Name: mass isolation and in vitro cultivation of intramolluscan stages of the human blood fluke schistosoma mansoni
Uploaded: 2018-01-14T13:00:00
Description: 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

Financiado em parte pela concessão de NIH RO1AI015503. Ratos infectados esquistossomíase foram fornecidos pela esquistossomose NIAID Resource Center, no Instituto de pesquisas biomédicas (Rockville, MD) através de HHSN272201000005I de contrato NIAID-NIH para distribuição por meio de recursos do BEI.

Todos os cuidados com animais e procedimentos experimentais foram aprovados pelo Comitê de uso da Universidade de Wisconsin-Madison, sob protocolo e cuidados institucionais de Animal nenhum. V005717. O seguinte protocolo envolve o trabalho em uma instituição de nível de biossegurança 2 (BSL2) para patógenos humanos, apesar de nenhum dos estágios esquistossomíase retratados no presente protocolo são infectantes aos seres humanos ou outros mamíferos. Segui as políticas institucionais para a manipulação de agen...

<ol>
	<li>Colley, D. G., Bustinduy, A. L., Secor, W. E., King, 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=Human+schistosomiasis.">Human schistosomiasis.</a> Lancet. 383, 2253-2264 (2014).</li><li>WHO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis:+number+of+people+treated+worldwide+in+2013.">Schistosomiasis: number of people treated worldwide in 2013.</a> Wkly. Epidemiol. Rec. 5 (90), 25-32 (2015).</li><li>Yoshino, T. P., Gourbal, B., Th&#233;ron, A., Jamieson, B. G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosoma+sporocysts.">Schistosoma sporocysts.</a> Schistosoma: biology, pathology and control. , 118-148 (2017).</li><li>Bayne, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+parasitism+of+vector+snail+Biomphalaria+glabrata+by+the+human+blood+fluke+(trematode)+Schistosoma+mansoni:+a+2009+assessment.">Successful parasitism of vector snail Biomphalaria glabrata by the human blood fluke (trematode) Schistosoma mansoni: a 2009 assessment.</a> Mol. Biochem. Parasitol. 165 (1), 8-18 (2009).</li><li>Yoshino, T. P., Coustau, C., Toledo, R., Fried, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunobiology+of+Biomphalaria-trematode+interactions.">Immunobiology of Biomphalaria-trematode interactions.</a> Biomphalaria snails and larval trematodes. , 159-189 (2011).</li><li>Coustau, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+gastropod+immunity+from+the+study+of+the+interaction+between+the+snail+Biomphalaria+glabrata+and+its+parasites:+A+review+of+research+progress+over+the+last+decade.">Advances in gastropod immunity from the study of the interaction between the snail Biomphalaria glabrata and its parasites: A review of research progress over the last decade.</a> Fish Shellfish Immunol. 46 (1), 5-16 (2015).</li><li>Bayne, C. J., Hahn, U. K., Bender, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+molluscan+host+resistance+and+of+parasite+strategies+for+survival.">Mechanisms of molluscan host resistance and of parasite strategies for survival.</a> Parasitology. 123, S159-S167 (2001).</li><li>McMullen, D. B., Beaver, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+schistosome+dermatitis.+IX.+The+life+cycles+of+three+dermatitis-producing+schistosomes+from+birds+and+a+discussion+of+the+subfamily+Bilharziellinae+(Trematoda:+Schistosomatidae).">Studies on schistosome dermatitis. IX. The life cycles of three dermatitis-producing schistosomes from birds and a discussion of the subfamily Bilharziellinae (Trematoda: Schistosomatidae).</a> Amer. J. Hyg. 42, 128-154 (1945).</li><li>Voge, M., Seidel, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+in+vitro+of+miracidia+of+Schistosoma+mansoni+and+S.+japonicum+into+young+sprocysts.">Transformation in vitro of miracidia of Schistosoma mansoni and S. japonicum into young sprocysts.</a> J. Parasitol. 58 (4), 699-704 (1972).</li><li>Eloi-Santos, S., Olsen, N. J., Correa-Oliveira, R., Colley, D. G. <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:+mortality,+pathophysiology,+and+susceptibility+differences+in+male+and+female+mice.">Schistosoma mansoni: mortality, pathophysiology, and susceptibility differences in male and female mice.