53 g calcium chloride dihydrate&amp;nbsp;</td><td>Mallinckrodt</td><td>4160</td><td>Slowly add calcium soln to the salt/sugar soln with&amp;nbsp;</td></tr><tr><td>0.05 g sodium bicarbonate&amp;nbsp;&amp;nbsp;</td><td>Fisher Scientific</td><td>S233-3</td><td>with constant mixing</td></tr><tr><td>1 g glucose</td><td>MP Biomedicals</td><td>152527</td><td>Adjust to pH 7.2 and filter sterilize using a&amp;nbsp;</td></tr><tr><td>1 g trehalose</td><td>Sigma-Aldrich</td><td>T0167</td><td>0.22 &amp;micro;m disposable bottle-top filter</td></tr><tr><td>10 mL 100X penicillin/streptomycin</td><td>Hyclone</td><td>SV30010</td><td>Add filtered penicillin and streptomycin soln&amp;nbsp;&lt;br/&gt; prior use</td></tr><tr><td>&lt;strong&gt;Incomplete Bge&amp;nbsp; medium (Ibge)&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;For 900 mL solution&lt;/strong&gt;</td></tr><tr><td>220 mL Schneider&amp;rsquo;s Drosophila medium modified</td><td>Lonza</td><td>04-351Q</td><td>Mix Schneider&#039;s medium with 680 mL ddH2O</td></tr><tr><td>4.5 g lactalbumin enzymatic hydrolysate</td><td>Sigma-Aldrich</td><td>L9010</td><td>Add lactalbumin hydrolysate and galactose</td></tr><tr><td>1.3 g galactose</td><td>Sigma-Aldrich</td><td>G0625</td><td>Adjust to pH 7.2 and filter sterilize using a 0.22 &amp;micro;m&lt;br/&gt; pre-sterilized disposable bottle-top filter</td></tr><tr><td>&lt;strong&gt;Complete Bge medium (cBge)&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;For 100 mL of solution&lt;/strong&gt;</td></tr><tr><td>90 mL Incomplete Bge medium</td><td></td><td></td><td>To heat-inactivate FBS: Incubate thawed FBS in&amp;nbsp;</td></tr><tr><td>9 mL heat-inact. fetal bovine serum (FBS) (Optima)</td><td>Atlanta Biologicals</td><td>S12450</td><td>waterbath at 60&amp;deg;C for 1 hr while gently&amp;nbsp;</td></tr><tr><td>1 mL 100X penicillin/streptomycin</td><td>Hyclone</td><td>SV30010</td><td>swirling the bottle every 10 min&lt;br/&gt; Aliquot heat-inactivated FBS into 15-mL tubes&amp;nbsp;&lt;br/&gt; and store at -20&amp;deg;C.&amp;nbsp; Mix medium + FBS and&lt;br/&gt; filter sterilize using a 0.22 &amp;micro;m pre-sterilized&amp;nbsp;&lt;br/&gt; disposable bottle-top filter&amp;nbsp;&lt;br/&gt; Add penicillin and streptomycin prior to use</td></tr><tr><td>&lt;strong&gt;Pond water (stock solution)&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;1L of stock solution&lt;/strong&gt;</td></tr><tr><td>12.5 g calcium carbonate</td><td>Fisher Scientific</td><td>C64-500</td><td>Mix all salts in 1L of ddH2O</td></tr><tr><td>1.25 g magnesium carbonate</td><td>Fisher Scientific</td><td>M27-500</td><td>Note that the salts will not have completely&amp;nbsp;</td></tr><tr><td>1.25 g&amp;nbsp; sodium chloride</td><td>Fisher Scientific</td><td>S271-3</td><td>dissolved.&amp;nbsp; Shake vigorously to suspend&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td></tr><tr><td>0.25 g&amp;nbsp; potassium chloride</td><td>Sigma-Aldrich</td><td>P5405</td><td>salts prior to making the working soln&amp;nbsp;&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Pond water (working solution)&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;1.5L of solution&lt;/strong&gt;</td></tr><tr><td>0.8 mL stock solution pond water (shake prior use) in&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td><td></td><td>Mix stock to ddH2O</td></tr><tr><td>1500 mL of ddH2O</td><td></td><td></td><td>Sterilize pond water by autoclaving (slow&amp;nbsp;cycle)</td></tr><tr><td>1.5 mL of 100X penicillin/streptomycin&amp;nbsp;</td><td>Hyclone</td><td>SV30010</td><td>Add penicillin and streptomycin prior use</td></tr><tr><td>&lt;strong&gt;Saline solution (1.2% NaCl)&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;1.5L of solution&lt;/strong&gt;</td></tr><tr><td>18 g sodium chloride in 1500 mL of ddH2O</td><td>Fisher Scientific</td><td>S271-3</td><td>Autoclave saline solution to sterilize</td></tr><tr><td>1.5 mL of 100X penicillin/ streptomycin</td><td>Hyclone</td><td>SV30010</td><td>Add penicillin and streptomycin prior use</td></tr><tr><td>&lt;strong&gt;Additional equipment and material:&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>7-L mouse euthanizing chamber</td><td></td><td></td><td>Following approved IACUC protocol no. V001551</td></tr><tr><td>Mice</td><td>Taconic Biosciences</td><td></td><td>Swiss-Webster, female, 6-wk old, murine&amp;nbsp;&amp;nbsp;</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; tank and regulator</td><td></td><td></td><td>pathogen-free</td></tr><tr><td>24-well tissue culture plate</td><td>TPP</td><td>92424</td><td></td></tr><tr><td>1-L volumetric flasks</td><td></td><td></td><td></td></tr><tr><td>Light source (150W)</td><td>Chiu Tech Corp</td><td>Model F0-150</td><td></td></tr><tr><td>Centrifuge, refrigerated, swinging bucket</td><td>Eppendorf</td><td>Model 5810R</td><td></td></tr><tr><td>Centrifuge bottles (250 mL)</td><td>Nalgene</td><td></td><td></td></tr><tr><td>15-mL centrifuge tubes, sterile</td><td>Corning</td><td>430053</td><td></td></tr><tr><td>Sterile disposable transfer pipets&amp;nbsp;</td><td>Fisher Scientific</td><td>1371120</td><td></td></tr><tr><td>0.22 &amp;micro;m pre-sterilized disposable bottle-top filter&amp;nbsp;</td><td>EMD Millipore</td><td>SCGPS05RE</td><td></td></tr><tr><td>Stainless steel blender</td><td>Waring Commercial</td><td>Model 51BL31</td><td></td></tr><tr><td>Blender cup, 100 mL capacity</td><td>Waring Commercial</td><td></td><td></td></tr><tr><td>Inverted compound microscope</td><td>Nikon Instruments&amp;nbsp;</td><td>Eclipse TE300</td><td></td></tr></tbody></table>

