Este proyecto fue financiado por los fondos de la Case Western Reserve University de inicio prevista para E. Jolly. Caracoles infectados fueron proporcionados por el Centro de Recursos esquistosoma, NIH-NIAID (NIAID Contrato No. HHSN272201000009I). También queremos agradecer a Chris King y Ronald Blanton de apoyo en la obtención de los caracoles infectados por esquistosomas y útil para los debates.

Las medidas de seguridad: Cercarias esquistosoma puede penetrar directamente en la piel sin las necesidades de un corte o los poros y causa la esquistosomiasis enfermedades crónicas. S. mansoni es un nivel de bioseguridad 2 organismo, por lo tanto, el equipo de protección personal y las medidas de seguridad se deben seguir cuando se trabaja con cercarias infecciosas. La mayoría de los equipos necesarios para crecer y transformar la esquistosomiasis es estándar, con algunas excepciones clave pocas indica en la tabla al final del protocolo. Todo el trabajo de cercarias se debe hacer en una habitación aislada. Schistosomules deben ser cultivadas en una campana estéril, aprobado bioseguridad. Dos capas de nitrilo o guantes de látex deben ser usados, así como una bata de laboratorio, resistente al agua un par de zapatos, pantalones sin agujeros o rasgones, y protectores a prueba de agua la manga para las muñecas y los brazos. En el caso de un derrame accidental, pulverizar inmediatamente el área contaminada con etanol al 70% o 10% de lejía, frotar vigorosamente si en su persona. Esto será suficiente para desactivar el parásito, pero todas las posibles infecciones deben ser informados y tomados en serio. Aunque schistosomules no son netamente infeccioso, las directrices institucionales varían, y debe ser consultado para determinar si BSL2 precauciones son necesarias durante las etapas de la vida no infecciosas. Desinfecte todas las superficies que entran en contacto con cualquier sustancia potencialmente infecciosos después de que el experimento se lleva a cabo y deseche los residuos en un contenedor de riesgo biológico aprobado. NOTA: la cubierta contenedores caracol 1 a 2 días antes de su vertimiento y protegerlo de la luz 1. La recolección de las cercarias caracol <ol><li> Llene un vaso limpio, libre de 500 ml de detergentes medio llena con agua destilada equilibrarse a temperatura ambiente. Si se trabaja con los caracoles huésped infectado con miracidios en fechas diferentes, utilizar un vaso para cada fecha de la infección (Esto es especialmente importante si los caracoles serán reutilizados para futuros experimentos). El tamaño del vaso también puede ser menor dependiendo de la experiencia del usuario. </li><li> Recoge los huéspedes infectados caracol que desea arrojar y depositar con cuidado en el vaso con un pez de acuario estándar de red y las pinzas de uso general, según sea necesario. </li><li> Colocar en un vaso de precipitados de emergencia para derrames bandeja de aproximadamente 8 a 12 pulgadas por debajo de una fuente de luz incandescente. Deje reposar durante 1-2 horas a la luz. Cercarias salir del caracol cuando se expone a la luz brillante. </li><li> Cuidadosamente filtra los caracoles de la mezcla de cercarias. Tome las precauciones de seguridad adicionales, como una mezcla es altamente infecciosa. Recuerde, cercarias directamente pueden penetrar la piel humana! </li><li> Vuelva a colocar la mezcla en la fuente de luz durante unos 15 minutos, permitiendo que cualquier material de desecho de partículas se asienten. </li><li> Transferencia de cercarias en un erlenmeyer limpio usando una pipeta, evitando la suciedad en el fondo del vaso. Colocar el matraz aproximadamente a un ángulo de 45 ° sobre el hielo en la oscuridad durante 45 - 60 minutos, lo que permite cercarias para establecerse en una esquina. </li><li> Utilice una pipeta de 50 mL para eliminar 75 a 90% del sobrenadante y depositarlo en el vaso que contiene lejía al 10% o 70% de