</a> Exp. Parasitol. 75 (2), 168-175 (1992).</li><li>Tucker, M. S., Karunaratne, L. B., Lewis, F. A., Freitas, T. C., Liang, Y. S. <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 Proto Immunol. 103, 19.1:19.1.1-19.1.58 (2013).</li><li>Basch, P. F., DiConza, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+miracidium-sporocyst+transition+in+Schistosoma+mansoni:+surface+changes+in+vitro+with+ultrastructural+correlation.">The miracidium-sporocyst transition in Schistosoma mansoni: surface changes in vitro with ultrastructural correlation.</a> J. Parasitol. 60 (6), 935-941 (1974).</li><li>Hansen, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+daughter+sporocysts+of+Schistosoma+mansoni:+their+occurrence+and+cultivation.">Secondary daughter sporocysts of Schistosoma mansoni: their occurrence and cultivation.</a> Ann. N. Y. Acad. Sci. 266, 426-436 (1975).</li><li>Stibbs, H. H., Owczarzak, O., Bayne, C. J., DeWan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosome+sporocyst-killing+amoebae+Isolated+from+Biomphalaria+glabrata.">Schistosome sporocyst-killing amoebae Isolated from Biomphalaria glabrata.</a> J. Invertebr. Pathol. 33, 159-170 (1979).</li><li>DiConza, J. J., 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=Axenic+cultivation+of+Schistosoma+mansoni+daughter+sporocysts.">Axenic cultivation of Schistosoma mansoni daughter sporocysts.</a> J. Parasitol. 60 (5), 757-763 (1974).</li><li>Hansen, E. L., Maramorosch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell+line+from+embryos+of+Biomphalaria+glabrata+(Pulmonata):+Establishment+and+characteristics.">A cell line from embryos of Biomphalaria glabrata (Pulmonata): Establishment and characteristics.</a> Invertebrate tissue culture: Research applications. , 75-97 (1976).</li><li>Yoshino, T. 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 (5), 714-722 (1995).</li><li>Ivanchenko, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+in+vitro+propagation+and+differentiation+of+cultures+of+the+intramolluscan+stages+of+the+human+parasite+Schistosoma+mansoni.">Continuous in vitro propagation and differentiation of cultures of the intramolluscan stages of the human parasite Schistosoma mansoni.</a> Proc. Natl. Acad. Sci. USA. 96, 4965-4970 (1999).</li><li>Yoshino, T. P., Bayne, C. J., Bickham, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molluscan+cells+in+culture:+primary+cell+cultures+and+cell+lines.">Molluscan cells in culture: primary cell cultures and cell lines.</a> Can. J. Zool. 91, 391-404 (2013).</li><li>Coustau, C., Yoshino, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flukes+without+snails:+Advances+in+the+in+vitro+cultivation+of+intramolluscan+stages+of+trematodes.">Flukes without snails: Advances in the in vitro cultivation of intramolluscan stages of trematodes.</a> Exp. Parasitol. 94, 62-66 (2000).</li><li>Milligan, J. N., Jolly, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cercarial+transformation+and+in+vitro+cultivation+of+Schistosoma+mansoni+schistosomules.">Cercarial transformation and in vitro cultivation of Schistosoma mansoni schistosomules.</a> J. Vis. Exp. (54), e3191 (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+mansoniin+vitro.+II+production+of+infertile+eggs+by+worm+pairs+cultured+from+cercariae.">Cultivation of Schistosoma mansoniin vitro. II production of infertile eggs by worm pairs cultured from cercariae.</a> J. Parasitol. 67 (2), 186-190 (1981).</li></ol>

Miracidia de S. mansoni, isolado e manipulados conforme descrito aqui, pode infectar somente o host intermediário de caracol e, portanto, não representam um risco biológico humano durante esta fase de desenvolvimento larval. No entanto, para evitar a exposição acidental/infecção de caracóis, tenha cuidado para realizar miracidial isolamentos em um local diferente de áreas onde espécies sensíveis de gênero Biomphalaria podem estar presentes ou mantida. Uma sala separada, registrada como espa?...

