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

Funded in part by NIH grant RO1AI015503. Schistosome-infected mice were provided by the NIAID Schistosomiasis Resource Center at the Biomedical Research Institute (Rockville, MD) through NIH-NIAID Contract HHSN272201000005I for distribution through BEI Resources.

All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Wisconsin-Madison under Protocol no. V005717. The following protocol involves working in a Biosafety Level 2 (BSL2) facility for human pathogens, although none of the schistosome stages depicted in this protocol are infective to humans or other mammals. Follow institutional policies for handling Risk Group 2 (RG2) human pathogens.
NOTE: We use female Swiss-Webster m...

<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 of S. mansoni, isolated and manipulated as described herein, can infect only the snail intermediate host, and therefore do not represent a human biohazard during this phase of larval development. However, to avoid accidental exposure/infection of snails, care should be taken to perform miracidial isolations in a different location from areas where susceptible Biomphalaria snail species may be present or maintained. A separate room, registered as BSL2 space, is highly recommended. In addition, ...

The human blood flukes Schistosoma spp., are the causative agents of schistosomiasis, a neglected tropical disease afflicting an estimated 230 million people worldwide1. The most widespread species, Schistosoma mansoni, is geographically distributed in South America, the Caribbean archipelago, the Middle East and sub-Saharan Africa2. The life cycle of S. mansoni, and other schistosomes, is complex, involving a mammalian definitive host and freshwater snails of the genus Biomphalaria that serve as obligate intermediate hosts.
S. mansoni male and female adult worm pairs living in the posterior mesenteric veins of the mammalian host reproduce, resulting in the release of embryonated eggs (ova) that become lodged in the small venules of the intestinal mucosa. Eggs then breaks through the vessel walls, migrate through mucosal tissue, and eventually enter the intestinal lumen where they are voided with the feces. When ova enter freshwater and free-swimming larvae (miracidia) hatch, they must, in short order, find a suitable Biomphalaria snail to infect in order to continue its life cycle. This infection process involves miracidial attachment to the snail&#39;s outer body surface followed by active penetration and entry of the larva into the host. Soon after entry, the miracidium begins to shed its outer ciliated epidermal plates as it forms a syncytium that will become the new outer surface (tegument) of the next larval stage, the primary or mother sporocyst. Through asexual reproduction, each primary sporocyst produces and releases a second generation of sporocysts, termed secondary or daughter sporocysts, which in turn, generate and release large numbers of the final intramolluscan stage, the cercaria3. Upon escape from the snail host, free-swimming cercariae are capable of attaching to and directly penetrating the skin of a human or other suitable mammalian host. Upon entry into their new host, cercariae transform to the parasitic schistosomula stage and invade the vascular system. They, in turn, migrate to the lung and then to the hepatic vein where they mature to adult worms, form male and female pairs and travel to the mesenteric veins to complete their life cycle.
Successful intramolluscan or &#34;snail phase&#34; development of larval schistosomes is essential to continuation of the cycle of human host-to-host transmission. Of critical importance is the period following entry of the free-swimming miracidium into the snail host and its early transformation to the primary sporocyst stage. Depending on the physiological and immunological compatibility between host and parasite, it is during this phase of larval development that initial success or failure to establish infections is determined4,5,6. Subsequent development of primary sporocysts capable of asexually producing secondary sporocysts, which then generate human-infective cercariae, further require a permissive physiological host environment that provides for all of the parasite&#39;s growth and reproductive needs.
To date, little is known about the underlying physiological, biochemical and molecular processes controlling schistosome-snail interactions, especially the molecules and pathways involved in regulating larval development and reproduction, or modulating host immune responses. Ready access to large numbers of these larval stages under in vitro culture conditions that permit experimental manipulation would greatly facilitate the discovery of developmental pathways and underlying mechanisms of cell growth, differentiation and reproduction. Critical parasite pathways or mechanisms, identified through in vitro experimentation, could then be used to disrupt larval growth/reproduction in the snail host, or to identify snail host immune mechanisms involved in recognition and elimination of miracidial or sporocyst stages.
In this article and video presentation, we provide a detailed description of a method for isolating large numbers of free-swimming S. mansoni miracidia for introduction into in vitro culture that may then be subjected to follow-up experimental manipulation (see Figure 1 for a schematic overview of the protocol workflow). Although similar procedures have been described previously7,8,9, we felt that a detailed protocol would be useful to researchers who wish to employ this model to address still unanswered questions related to intramolluscan larval development, cellular proliferation, and schistosome-snail immune interactions. In addition, this approach could easily be adapted for isolation of eggs from other medically important trematodes such as bladder-dwelling S. haematobium, liver flukes or lung flukes.

<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

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.

The vast majority of miracidia typically will have been collected in the first two &#34;harvests&#34;. Incubation of isolated miracidia in CBSS+ triggers the miracidium-to-sporocyst transformation process (Figure 4A-C). Within the first hour following transfer to culture wells containing CBSS+, the majority of miracidia cease swimming (Figure 4A). At 6 h in culture, miracidia are in the process of actively sheddi...

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

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.

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 ...

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