Schistosomiasis

Matthew S. Tucker1, Laksiri B. Karunaratne1, Fred A. Lewis1, Tori C. Freitas2, Yung‐san Liang1

1 Biomedical Research Institute, Rockville, Maryland, 2 Myriad RBM, Inc., Saranac Lake, New York
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 19.1
DOI:  10.1002/0471142735.im1901s103
Online Posting Date:  November, 2013
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Schistosomiasis is the second most important parasitic disease in the world in terms of public health impact. Globally, it is estimated that the disease affects over 200 million people and is responsible for 200,000 deaths each year. The three major schistosomes infecting humans are Schistosoma mansoni, S. japonicum, and S. haematobium. Much immunological research has focused on schistosomiasis because of the pathological effects of the disease, which include liver fibrosis and bladder dysfunction. This unit covers a wide range of aspects with respect to maintaining the life cycles of these parasites, including preparation of schistosome egg antigen, maintenance of intermediate snail hosts, infection of the definitive and intermediate hosts, and others. The unit primarily focuses on S. mansoni, but also includes coverage of S. japonicum and S. haematobium life cycles. Curr. Protoc. Immunol. 103:19.1.1‐19.1.58. © 2013 by John Wiley & Sons, Inc.

Keywords: schistosomiasis; snail; mansoni; japonicum; haematobium

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Biohazard Considerations
  • Basic Protocol 1: Percutaneous Exposure of Mice to Schistosoma Mansoni Cercariae via the Tail
  • Alternate Protocol 1: Abdominal Percutaneous Exposure of Mice to Schistosoma Mansoni Cercariae
  • Alternate Protocol 2: Injection of Mice with Schistosoma Mansoni Cercariae
  • Support Protocol 1: Collecting Schistosoma Mansoni Cercariae from Infected Snails
  • Basic Protocol 2: Abdominal Percutaneous Exposure of Mice to Schistosoma Japonicum Cercariae
  • Support Protocol 2: Collecting Schistosoma Japonicum Cercariae from Infected Oncomelania Hupensis ssp. Snails
  • Basic Protocol 3: Abdominal Percutaneous Exposure of Hamsters to Schistosoma Haematobium Cercariae
  • Support Protocol 3: Collecting Schistosoma Haematobium Cercariae from Infected Snails
  • Support Protocol 4: Snail Propagation and Maintenance
  • Support Protocol 5: Propogation and Maintenance of Oncomelania Hupensis ssp. as a Host for S. Japonicum
  • Support Protocol 6: Collecting, Isolating, and Hatching Oncomelania Hupensis ssp. Eggs
  • Support Protocol 7: Changing Containers of Snails
  • Support Protocol 8: Infection of Snails with Schistosoma spp. Miracidia
  • Basic Protocol 4: In Vitro Transformation of Schistosoma spp. Cercariae to Schistosomules by Vortexing
  • Alternate Protocol 3: In Vitro Transformation of Schistosoma spp. Cercariae to Schistosomules by Needle and Syringe
  • Alternate Protocol 4: Collection of Schistosoma Mansoni and Schistosoma Japonicum Schistosomules from Mouse Lungs
  • Basic Protocol 5: Culturing Schistosomules
  • Basic Protocol 6: Cryoperserving and Thawing Schistosomules
  • Basic Protocol 7: Collection of Adult Schistosome Worms from the Portal Venous Systems of Mice
  • Support Protocol 9: Perfusion of Adult Schistosoma Haematobium Worms from Hamsters
  • Basic Protocol 8: Enumeration of Schistosome Eggs from Mouse Tissues
  • Basic Protocol 9: Collection of Adult Schistosome Eggs and Injection into Mice to Induce Pulmonary Granuloma Formation
  • Basic Protocol 10: Preparation of Schistosoma spp. Crude Soluble Egg Antigen
  • Support Protocol 10: Preparation of Snail Food
  • Support Protocol 11: Preparation of Mud Base Medium for Growth of Nostoc sp. Algae and Navicula Pelliculosa Diatoms
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Percutaneous Exposure of Mice to Schistosoma Mansoni Cercariae via the Tail

  Materials
  • Cercariae (see protocol 4)
  • Conditioned water (see recipe)
  • Mice
  • 12 × 75–mm glass or plastic test tubes
  • Exposure racks: e.g., test tube racks of height such that 12 × 75–mm tubes are flush with top of rack (see Fig. )
  • Broome plastic restraining devices for mice (Harvard Apparatus; see Fig. )
  • Adhesive tape (Zonas Porous from Johnson & Johnson, 0.5‐in. width,)
  • Counting dish: 90 × 50–mm glass evaporating dish (Pyrex 3180) scored with a diamond pen
  • Dissecting microscope
  • Additional reagents and equipment for mouse handling and restraint (unit ) and iodine staining and counting of cercariae (see protocol 4)
CAUTION: Schistosomes are a biohazard. Workers should wear latex gloves at all times when handling schistosomal suspensions, infected snails, or any material associated with infected snails. Carefully review the discussion of Biohazard Considerations at the beginning of this unit before proceeding.