O ser humano sangue vermes Schistosoma spp., são os agentes causadores da esquistossomose, uma doença tropical negligenciada que afligem um estimado de 230 milhões de pessoas em todo o mundo1. A espécie mais difundida, Schistosoma mansoni, é geograficamente distribuída na América do Sul, o arquipélago das Caraíbas, o Médio Oriente e de África subsahariana2. O ciclo de vida de S. mansonie outros schistosomes, é complexo, envolvendo um mamíferos hospedeiro definitivo e caramujos de água doce do gênero Biomphalaria que servem como hospedeiros intermediários obrigatórios.
Adulto masculino e feminino de S. mansoni verme pares vivem nas veias mesentéricas posteriores de reproduzir o anfitrião dos mamíferos, resultando na liberação de ovos embrionados (óvulos) que se alojaram nas pequenas vênulas da mucosa intestinal. Ovos, em seguida, rompe as paredes dos vasos, migram através do tecido da mucosa e acabaram por inserir o lúmen intestinal onde eles são anulados com as fezes. Quando óvulos digite escotilha de larvas de água doce e livres (miracidia), eles devem, na ordem curta, encontrar um caracol de Biomphalaria apropriado para infectar a fim de continuar seu ciclo de vida. Esse processo de infecção envolve miracidial apego à superfície do corpo exterior o caracol seguido por penetração ativa e entrada da larva no hospedeiro. Logo após a entrada, o miracidium começa a derramar suas placas epidérmicas ciliadas exteriores como forma um sincício que se tornará a nova superfície externa (tegumento) do próximo estágio larval, o sporocyst primário ou mãe. Através de reprodução assexuada, cada sporocyst primário produz e libera uma segunda geração de sporocysts, denominados secundários ou sporocysts filha, que por sua vez, geram e liberar um grande número de fase intramolluscan final, a cercaria3 . Após a fuga do host de caracol, cercárias livres são capazes de anexar a e penetrando diretamente na pele de um humano ou outro hospedeiro adequado mamífera. Com a entrada em seu novo hospedeiro, cercárias transformam-se para a fase de parasitas schistosomula e invadem o sistema vascular. Eles, por sua vez, migraram para o pulmão e depois para a veia hepática onde amadurecem aos vermes adultos, formam pares masculinos e femininos e viajar para as veias mesentéricas para completar seu ciclo de vida.
Intramolluscan de sucesso ou desenvolvimento de "fase de caracol" de schistosomes larvas é essencial para a continuação do ciclo de transmissão humana de host para host. De importância crítica é a seguinte entrada de período do miracidium livres para o host de caracol e a sua transformação precoce à fase primária sporocyst. Dependendo da compatibilidade fisiológica e imunológica entre hospedeiro e parasita, é durante esta fase de desenvolvimento larval esse sucesso inicial ou falha estabelecer infecções é determinado4,5,6 . Desenvolvimento subsequente de primários sporocysts capazes de produzir assexuadamente sporocysts secundários, o que geram humanos-infectantes cercárias, mais exigem um ambiente de host fisiológicas permissiva que fornece para todo o crescimento do parasita e precisa de reprodutiva.
Até à data, pouco é conhecido sobre a base fisiológica, bioquímica e moleculares processos controlar interações esquistossomíase-caracol, especialmente as moléculas e vias envolveram na regulação da reprodução e desenvolvimento larval, ou modulando respostas imunes de anfitrião. Pronto acesso a um grande número destes estágios larval condições em vitro cultura que permitem a manipulação experimental que iria facilitar muito a descoberta de caminhos do desenvolvimento e os mecanismos subjacentes do crescimento celular, diferenciação e reprodução. Caminhos críticos parasita ou mecanismos, identificados através de experimentos em vitro , poderiam então ser usados para interromper o crescimento larval/reprodução do acolhimento de caracol, ou para identificar mecanismos imunes de anfitrião de caracol envolvidos no reconhecimento e eliminação de estágios miracidial ou sporocyst.