mass isolation and in vitro cultivation of intramolluscan stages of the human blood fluke schistosoma mansoni

Humanas sangre trematodos, Schistosoma spp., tienen un ciclo de vida complejo que comprende las fases del desarrollo asexuales y sexuales dentro de un caracol intermedio y mamífero huésped definitivo, respectivamente. La capacidad para aislar y sostener las etapas del ciclo de vida diferente bajo condiciones de cultivo en vitro ha facilitado enormemente las investigaciones de los mecanismos celulares, bioquímicos y moleculares de regulación de host, el desarrollo y crecimiento del parásito interacciones. Transmisión de la esquistosomiasis requiere reproducción asexual y el desarrollo de múltiples etapas larvales en el host de caracol; desde el miracidium contagiosa, a través de esporocisto primaria y secundaria, a la etapa final de cercarial que es contagioso a los seres humanos. En este trabajo presentamos un protocolo paso a paso para la eclosión total y aislamiento del mansoni del schistosoma miracidia de huevos obtenidos de hígados de ratones infectados y su posterior introducción en cultivo en vitro . Se prevé que el protocolo detallado anime a nuevos investigadores y ampliar este importante campo de investigación de esquistosoma.

La gran mayoría de miracidia normalmente habrán sido recogida en las dos primeras "cosechas". Incubación de miracidia aislado en CEMB + desencadena el proceso de transformación de miracidio-sporocyst (Figura 4A- C). En la primera hora siguiente traslado a cultura pozos que contienen CBSS +, la mayoría de miracidia deje nadar (Figura 4A). A las 6 h en cultura, miracidia estamos activamente vertimiento sus pla...