Alternate Protocol 1: Abdominal Percutaneous Exposure of Mice to Schistosoma Mansoni Cercariae

  Materials
  • Mice
  • Scale to weigh mice
  • Anesthetic (sodium pentobarbital) for cercarial exposure (see recipe)
  • Animal clippers, fitted with a #40 blade
  • Conditioned water (see recipe)
  • Gauze sponges
  • 10‐cm watch glass or custom‐made slotted boards for abdominal skin exposure (slots ∼1‐in. width)
  • Cercariae (see protocol 4)
  • Dissecting microscope
  • Sieve (made from PVC tubing measuring 10 mm in diameter × 20 mm high glued to a stainless steel wire mesh of 45‐µm size, Newark Wire, http://www.newarkwire.com/)
  • Petri dish (60 × 15 mm)
  • 18‐mm high × 10‐mm wide stainless steel ring (7 mm i.d.; can be obtained from standard plumbing supply vendors) (optional)
  • Strong light source (desk lamp)
  • Additional reagents and equipment for intraperitoneal injection of mice (unit )
CAUTION: Schistosomes are a biohazard. Workers should wear latex gloves at all times when handling schistosomal suspensions, infected snails, or any material associated with infected snails or infected animals. Carefully review the discussion of Biohazard Considerations at the beginning of this unit before proceeding.

Alternate Protocol 2: Injection of Mice with Schistosoma Mansoni Cercariae

  Materials
  • Cercariae (see protocol 4)
  • Mice
  • 1‐ml plastic syringes and 21‐G disposable hypodermic needles
  • Counting dish: 90 × 50–mm glass evaporating dish (Pyrex 3180) scored with a diamond pen
  • Dissecting microscope
  • Additional reagents and equipment for iodine staining and counting of cercariae (see protocol 4) and injection of mice (unit )
CAUTION: Schistosomes are a biohazard. Workers should wear latex gloves at all times when handling schistosomal suspensions, infected snails, or any material associated with infected snails. Carefully review the discussion of Biohazard Considerations at the beginning of this unit before proceeding.

Support Protocol 1: Collecting Schistosoma Mansoni Cercariae from Infected Snails

  Materials
  • Infected Biomphalaria glabrata snails (see protocol 13)
  • Conditioned water (see recipe)
  • Iodine solution (see recipe)
  • Mice
  • 100‐ml beakers
  • Incubator fitted with a strong light source
  • Featherweight forceps (Ward's Biological Supply, #14 V 0520) or small fish net
  • Filtration screen apparatus (if available) consisting of a 300‐ml funnel with glass support (Millipore, cat no XX1004703) and a 47‐mm‐diameter, 47‐µm mesh size stainless steel support screen (see Fig. )
  • Eppendorf blue 101‐ to 1000‐µl plastic pipet tips (or equivalent universal tip; VWR, cat. no. 83007‐376) and 100 to 1000 µl pipettor (e.g., Eppendorf Research)
  • Counting dish: 90 × 50–mm glass evaporating dish (Pyrex 3180) scored with a diamond pen
  • Dissecting microscope

Basic Protocol 2: Abdominal Percutaneous Exposure of Mice to Schistosoma Japonicum Cercariae

  Materials
  • Mice
  • Anesthetic (sodium pentobarbital) for cercarial exposure (see recipe)
  • Conditioned water (see recipe)
  • Scale to weigh mice
  • Animal clippers, fitted with a no. 40 blade
  • Gauze sponges
  • 10‐cm watch glass or custom‐made slotted boards for abdominal skin exposure (slots ∼1‐in. width)
  • Cercariae (see protocol 6)
  • Dissecting microscope
  • Fine forceps or small hypodermic needle (e.g., 25‐G) placed on a syringe
  • Petri dishes (60 × 15 mm)
  • Pasteur pipets
  • Hairloop (approximately 2 × 4 mm; see recipe) attached to a 23‐G needle fitted onto a 1‐ml syringe
  • Strong light source (desk lamp)
  • Additional reagents and equipment for intraperitoneal injection of mice (unit )

Support Protocol 2: Collecting Schistosoma Japonicum Cercariae from Infected Oncomelania Hupensis ssp. Snails