Neste artigo e o vídeo de apresentação, nós fornecemos uma descrição detalhada de um método para isolar um grande número de livres de S. mansoni miracidia para introdução em vitro de cultura que pode ser sujeito a acompanhamento experimental manipulação (ver Figura 1 para uma visão esquemática do fluxo de trabalho do protocolo). Embora os procedimentos semelhantes têm sido descritos anteriormente7,8,9, nós sentimos que um protocolo detalhado seria útil para pesquisadores que desejam empregar este modelo para abordar questões ainda sem resposta relacionadas com intramolluscan desenvolvimento larval, proliferação celular e interações imune esquistossomíase-caracol. Além disso, esta abordagem pode facilmente ser adaptada para isolamento de ovos provenientes de outras tremátodes medicamente importantes como bexiga-moradia S. haematobium, solhas de fígado ou acasos de pulmão.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chernin&#39;s balanced salt solution (CBSS+)</td>
			<td></td>
			<td></td>
			<td>For 1L of solution</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&#160;</td>
		</tr>
		<tr>
			<td>0.15 g of potassium chloride&#160;</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&#160;</td>
		</tr>
		<tr>
			<td>0.45 g magnesium sulfate heptahydrate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>M1880</td>
			<td>mL ddH2O</td>
		</tr>
		<tr>
			<td>0.53 g calcium chloride dihydrate&#160;</td>
			<td>Mallinckrodt</td>
			<td>4160</td>
			<td>Slowly add calcium soln to the salt/sugar soln with&#160;</td>
		</tr>
		<tr>
			<td>0.05 g sodium bicarbonate&#160;&#160;</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&#160;</td>
		</tr>
		<tr>
			<td>1 g trehalose</td>
			<td>Sigma-Aldrich</td>
			<td>T0167</td>
			<td>0.22 &#181;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&#160; 
			prior use</td>
		</tr>
		<tr>
			<td>Incomplete Bge&#160; medium (Ibge)</td>
			<td></td>
			<td></td>
			<td>For 900 mL solution</td>
		</tr>
		<tr>
			<td>220 mL Schneider&#8217;s Drosophila medium modified</td>
			<td>Lonza</td>
			<td>04-351Q</td>
			<td>Mix Schneider&#39;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 &#181;m 
			pre-sterilized disposable bottle-top filter</td>
		</tr>
		<tr>
			<td>Complete Bge medium (cBge)</td>
			<td></td>
			<td></td>
			<td>For 100 mL of solution</td>
		</tr>
		<tr>
			<td>90 mL Incomplete Bge medium</td>
			<td></td>
			<td></td>
			<td>To heat-inactivate FBS: Incubate thawed FBS in&#160;</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&#176;C for 1 hr while gently&#160;</td>
		</tr>
		<tr>
			<td>1 mL 100X penicillin/streptomycin</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td>swirling the bottle every 10 min 
			Aliquot heat-inactivated FBS into 15-mL tubes&#160; 
			and store at -20&#176;C.&#160; Mix medium + FBS and 
			filter sterilize using a 0.22 &#181;m pre-sterilized&#160; 
			disposable bottle-top filter&#160; 
			Add penicillin and streptomycin prior to use</td>
		</tr>
		<tr>
			<td>Pond water (stock solution)</td>
			<td></td>
			<td></td>
			<td>1L of stock solution</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&#160;</td>
		</tr>
		<tr>
			<td>1.25 g&#160; sodium chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td>dissolved.&#160; Shake vigorously to suspend&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
		</tr>
		<tr>
			<td>0.25 g&#160; potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P5405</td>
			<td>salts prior to making the working soln&#160;&#160;</td>
		</tr>
		<tr>
			<td>Pond water (working solution)</td>
			<td></td>
			<td></td>
			<td>1.5L of solution</td>
		</tr>
		<tr>
			<td>0.8 mL stock solution pond water (shake prior use) in&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</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&#160;cycle)</td>
		</tr>
		<tr>
			<td>1.5 mL of 100X penicillin/streptomycin&#160;</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td>Add penicillin and streptomycin prior use</td>
		</tr>
		<tr>
			<td>Saline solution (1.2% NaCl)</td>
			<td></td>
			<td></td>
			<td>1.5L of solution</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>Additional equipment and material:&#160;</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&#160;&#160;</td>
		</tr>
		<tr>
			<td>CO2 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&#160;</td>
			<td>Fisher Scientific</td>
			<td>1371120</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#181;m pre-sterilized disposable bottle-top filter&#160;</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&#160;</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