etanol, teniendo cuidado de no dejar que las gotas de agua que contienen cercarias por goteo de la pipeta. </li><li> Agitar el matraz Erlenmeyer que contiene cercarias suavemente para volver a suspender las cercarias en la solución. Use una pipeta para transferir la muestra a la estéril tubos de 50 ml cónicos. Coloque los tubos de nuevo en hielo durante 20 minutos en la oscuridad. Cercarias se depositarán en el fondo del tubo en la oscuridad. </li></ol> 2. Transformación manual de cercarias al schistosomules Antes de empezar: Asegúrese RPMI 1640 completo (RPMI, el 5% de suero fetal bovino, 1X penicilina / estreptomicina) y calentar a 37 º C. (Es muy importante que el suero fetal bovino utilizado ha sido inactivada por incubación a 56 ° C durante 1 hora como esquistosomas pueden ser sensibles al complemento). <ol><li> En una campana de riesgo biológico, eliminar el sobrenadante a un 5 a 10 ml de solución en cada uno cónico de 50 ml con una pipeta o un dispositivo de vacío. </li><li> Adjunte una de calibre 22 de doble punta, luer lok emulsionante aguja a una jeringa de tamaño adecuado. </li><li> Dibujar lentamente la mezcla en la jeringa. Recoger cercarias de todos los tubos de 50 ml en una jeringa. Esté atento de goteo. </li><li> Con cuidado, gira la jeringa sobre manera que el extremo aguja separada está para arriba. Añada otra jeringa a este lado de la aguja emulsionante. </li><li> Pase la mezcla a través de la aguja de un total de 40 veces (20 veces de ida y vuelta) para transformar las cercarias. </li><li> La posición de la jeringa verticalmente de manera que la mezcla está contenida en la jeringa y la parte inferior de la jeringa superior está vacía. Retire la jeringa encima y desinfección en el 70% de etanol. </li><li> Fuerte caída de la mezcla a gota en un recipiente limpio de alta amurallada plato 100mm x 50mm pyrex o cristalizador. Desinfectar agujas y jeringas en un 70% de etanol. </li><li> Añadir 37 ° C medio RPMI 1640 completo de manera que elparte inferior de la placa de Petri está totalmente cubierta, como si el vertido de un plato de caldo de Luria. </li><li> Hágalo girar suavemente para recoger schistosomules en el centro de la placa de Petri. Evitar salpicaduras. Nota: Puede ser útil para apagar la luz de campana para dar este paso para visualizar más adecuadamente la schistosomules en el centro. </li><li> Inmediatamente depósito schistosomules concentrado en un nuevo máximo de 12 y placa de cultivo celular y mediante un gotero estéril. </li><li> Repetir 2.9 y 2.10 hasta schistosomules ya no están presentes en el centro del plato (aproximadamente 10 veces). Depósito de cada uno posterior recogida en un lugar bien fresco. </li><li> Llene cada uno bien hasta la mitad (alrededor de 1,5 ml) precalentado con medio RPMI completo. </li><li> Visualizar en un microscopio invertido para verificar la presencia de schistosomules y la ausencia de cercarias. </li><li> Crecimiento de la schistosomules en una incubadora de CO 2 fijado a 37 ° C y 5% de CO 2. </li></ol> 3. Los resultados representativos: Transferencia pendiente de schistosomules transformado en medios de cultivo, la visualización en una brillante invertido microscopio de campo (ver tabla de equipos para un ejemplo) debería dar una imagen similar a la Figura 1, en el que sólo schistosomules transformado están presentes en el pozo. Los contaminantes potenciales podrían incluir cercarias intacto, cortó las colas de cercarias, u otros desechos. Si no se dispone de microscopio invertido, un microscopio estándar o incluso un alcance disección será suficiente para visualizar el schistosomules. Tenga en cuenta que este procedimiento no siempre es transformar el 100% de cercarias. <img src="/files/ftp_upload/3191/3191fig1.jpg" alt="Figura 1"/> Figura 1: Transformada schistosomulum <img src="/files/ftp_upload/3191/3191fig2.jpg" alt="Figura 2"/> Figura 2: Ruptura de las colas y Cercariae

