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

Summary

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.

Abstract

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.

Introduction

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'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 "snail phase" 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 determined^4,5,6. 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'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 previously^7,8,9, 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.

Protocol

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 mice (6-week old) due to their higher susceptibility to infection¹⁰, thereby yielding larger egg burdens. Tucker et al. describe the mouse and snail infection protocols used in this model system¹¹. The Table of Materials lists all of the specific equipment, materials, reagents and animal sources needed to carry out this experimental protocol.

1. Processing of infected livers

1. Euthanize mice, 6-7 weeks post-infection, by CO₂ asphyxiation (rate of 10-30% of the chamber's volume per minute). Monitor mice for cessation of respiration and heartbeat.
   NOTE: Typically, up to 20 mice may be processed at a given time.
2. After transferring euthanized mice to a biosafety cabinet, place mice on their backs and spray their ventral surfaces with 70% ethanol. Let soak for 1 min.
3. Pinch and pull up skin of the lower abdomen and cut across the base of the pinched skin closest to the abdomen with sterile surgical scissors. Pull the cut skin anteriorly (i.e., toward the head) to reveal the liver. Note that the liver should be enlarged and speckled due to an egg-induced granulomatous response (Figure 2A).
4. Remove the liver and place it in a beaker containing 200 mL of sterile 1.2% NaCl solution containing pen/strep.
5. Repeat steps 1.3 and 1.4 with remaining mice.
   NOTE: Twenty mice are usually processed in a single harvest.
6. Place the livers in a sterile Petri dish and remove non-liver fat/connective tissues with sterile forceps.
7. Transfer trimmed/cleaned livers to a sterile 250-mL centrifuge bottle containing ~200 mL of sterile 1.2% NaCl solution with pen/strep.
8. Vigorously shake the bottle containing the livers and, using a sterile disposable pipette, remove the excess surface foam/floating debris. Slowly pour off the remaining saline solution into a container or sink containing 1% bleach (disinfectant).
9. Add ~200 mL of fresh saline solution to the livers and repeat step 1.8 three more times.

2. Liver extraction and miracidial isolation

1. Place the livers into a small sterile stainless-steel blender cup and add ~2 mL of 1.2% NaCl solution (Figure 2B).
2. Cover the cup with foil and a petri dish to prevent spills, and blend for 1 min by alternating low and high speed (20 s low speed/10 s high speed; repeat) (Figure 2C).
3. Evenly distribute the blended liver suspension into sterile 250-mL centrifuge bottles (for 20 livers use 4 bottles).
4. Add sterile saline with antibiotics to ~200 mL total volume/bottle. Weigh/balance bottles prior to centrifugation.
5. Centrifuge the blended livers at 290 x g for 15 min at 4 ^oC. Gently pour off supernatants into a container with bleach prior to discarding.
6. Repeat steps 2.4-2.5 once. Be sure to thoroughly resuspend liver sediment prior to centrifugation.
7. After discarding the last saline wash (Step 2.6), add 200 mL sterile pond water with pen/strep to the pelleted liver tissue, shake to resuspend sediment, and transfer the suspension to a sterilized 1-L volumetric flask that has been completely covered with aluminum foil, except for the top 3 cm of the neck (Figure 2D).
   NOTE: Use two bottles (= 10 livers) per 1-L flask. Pond water simulates the natural freshwater environment needed to stimulate miracidial hatching from eggs.
8. Using sterile pond water, fill up the flask to approximately 3 cm above the foil covering on neck. Then, without delay (as miracidia soon begin to hatch), remove residual foam and liver tissue floating to the surface by quickly pipetting up ~12 mL of pond water containing debris and discarding it into a separate discard tube. Replace with fresh sterile pond water.
9. Repeat cleaning Step 2.8, three more times, checking for presence of miracidia in the discard tube. Cease repetition of cleaning step if miracidia appear in the wash solution.
10. After the last wash, slowly add ~12 mL of warm sterile pond water (pre-warmed to 30 ^oC) to create a temperature gradient with clean, sterile warm water on top.
    NOTE: This is best accomplished by slowly discharging water along the side of the flask neck to avoid mixing.
11. Place a small Petri dish cover over the flask opening and shine a bright light across the top of the flask above the foil cover to illuminate the upper surface of water.
    NOTE: Typically, within 5-10 min, swimming miracidia, attracted to the light, will concentrate in large numbers within the first 2-3 cm of pond water (Figure 3A).