Acasos de sangue humanos, Schistosoma spp., têm um ciclo de vida complexo que envolve as fases do desenvolvimento sexuais e assexuadas dentro de um caracol intermediário e mamífero hospedeiro final, respectivamente. A capacidade de isolar e sustentar os estágios do ciclo de vida diferentes condições em vitro cultura facilitou grandemente as investigações dos mecanismos celulares, bioquímicos e moleculares de regulação, host, desenvolvimento e crescimento do parasita interações. Transmissão da esquistossomose exige reprodução assexuada e o desenvolvimento de múltiplos estágios larvas dentro do anfitrião do caracol; o miracidium infecciosa, através de sporocysts primárias e secundárias, para a fase final do cercarial que é infecciosa para os seres humanos. Neste trabalho apresentamos um protocolo passo a passo para incubação em massa e isolamento de Schistosoma mansoni miracidia de ovos obtidos a partir de fígados de ratos infectados e sua subsequente introdução na cultura em vitro . Prevê-se que o protocolo detalhado irá incentivar novos pesquisadores a participar e ampliar este importante campo de pesquisa esquistossomíase.

A grande maioria dos miracidia normalmente será foram coletada os dois primeiros "colheitas". Incubação de miracidia isolado na CBSS + desencadeia o processo de transformação de miracidium-para-sporocyst (Figura 4A- C). Dentro da primeira hora seguinte transferência de cultura poços contendo CBSS +, a maioria dos miracidia cessar natação (Figura 4A). Às 6 h em cultura, miracidia estão ativamente derram...

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 artigo descreve um protocolo para o isolamento de axénica em larga escala e a coleção de miracidia livres de sangue fascíola Schistosoma mansoni e seu posterior processamento para introdução na cultura em vitro .

Isolamento em massa e cultivo In Vitro de Intramolluscan fases do sangue fascíola Schistosoma Mansoni