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis.">Schistosomiasis.</a> N Engl J Med. 346, 1212-1220 (2002).</li><li>Steinmann, P., Keiser, J., Bos, R., Tanner, M., Utzinger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis+and+water+resources+development:+systematic+review,+meta-analysis,+and+estimates+of+people+at+risk.">Schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk.</a> Lancet Infect Dis. 6, 411-425 (2006).</li><li>Savioli, L., Albonico, M., Engels, D., Montresor, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+the+prevention+and+control+of+schistosomiasis+and+soil-transmitted+helminthiasis.">Progress in the prevention and control of schistosomiasis and soil-transmitted helminthiasis.</a> Parasitol Int. 53, 103-113 (2004).</li><li>King, C. 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Parasitology.</a> 124, 153-167 (2002).</li><li>Grabe, K., Haas, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Navigation+within+host+tissues:+cercariae+orientate+towards+dark+after+penetration.">Navigation within host tissues: cercariae orientate towards dark after penetration.</a> Parasitol Res. 93, 111-113 (2004).</li><li>Shiff, C. J., Cmelik, S. H., Ley, H. E., Kriel, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+human+skin+lipids+on+the+cercarial+penetration+responses+of+Schistosoma+haematobium+and+Schistosoma+mansoni.">The influence of human skin lipids on the cercarial penetration responses of Schistosoma haematobium and Schistosoma mansoni.</a> J Parasitol. 58, 476-480 (1972).</li><li>Saladin, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosoma+mansoni:+cercarial+responses+to+irradiance+changes.">Schistosoma mansoni: cercarial responses to irradiance changes.</a> J Parasitol. 68, 120-124 (1982).</li><li>Chai, M., McManus, D. P., McInnes, R., Moertel, L., Tran, M., Loukas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+profiling+of+lung+schistosomula,+in+vitro+cultured+schistosomula+and+adult+Schistosoma+japonicum.">Transcriptome profiling of lung schistosomula, in vitro cultured schistosomula and adult Schistosoma japonicum.</a> Cell Mol Life Sci. 63, 919-929 (2006).</li><li>Gobert, G. N., Tran, M. H., Moertel, L., Mulvenna, J., Jones, M. K., McManus, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+changes+in+Schistosoma+mansoni+during+early+schistosomula+development+and+in+the+presence+of+erythrocytes.">Transcriptional changes in Schistosoma mansoni during early schistosomula development and in the presence of erythrocytes.</a> PLoS Negl Trop Dis. 4, (2011).</li><li>Basch, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+Schistosoma+mansoni+in+vitro.+I.+Establishment+of+cultures+from+cercariae+and+development+until+pairing.">Cultivation of Schistosoma mansoni in vitro. I. Establishment of cultures from cercariae and development until pairing.</a> J Parasitol. 67, 179-185 (1981).</li><li>Yoshino, T. P. &. a. m. p. ;. a. m. p., Laursen, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+Schistosoma+mansoni+daughter+sporocysts+from+mother+sporocysts+maintained+in+synxenic+culture+with+Biomphalaria+glabrata+embryonic+(Bge)+cells.">Production of Schistosoma mansoni daughter sporocysts from mother sporocysts maintained in synxenic culture with Biomphalaria glabrata embryonic (Bge) cells.</a> J Parasitol. 81, 714-722 (1995).</li><li>Colley, D. G., Wikel, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosoma+mansoni:+simplified+method+for+the+production+of+schistosomules.">Schistosoma mansoni: simplified method for the production of schistosomules.</a> Exp Parasitol. 35, 44-51 (1974).</li><li>Lazdins, J. K., Stein, M. J., David, J. R., Sher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosoma+mansoni:+rapid+isolation+and+purification+of+schistosomula+of+different+developmental+stages+by+centrifugation+on+discontinuous+density+gradients+of+Percoll.">Schistosoma mansoni: rapid isolation and purification of schistosomula of different developmental stages by centrifugation on discontinuous density gradients of Percoll.</a> Exp Parasitol. 53, 39-44 (1982).</li><li>Cousin, C. E., Stirewalt, M. A., Dorsey, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosoma+mansoni:+ultrastructure+of+early+transformation+of+skin-+and+shear-pressure-derived+schistosomules.">Schistosoma mansoni: ultrastructure of early transformation of skin- and shear-pressure-derived schistosomules.</a> Exp Parasitol. 51, 341-365 (1981).</li><li>Lewis, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis.">Schistosomiasis.</a> Curr Protoc Immunol. Chapter 19, 1-1 (2001).</li></ol>