Watch this Scientific Journal Video about Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni at JoVE.com

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

Este artículo describe un protocolo para el aislamiento axénico a gran escala y la colección de miracidia nadan de la humana platija de la sangre mansoni de Schistosoma y su posterior procesado para la introducción en cultivo en vitro .

Total aislamiento y cultivo In Vitro de Intramolluscan etapas de la humano platija de la sangre Schistosoma Mansoni

Human blood flukes, Schistosoma spp., have a complex life cycle that involves asexual and sexual developmental phases within a snail intermediate and ...

mass-isolation-vitro-cultivation-intramolluscan-stages-human-blood

Research

JoVE Journal

Developmental Biology

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

9.1K vistas.  University of Wisconsin, School of Veterinary Medicine. Este artículo describe un protocolo para el aislamiento axénico a gran escala y la colección de miracidia nadan de la humana platija de la sangre mansoni de Schistosoma y su posterior procesado para la introducción en cultivo en vitro .

Video: Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

Protocolo para la producción de un cruce genético de los parásitos de la malaria de roedores

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

Investigación

Inmunología e Infección

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ranks second to malaria among parasitic diseases in terms of public health and socio-economic impact (1-4). Schistosome parasites are trematode worms with a complex life cycle interchanging between a parasitic life in molluscan and mammalian hosts with intervening free-swimming stages. Briefly, free-swimming cercariae infect a mammalian host by penetrating the skin with the aid of secreted proteases, during which time the cercariae lose their tails, transforming into schistosomules. The schistosomules must now evade the host immune system, develop a gut for digestion of red blood cells, and migrate though the lungs and portal circulation en route to their final destination in the hepatic portal system and eventually the mesenteric veins (for S. mansoni) where male and female worms pair and mate, producing hundreds of eggs daily. Some of the eggs are excreted from the body into fresh water, where the eggs hatch into free-swimming miracidia (5-10). The miracidia infect specific snail species and transform into mother and daughter sporocysts, which in turn, produce infective cercariae, completing the life cycle. Unfortunately, the entire schistosome life cycle cannot be cultured in vitro, but infective cercariae can be transformed into schistosomules, and the schistosomules can be cultured for weeks for the analysis of schistosome development in vitro or microarray analysis. In this protocol, we provide a visual description of cercarial transformation and in vitro culturing of schistosomules. We shed infectious cercariae from the snail host Biomphalaria glabrata and manually transform them into schistosomules by detaching their tails using an emulsifying double-ended needle. The in vitro cercarial transformation and schistosomules culture techniques described avoid the use of a mammalian host, which simplifies visualization of schistosomes and facilitates the collection of the parasite for experimental analysis. in vitro transformation and culturing techniques of schistosomes have been done for years (11, 12), but no visual protocols have been developed that are available to the entire community.

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

We describe an in vitro method for culturing schistosomula of the flatworm parasite Schistosoma mansoni, via the harvesting and transformation of infective cercariae from the fresh water snail intermediate host Biomphalaria glabrata.

Transformación y cercarias In vitro El cultivo de la Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ...

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

Con óvulos de<em&gt; Schistosoma mansoni</em&gt; Como un<em&gt; En vivo</em&gt; Modelo de la inflamación pulmonar inducida por helmintos

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

Transient Transduction of the Strobilated Forms of Echinococcus granulosus

Cystic echinococcosis or hydatid disease is one of the most important zoonotic parasitic diseases caused by Echinococcus granulosus, a small tapeworm harbored in the intestine of canines. There is an urgent need for applied genetic research to understand the mechanisms of pathogenesis and disease control and prevention. However, the lack of an effective gene evaluation system impedes direct interpretation of the functional genetics of cestode parasites, including the Echinococcus species. The present study demonstrates the potential of lentiviral gene transient transduction in the metacestode and strobilated forms of E. granulosus. Protoscoleces (PSCs) were isolated from hydatid cysts and transferred to specific biphasic culture media to develop into strobilated worms. The worms were transfected with harvested third-generation lentivirus, along with HEK293T cells as a transduction process control. A pronounced fluorescence was detected in the strobilated worms over 24 h and 48 h, indicating transient lentiviral transduction in E. granulosus. This work presents the first attempt at lentivirus-based transient transduction in tapeworms and demonstrates the promising outcomes with potential implications in experimental studies on flatworm biology.