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

In this video, we describe a detailed protocol for the large scale hatching and isolation of free swimming Schistosoma mansoni miracidia and the subsequent introduction into in vitro culture. Schistosoma mansoni is one of four major blood fluke species that infect over two hundred million people worldwide causing the neglected tropical disease known as Schistosomiasis. The schistosome life cycle is complex involving snails as intermediate hosts and mammals including humans as the definitive hosts.In vitro cultivation of larval and adult schistosomes represents an invaluable tool for advancing research towards control and elimination of this parasitic disease. In this video, using a mouse model to produce S.mansoni eggs containing the snail-infected miracidial stage, we illustrate a step by step procedure for mass isolation of miracidia hatched from eggs recovered from livers and their in vitro cultivation. Assisting me in this demonstration is Nathalie Dinguirard and Codie Heinemann.The following is a workflow diagram illustrating the three major steps to be covered in the accompanying video. In the first segment, we illustrate the processing of infected livers. Using mice infected with Schistosoma mansoni adults, we will describe how to remove their infected livers and the steps used to prepare them for tissue blending including removing a non-liver tissue and subsequent washing in sterile saline solution.In the second segment, we will show how eggs are extracted from livers and miracidia are isolated. Eggs are released from livers by mechanical blending followed by the repeated washing of the homogenized liver tissue in saline by centrifugation. Pelleted liver tissue containing eggs are then re-suspended in sterile artificial pond water and carefully transferred to foil covered volumetric flasks.Excess foam and floating liver debris is eliminated by the rapid sequential removal and addition of approximately 12 mills of pond water to the flask. After the last wash, pre-warmed pond water is gently added to the flask followed by illumination of the top few centimeters of the flask neck with the bright light source to attract miracidia. Once the miracidia have accumulated at the light source, in the third and final segment of the video, we will demonstrate the collection of these larvae and their cultivation.Miracidia are transferred from the flask to 15 milliliters centrifuged tubes. Then immobilized on ice and concentrated by centrifugation. In the final steps, miracidia are pulled into a single tube.The pond water is replaced with Chernin's balanced salt solution and miracidia are allocated into wells of the 24 well tissue culture plate for in vitro cultivation at 26 degrees centigrade. After euthanizing infected mice following institutional animal care approved protocols, they are placed on their back and 70%ethanol is sprayed on the ventral surfaces. To remove the liver, pinch and pull up skin of the lower abdomen and cut across the pinched skin with sterile surgical scissors.Pull the skin up to reveal the liver and gently remove it by hand. Livers from infected mice appear enlarged and speckled due to an egg-induced granulomatous response. Once the liver's removed, place it in a beaker containing 200 mills of sterile 1.2%saline solution containing Pen Strep and proceed to remover livers from the remaining mice.Livers are then transferred to a sterile Petri dish and any non-liver fat and connective tissues are removed with sterile forceps. Once the trimming is complete, livers are transferred to sterile 250 milliliter centrifuge bottles containing approximately 200 mills of sterile saline and vigorously shaken to suspend the extraneous tissues and excess blood. Using a sterile disposable pipette, remove the surface foam and any floating tissue debris.Recap the bottle and vigorously shake a second time followed by, again, removing foam and debris and then carefully decanting and discarding the saline wash solution. Add 200 milliliters of fresh saline solution to the livers and repeat the process three more times. You should see a noticeable clearing of the discarded wash solution.Place the washed livers into a small sterile stainless steel blender cup and add approximately two mills of saline with antibiotics. Cover the cup with foil and a Petri dish to prevent spills and blend for one minute by alternating low and high speeds. After blending, evenly distribute the liver suspension into sterile 250 milliliter centrifuged bottles.Typically, four bottles are used when processing 20 livers. Fill each bottle with approximately 200 milliliters of sterile saline. Weigh or balance the bottles and shake each one prior to centrifuging the blended livers.Following centrifugation, gently decant and discard the wash saline. You should observe the pelleted liver sediment containing the eggs at the bottom of the bottle and then repeat this step once more by adding 200 milliliters of fresh saline to each bottle and shaking vigorously to thoroughly re-suspend the liver sediment prior to centrifuging. During the centrifugation step, prepare two sterile one liter volumetric flasks by covering them completely with aluminum foil except for the top three centimeters.Use separate pieces of foil to cover the body and neck of the flask to facilitate washing steps that will be described in the next segment. Okay, back to the livers. After centrifugation and discarding the last saline wash, add 200 milliliters of sterile artificial pond water containing Pen Strep to the pelleted liver tissue and again shake to completely re-suspend the sediment.Carefully transfer the pond water liver suspension to the volumetric flasks that are now covered with aluminum foil. Using sterile pond water, fill up the flask including the neck and without delay, remove residual foam and floating liver tissue by quickly pipetting up approximately 12 mills of pond water containing debris and discarding it into a separate tube. Replace the fresh pond water and repeat this cleaning step three more times checking for the presence of miracidia in the discard tube.Seize additional cleaning steps if miracidia appear swimming in the discard tube or when no more tissue debris is observed. After the last wash, slowly add eight milliliters of pre-warmed sterile pond water to create a temperature gradient at the top of the flask. This is best accomplished by slowly discharging water along the side of the flask neck to avoid mixing.Place a small Petri dish to cover the flask opening and shine a bright light across the top of the neck above the foil cover to illuminate the upper surface of the water. Typically, within five to 10 minutes, swimming miracidia attracted to the light will concentrate in large numbers within the top two to three centimeters of the pond water surface. When miracidia have amassed at the surface, using a sterile pipette, remove and transfer approximately eight mills of pond water to a sterile 15 mill centrifuged tube.Place the tube containing miracidia on ice. Add eight mills of warm, sterile pond water along the side of the volumetric flask and wait for another 10 minutes. Repeat the collection of miracidia three more times using new 15 mill tubes each time.After the fourth collection, keep the final tube on ice for 10 minutes to allow immobilized miracidia to settle. This first set of isolated parasites typically represents approximately 80%of the total miracidial harvest. Pellet miracidia by centrifuging tubes for one minute in a pre-cooled refrigerated centrifuge.Following centrifugation, immediately remove most of the supernatant, being careful not to disturb the parasite pellets. This step should be done quickly as miracidia will soon begin to start swimming. Gently re-suspend the miracidia and transfer them to a single 15 mill tube.Rinse each of the original tubes with approximately one mill of sterile pond water and add to the pooled tube. Allow parasites to settle on ice for about five minutes and again, pellet them by centrifugation. Carefully remove the supernatant without disturbing the pellet and add six to eight milliliters of sterile Chernin's balanced salt solution or CBSS containing antibiotics.Gently re-suspend the miracidia in CBSS and allocate them into a 24 well tissue culture plate. Incubate parasites at 26 degrees centigrade under normoxic conditions. As you can see, miracidia will actively swim in wells following initial incubation and most will start to settle at the bottom of the well to begin transforming to the primary sporocyst stage.Larvae will start shedding the ciliated epidermal plates after two to three hours in CBSS culture. After 24 hours, the vast majority of miracidia will have shed their plates and completed their transformation to sporocysts. In vitro cultivation of parasitic elements, especially those of medical and veterinary importance, represents an invaluable research tool, not only for identifying and characterizing unique pathways, regulating growth, development, reproduction, and host immune interactions, but also by providing practical methods for screening identified molecules and pathways for potential therapeutic intervention.Looking to the future with development and continual refinement of tools currently available for manipulating genes and gene expression in vitro through transgenic RNA interference and genome editing approaches, it is envisioned that efficient methods for large scale isolation and cultivation of miracidia from other important schistosome species, for example, liver flukes or lung flukes will be needed to further research advancements in this field. We anticipate that the basic approach for the isolation of larval schistosomes presented here could be easily adapted to accommodate these and other helmin species.

Research

JoVE Journal

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

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