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 fema....

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.

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 shedding their.......

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