Transient Transduction of the Strobilated Forms of Echinococcus granulosus

We describe a rapid transient transduction technique in different developmental stages of Echinococcus granulosus using third-generation lentiviral vectors.

Transducción transitoria de las formas estrobiladas de Echinococcus granulosus

Cystic echinococcosis or hydatid disease is one of the most important zoonotic parasitic diseases caused by Echinococcus granulosus, a small tapeworm ...

Genética

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

Establecimiento y Optimización de una instalación de alto rendimiento para el Estudio<em&gt; Staphylococcus epidermidis</em&gt; Y<em&gt; Mycobacterium marinum</em&gt; La infección como modelo para el descubrimiento de fármacos

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

El aislamiento de las células mieloides de ratón de la piel y el drenaje de ganglios linfáticos Siguiendo intradérmica La inmunización con viva atenuada<em&gt; Plasmodium</emLos esporozoitos&gt;

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

Una estrategia rápida para el aislamiento de nuevos Faustoviruses de muestras ambientales Uso<em&gt; vermiformis Vermamoeba</em

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

Aislamiento y caracterización de las vesículas extracelulares de adultos Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...

Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In particular, small extracellular vesicles (sEVs) act as nano-carriers transferring natural antigens, which are critical in host immunomodulation and parasite survival. This article presents a step-by-step protocol to isolate sEVs from larval stage cultures of Echinococcus granulosus and analyzes their uptake by dendritic cells obtained from murine bone marrow, which acquire adhesion and antigen presentation capacity during their maturation after one week of in vitro culture. This article provides comprehensive information for generating, purifying, and quantifying sEVs using ultracentrifugation alongside parallel analyses of dynamic light scattering and transmission electron microscopy. Additionally, a detailed experimental protocol is outlined for isolating and cultivating mouse bone marrow cells and driving their differentiation into dendritic cells using Flt3L. These dendritic cells can present antigens to na&#239;ve T cells, thereby modulating the type of immune response in vivo. Thus, alternative protocols, including confocal microscopy and flow cytometry analysis, are proposed to check the acquired maturational phenotype of dendritic cells previously exposed to parasitic sEVs. Finally, it is worth noting that the described protocol can be applied as a whole or in individual parts to carry out parasite in vitro culture, isolate extracellular vesicles, generate bone marrow-derived dendritic cell cultures, and perform uptake assays with these cells.

Here, we describe in vitro culture conditions, isolation, and increased generation of extracellular vesicles (EVs) from Echinococcus granulosus. The small EVs were characterized by dynamic light scattering and transmission electron microscopy. The uptake by bone marrow-derived dendritic cells and their phenotypic modulation were studied using confocal microscopy and flow cytometry.

Aislamiento y absorción de células dendríticas de pequeñas vesículas extracelulares de Echinococcus granulosus

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In ...

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a &#39;whole mount&#39; tissue preparation, or developmental stage (such as an embryo or larva) of interest. WMISH is extremely powerful because it can significantly contribute to the functional characterization of complex metazoan genomes, a challenge that is becoming more of a bottleneck with the deluge of next generation sequence data. Despite the conceptual simplicity of the technique much time is often needed to optimize the various parameters inherent to WMISH experiments for novel model systems; subtle differences in the cellular and biochemical properties between tissue types and developmental stages mean that a single WMISH method may not be appropriate for all situations. We have developed a set of WMISH methods for the re-emerging gastropod model Lymnaea stagnalis that generate consistent and clear WMISH signals for a range of genes, and across all developmental stages. These methods include the assignment of larvae of unknown chronological age to an ontogenetic window, the efficient removal of embryos and larvae from their egg capsules, the application of an appropriate Proteinase-K treatment for each ontogenetic window, and hybridization, post-hybridization and immunodetection steps. These methods provide a foundation from which the resulting signal for a given RNA transcript can be further refined with probe specific adjustments (primarily probe concentration and hybridization temperature).

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

The goal of this protocol is to provide users with a set of methods for the high-throughput decapsulation of Lymnaea stagnalis embryos and larvae in preparation for whole mount in situ hybridization, and for subsequent pre- and post-hybridization treatments.

Total aislamiento y cultivo In Vitro de Intramolluscan etapas de la humano platija de la sangre Schistosoma Mansoni

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