No hay conflictos de interés declarado.

Varios posibles problemas podrían surgir durante el proceso de transformación, sin embargo, este protocolo está diseñado para evitar la mayoría de estos problemas. Un peligro potencial es la obstrucción de la aguja emulsionantes, que se presenta generalmente debido a los escombros sobrantes de la cosecha en el paso 1.3. Asegúrese de permitir todas las partículas que se asientan en el paso 1.5 y la transferencia de las cercarias con cuidado en el paso 1.6 para evitar este problema. Además, como se ha descrito antes, trabajando dentro de una cabina de seguridad biológica, la utilización de equipos de protección personal (incluso protectores para la cara) y evitar una presión excesiva sobre las jeringas es aconsejable. Las colas cercarias pueden ser visualizados en los medios de comunicación, lo cual es indicativo de la separación de los pobres durante el paso 2.9. Las colas muestran un movimiento similar al espasmo y tienen una forma distinta (ver Figura 2), y thusly se distinguen fácilmente de las schistosomules. Las colas pueden ser removidos por el lavado de la schistosomules de más de medio RPMI completo que permite la schistosomules a un acuerdo. Las colas se quedan flotando y se puede aspirar fuera. Si cercarias intacto están presentes en los medios de comunicación, la transformación en el paso 2.5 no se ha ejecutado correctamente. Una imagen de una cercaria intacta se muestra en la Figura 3b de referencia. Si se produce este problema, asegúrese de que la aguja emulsionante que se utiliza es del tamaño adecuado. Además, el número de veces que se pasa la mezcla cercarias través de la aguja se puede aumentar, aunque el 40 veces suele ser mucho más que suficiente para alcanzar el 100% de corte de las colas de cercarias. Transformación de la cercaria en schistosomules fue descrita por primera vez en 1974 por Colley y Wikel (13). Desde entonces, la preparación de schistosomules se ha hecho utilizando la estrategia de corte con una aguja y una jeringa como se describe en este protocolo, o utilizando un enfoque de agitación y se separa con gradientes de Percoll (14, 15) o girando como se demuestra en este trabajo. Estas dos técnicas para la preparación de schistosomules están muy bien descritas por Fred Lewis (16) y más información para el cultivo de más de schistosomules de crecimiento a largo plazo se puede encontrar en el NIAID recursos esquistosoma Center ( <a href="http://www.schisto-resource.org/">www.schisto-resource.org</a> ).

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th></tr><tr><td> 22g x 2-7/8 &quot;de aguja emulsionante </td><td> Fisher Scientific </td><td> 14 a 825-17G </td></tr><tr><td> RPMI 1640 1X (500ml) </td><td> Invitrogen </td><td> 11835030 </td></tr><tr><td> De suero fetal bovino (FBS) inactivado por calor </td><td> Invitrogen </td><td> 10082139 </td></tr><tr><td> Penicilina / estreptomicina (100 veces) </td><td> Invitrogen </td><td> 15070063 </td></tr><tr><td> Cristalizador (100 mm) </td><td> Fisher Scientific </td><td> 08-741D </td></tr><tr><td> Pinzas de uso general </td><td> Fisher Scientific </td><td> 10-316C </td></tr><tr><td> Petco Acuario neto de camarones en salmuera </td><td> Petco </td><td> SKU: 1190954 </td></tr></tbody></table>

cercarial transformation and in vitro cultivation of schistosoma mansoni schistosomules

Parásitos esquistosoma son los agentes causantes de la esquistosomiasis, una enfermedad crónica debilitante que afecta a más de 200 millones de personas a nivel mundial y ocupa el segundo lugar de la malaria entre las enfermedades parasitarias en términos de salud pública y el impacto socio-económico (1-4). Parásitos esquistosoma son gusanos trematodos con un ciclo de vida complejo intercambio entre una vida parasitaria en los ejércitos de moluscos y mamíferos, con intervención de nado libre etapas. En pocas palabras, libre cercarias infectar a un huésped mamífero, penetrando la piel con la ayuda de proteasas secretadas, tiempo durante el cual las cercarias perder sus colas, transformándose en schistosomules. El schistosomules ahora deben evadir el sistema inmune del huésped, desarrollar un intestino para la digestión de las células rojas de la sangre, y aunque la migración de los pulmones y la circulación portal en ruta hacia su destino final en el sistema porta hepática y, finalmente, las venas mesentéricas (por S. mansoni) en par de gusanos machos y hembras y aparearse, produciendo cientos de huevos cada día. Algunos de los huevos son excretados del cuerpo en el agua dulce, donde los huevos se transforman en miracidios que nadan libremente (5-10). Los miracidios infectar a especies concretas de caracol y se transforman en esporocistos madre e hija, que a su vez, producen cercarias infectantes, completando el ciclo de vida. Por desgracia, todo el ciclo de vida de esquistosomas no pueden ser cultivados in vitro, pero cercarias infecciosas se pueden transformar en schistosomules, y el schistosomules pueden ser cultivadas durante semanas para el análisis del desarrollo de esquistosoma en la vitro o el análisis de microarrays. En este protocolo, se presenta una descripción visual de la transformación de cercarias y cultivo in vitro de schistosomules. Nos despojamos de cercarias infecciosas del caracol Biomphalaria glabrata de acogida y de forma manual los transforman en schistosomules separando la cola con un emulsionante aguja de doble punta. El in vitro y técnicas de transformación de cercarias schistosomules cultura describe evitar el uso de un huésped mamífero, lo que simplifica la visualización de los esquistosomas y facilita la recolección de los parásitos para el análisis experimental. Transformación in vitro y técnicas de cultivo de los esquistosomas se han hecho durante años (11, 12), pero no existen protocolos visuales han sido desarrollados que están disponibles para toda la comunidad.