3. Miracidial harvesting and cultivation

1. When miracidia have amassed at the surface after 10 min, using a sterile transfer pipet, remove ~8 mL of pond water and transfer to a sterile 15-mL centrifuge tube. Place the tube containing miracidia on ice.
2. Add back 8 mL of warm sterile pond water along the inside of the volumetric flask and wait another 10 min.
3. Using new tubes each time, repeat steps 3.1 and 3.2 three more times. After the 4^th collection, keep the final tube on ice for 10 min to allow the miracidia to settle. This set of tubes comprises the first harvest.
4. Centrifuge tubes containing miracidia at 290 x g for 1 min at 4 ^oC in a pre-cooled refrigerated centrifuge.
5. Immediately remove most of the supernatant (~7 mL) being careful not to disturb the parasite pellet (Figure 3B).
6. Pool all the miracidia from this first harvest into one tube, then rinse all of the original tubes with ~1 mL sterile pond water and add to the "pooled" tube.
7. Allow parasites to settle on ice for ~5 min, and again centrifuge the parasites at 290 x g for 1 min at 4 ^oC.
8. Carefully remove the supernatant and add 6-8 mL sterile Chernin's balanced salt solution (CBSS+; see Table of Materials) containing pen/strep.
9. Gently suspend the miracidia and aliquot them into a 24-well culture plate (1 mL per well), followed by rinsing the tube in 6-8 mL CBSS+ and adding 1 mL of rinse to each well. Incubate parasites at 26 ^oC under normoxic conditions. Concentrations of isolated miracidia will vary, but will usually average ~7000/mL.
10. If parasites are still concentrating at the light source, repeat Steps 3.1 to 3.9 to continue harvesting late-hatching miracidia but increase the time interval between collections to 15-20 min.
    NOTE: This new set of tubes represents the second harvest. However, because miracidial numbers typically are low, larvae from all tubes usually are pooled into a single culture well containing 2 mL CBSS+.
11. At 24 h post-harvest and cultivation, gently resuspend sporocysts in their wells by pipetting.
12. Wait a few min to allow parasites to settle to the bottom of the wells. Once they have settled, carefully remove the supernatant (~1.5 mL), which will contain most of the ciliated epidermal plates shed by miracidia during larval transformation. This "transformation" supernatant may either be discarded or incorporated into follow-up studies of larval secretory products.
13. Add 1.5 mL of fresh CBSS+ to each well and gently pipette to resuspend sporocysts. Repeat step 3.12 if further washing or changing to a different medium is desired.

Results

The vast majority of miracidia typically will have been collected in the first two "harvests". 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...

Discussion

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, ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chernin's balanced salt solution (CBSS+)			For 1L of solution
2.8 g sodium chloride	Fisher Scientific	S271-3	Dissolve salts, except calcium chloride, and
0.15 g of potassium chloride	Sigma-Aldrich	P5405	sugars in 800 mL ddH2O
0.07 g sodium phosphate, dibasic anhydrous	Fisher Scientific	S374-500	Dissolve calcium chloride separately in 200
0.45 g magnesium sulfate heptahydrate	Sigma-Aldrich	M1880	mL ddH2O
0.53 g calcium chloride dihydrate	Mallinckrodt	4160	Slowly add calcium soln to the salt/sugar soln with
0.05 g sodium bicarbonate	Fisher Scientific	S233-3	with constant mixing
1 g glucose	MP Biomedicals	152527	Adjust to pH 7.2 and filter sterilize using a
1 g trehalose	Sigma-Aldrich	T0167	0.22 µm disposable bottle-top filter
10 mL 100X penicillin/streptomycin	Hyclone	SV30010	Add filtered penicillin and streptomycin soln  prior use
Incomplete Bge  medium (Ibge)			For 900 mL solution
220 mL Schneider’s Drosophila medium modified	Lonza	04-351Q	Mix Schneider's medium with 680 mL ddH2O
4.5 g lactalbumin enzymatic hydrolysate	Sigma-Aldrich	L9010	Add lactalbumin hydrolysate and galactose
1.3 g galactose	Sigma-Aldrich	G0625	Adjust to pH 7.2 and filter sterilize using a 0.22 µm pre-sterilized disposable bottle-top filter
Complete Bge medium (cBge)			For 100 mL of solution
90 mL Incomplete Bge medium			To heat-inactivate FBS: Incubate thawed FBS in
9 mL heat-inact. fetal bovine serum (FBS) (Optima)	Atlanta Biologicals	S12450	waterbath at 60°C for 1 hr while gently
1 mL 100X penicillin/streptomycin	Hyclone	SV30010	swirling the bottle every 10 min Aliquot heat-inactivated FBS into 15-mL tubes  and store at -20°C.  Mix medium + FBS and filter sterilize using a 0.22 µm pre-sterilized  disposable bottle-top filter  Add penicillin and streptomycin prior to use
Pond water (stock solution)			1L of stock solution
12.5 g calcium carbonate	Fisher Scientific	C64-500	Mix all salts in 1L of ddH2O
1.25 g magnesium carbonate	Fisher Scientific	M27-500	Note that the salts will not have completely
1.25 g  sodium chloride	Fisher Scientific	S271-3	dissolved.  Shake vigorously to suspend
0.25 g  potassium chloride	Sigma-Aldrich	P5405	salts prior to making the working soln
Pond water (working solution)			1.5L of solution
0.8 mL stock solution pond water (shake prior use) in			Mix stock to ddH2O
1500 mL of ddH2O			Sterilize pond water by autoclaving (slow cycle)
1.5 mL of 100X penicillin/streptomycin	Hyclone	SV30010	Add penicillin and streptomycin prior use
Saline solution (1.2% NaCl)			1.5L of solution
18 g sodium chloride in 1500 mL of ddH2O	Fisher Scientific	S271-3	Autoclave saline solution to sterilize
1.5 mL of 100X penicillin/ streptomycin	Hyclone	SV30010	Add penicillin and streptomycin prior use
Additional equipment and material:
7-L mouse euthanizing chamber			Following approved IACUC protocol no. V001551
Mice	Taconic Biosciences		Swiss-Webster, female, 6-wk old, murine
CO2 tank and regulator			pathogen-free
24-well tissue culture plate	TPP	92424
1-L volumetric flasks
Light source (150W)	Chiu Tech Corp	Model F0-150
Centrifuge, refrigerated, swinging bucket	Eppendorf	Model 5810R
Centrifuge bottles (250 mL)	Nalgene
15-mL centrifuge tubes, sterile	Corning	430053
Sterile disposable transfer pipets	Fisher Scientific	1371120
0.22 µm pre-sterilized disposable bottle-top filter	EMD Millipore	SCGPS05RE
Stainless steel blender	Waring Commercial	Model 51BL31
Blender cup, 100 mL capacity	Waring Commercial
Inverted compound microscope	Nikon Instruments	Eclipse TE300