Watch this Scientific Journal Video about Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules at JoVE.com

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Se describe un<em&gt; In vitro</em&gt; Método para esquistosómula cultivo del parásito platelminto<em&gt; Schistosoma mansoni</em&gt;, A través de la recolección y transformación de cercarias infecciosas del caracol de agua dulce huésped intermedio glabrata Biomphalaria.

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 ...

cercarial-transformation-vitro-cultivation-schistosoma-mansoni

Research

JoVE Journal

Immunology and Infection

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

18.0K vistas.  Case Western Reserve University. Se describe un In vitro Método para esquistosómula cultivo del parásito platelminto Schistosoma mansoni, A través de la recolección y transformación de cercarias infecciosas del caracol de agua dulce huésped intermedio glabrata Biomphalaria.

Video: Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

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 ...

Investigación

Inmunología e Infección

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

Human blood flukes, Schistosoma spp., have a complex life cycle that involves asexual and sexual developmental phases within a snail intermediate and mammalian final host, respectively. The ability to isolate and sustain the different life cycle stages under in vitro culture conditions has greatly facilitated investigations of the cellular, biochemical and molecular mechanisms regulating parasite growth, development and host interactions. Transmission of schistosomiasis requires asexual reproduction and development of multiple larval stages within the snail host; from the infective miracidium, through primary and secondary sporocysts, to the final cercarial stage that is infective to humans. In this paper we present a step-by-step protocol for mass hatching and isolation of Schistosoma mansoni miracidia from eggs obtained from livers of infected mice, and their subsequent introduction into in vitro culture. It is anticipated that the detailed protocol will encourage new researchers to engage in and broaden this important field of schistosome research.

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

This article describes a protocol for the large-scale axenic isolation and collection of free-swimming miracidia of the human blood fluke Schistosoma mansoni and their subsequent processing for introduction into in vitro culture.

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 ...

Biología del Desarrollo

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

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

El uso de<em&gt; Flexneri Shigella</em&gt; Para estudiar las interacciones Autophagy Citoesqueleto

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

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 ...

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and Strongyloides ratti. S.&#160;stercoralis is a human-parasitic, skin-penetrating nematode that infects approximately 610 million people, while the rat parasite S.&#160;ratti is closely related to S.&#160;stercoralis and is often used as a laboratory model for S.&#160;stercoralis. Both S.&#160;stercoralis and S.&#160;ratti are easily amenable to the generation of transgenics and knockouts through the exogenous nucleic acid delivery technique of intragonadal microinjection, and as such, have emerged as model systems for other parasitic helminths that are not yet amenable to this technique.
Parasitic Strongyloides adults inhabit the small intestine of their host and release progeny into the environment via the feces. Once in the environment, the larvae develop into free-living adults, which live in feces and produce progeny that must find and invade a new host. This environmental generation is unique to the Strongyloides species and similar enough in morphology to the model free-living nematode Caenorhabditis elegans that techniques developed for C. elegans can be adapted for use with these parasitic nematodes, including intragonadal microinjection. Using intragonadal microinjection, a wide variety of transgenes can be introduced into Strongyloides. CRISPR/Cas9 components can also be microinjected to create mutant Strongyloides larvae. Here, the technique of intragonadal microinjection into Strongyloides, including the preparation of free-living adults, the injection procedure, and the selection of transgenic progeny, is described. Images of transgenic Strongyloides larvae created using CRISPR/Cas9 mutagenesis are included. The aim of this paper is to enable other researchers to use microinjection to create transgenic and mutant Strongyloides.

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

The functional genomic toolkit for the parasitic nematodes Strongyloides stercoralis and Strongyloides ratti includes transgenesis, CRISPR/Cas9-mediated mutagenesis, and RNAi. This protocol will demonstrate how to use intragonadal microinjection to introduce transgenes and CRISPR components into S.&#160;stercoralis and S.&#160;ratti.

Generación de transgénicos y knockouts en especies de Strongyloides por microinyección

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and ...

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of Plasmodium deposited by mosquitoes in the skin of vertebrate hosts undergoes a phase of mandatory development in the liver before initiating clinical malaria. We know little about the biology of Plasmodium development in the liver; access to the sporozoite stage and the ability to genetically modify such sporozoites are critical tools for studying the nature of Plasmodium infection and the resulting immune response in the liver. Here, we present a comprehensive protocol for the generation of transgenic Plasmodium berghei sporozoites. We genetically modify blood-stage P. berghei and use this form to infect Anopheles mosquitoes when they take a blood meal. After the transgenic parasites undergo development in the mosquitoes, we isolate the sporozoite stage of the parasite from the mosquito salivary glands for in vivo and in vitro experimentation. We demonstrate the validity of the protocol by generating sporozoites of a novel strain of P. berghei expressing the green fluorescent protein (GFP) subunit 11 (GFP11), and show how it could be used to investigate the biology of liver-stage malaria.

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is transmitted through inoculation of the sporozoite stage of Plasmodium by infected mosquitoes. Transgenic Plasmodium has allowed us to understand the biology of malaria better and has contributed directly to malaria vaccine development efforts. Here, we describe a streamlined methodology to generate transgenic Plasmodium berghei sporozoites.

Generación de esporozoítos de Plasmodium berghei modificados genéticamente

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage 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 ...

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Transfection is a technical process through which genetic material, such as DNA and double-stranded RNA, are delivered into cells to modify the gene of interest. Currently, transgenic technology is becoming an indispensable tool for the study of Eimeria, the causative agents of coccidiosis in poultry and livestock. This protocol provides a detailed description of stable transfection in eimerian parasites: purification and nucleofection of sporozoites or second-generation merozoites, and in vivo propagation of transfected parasites. Using this protocol, we achieved transfection in several species of Eimeria. Taken together, nucleofection is a useful tool to facilitate genetic manipulation in eimerian parasites.

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Here, we provided a method to achieve stable transfection of chicken Eimeria parasites by nucleofecting sporozoites or second-generation merozoites. Genetically modified eimerian parasites expressing heterologous antigenic genes could be used as vaccine delivery vehicles.

Nucleofección y Propagación In Vivo de Parásitos de La Eimeria de Pollo

Transfection is a technical process through which genetic material, such as DNA and double-stranded RNA, are delivered into cells to modify the gene of ...

Biología

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Malaria remains one of the most important public health problems, causing significant morbidity and mortality. Malaria is a mosquito borne disease transmitted through an infectious bite from the female Anopheles mosquito. Malaria control will eventually rely on a multitude of approaches, which includes ways to block transmission to, through and from mosquitoes. To study mosquito stages of malaria parasites in the laboratory, we have optimized a protocol to culture highly infectious Plasmodium falciparum gametocytes, a parasite stage required for transmission from the human host to the mosquito vector. P. falciparum gametocytes mature through five morphologically distinct steps, which takes approximately 1-2 weeks. Gametocyte culture described in this protocol is completed in 15 days and are infectious to mosquitoes from days 15-18. These protocols were developed to maintain a continuous cycle of infection competent gametocytes and to maintain uninterrupted supply of mosquito stages of the parasite. Here, we describe the methodology of gametocyte culture and how to infect mosquitoes with these parasites using glass membrane feeders.

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Detailed investigations on mosquito stages of malaria parasites are critical to design effective transmission blocking strategies. This protocol demonstrates how to effectively culture infectious gametocytes and then feed these gametocytes to mosquitoes to generate mosquito stages of P. falciparum.

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

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