  Materials
  • Infected Oncomelania hupensis ssp. snails (see protocol 13)
  • Conditioned water (see recipe)
  • Mice
  • Petri dish (60 × 15 mm)
  • Featherweight forceps for handling snails (Ward's Biological Supply, cat. no. 14 V 0520)
  • Dissecting microscope
  • Strong light source (desk lamp)
  • Fine‐tip dissecting forceps and a small‐gauge needle

Basic Protocol 3: Abdominal Percutaneous Exposure of Hamsters to Schistosoma Haematobium Cercariae

  Materials
  • Hamsters
  • Anesthetic (sodium pentobarbital) for cercarial exposure (see recipe)
  • Conditioned water (see recipe)
  • Cercariae (see protocol 8)
  • Scale to weigh hamster and tissues
  • Animal clippers, fitted with no. 40 blade
  • Gauze sponges
  • 10‐cm watch glass
  • Strong light source (desk lamp)
  • Sieve (made from PVC tubing, measuring 10 mm in diameter × 20‐mm‐high glued to a stainless steel wire mesh of 20‐µm size; Newark Wire, http://www.newarkwire.com/)
  • 18‐mm high × 10‐mm wide stainless steel ring (7 mm i.d.; can be obtained from standard plumbing supply vendors; optional)
  • Petri dishes (100 × 25 mm)
  • Dissecting microscope
  • Additional reagents and equipment for intraperitoneal injection (unit )

Support Protocol 3: Collecting Schistosoma Haematobium Cercariae from Infected Snails

  Materials
  • Infected Bulinus spp. snails (see protocol 13)
  • Conditioned water (see recipe)
  • Hamsters
  • Plastic container with wire mesh glued on (3 mm × 3 mm square openings) to the top of the container and lid used for cleaning snails; the top and mesh‐covered lid are used for cleaning Bulinus snails prior to drying and also for rinsing snails in general (to remove rotifers); the snails are restrained between the two layers of wire mesh during rinsing (see protocol 8).
  • Featherweight forceps (Ward's Biological Supply, cat. no. 14 V 0520) or small fish net for manipulating snails
  • Sieve (made from PVC tubing measuring 10 mm in diameter × 20 mm high glued to a 20‐µm stainless steel wire mesh; Newark Wire, http://www.newarkwire.com/)
  • Spray apparatus (2‐gal deck sprayer, pump‐type, typically found in hardware stores)
  • Petri dish (100 × 25 mm)
  • Strong light source (desk lamp)

Support Protocol 4: Snail Propagation and Maintenance

  Materials
  • Biomphalaria glabrata snails (see above for source)
  • Conditioned water (see recipe)
  • Romaine lettuce
  • Cyanobacteria (Nostoc spp.; protocol 25)
  • Autoclaved mud, as nutrient source for growth of Nostoc ( protocol 25, optional)
  • Prepared snail food ( protocol 24, optional)
  • 10‐ or 30‐gallon (equivalent to 45‐ or 135‐liter) aquaria with under‐gravel filters and standard immersible aquarium heaters
  • Plastic mouse cages (polycarbonate, 11″ L × 9″ W × 6″ H , equivalent to 28 × 23 × 15 cm or 18.5″ L × 10″ W × 6″ H, equivalent to 47 × 25 × 15 cm) or small plastic snail‐rearing containers (plastic pans; 12″ L × 8.5″ W × 2.5″ H in., equivalent to 30.5 × 22 × 6.5 cm)
  • 400‐ml beakers
CAUTION: Schistosomes are a biohazard. Workers should wear latex gloves at all times when handling schistosomal suspensions, infected snails, or any material associated with infected snails. Carefully review the discussion of Biohazard Considerations at the beginning of this unit before proceeding.

Support Protocol 5: Propogation and Maintenance of Oncomelania Hupensis ssp. as a Host for S. Japonicum

  Materials
  • Conditioned water
  • Petri dish full of Nostoc sp. and mud ( protocol 25)
  • Petri dish full of Navicula pelliculosa diatoms ( protocol 25)
  • Lime (pulverized limestone)
  • Oncomelania hupensis ssp. snails (20 to 30 pairs, male plus female)
  • Suitable shallow plastic pans (or aquaria) for maintenance of snails (see above)
  • Glass lids for snail containers
  • Children's clay
  • Additional reagents and equipment for changing containers of snails ( protocol 12)

Support Protocol 6: Collecting, Isolating, and Hatching Oncomelania Hupensis ssp. Eggs

  Materials
  • Container of Oncomelania hupensis ssp. snails (20 to 30 pairs, male plus female; protocol 10)
  • Petri dish of Nostoc sp. and mud ( protocol 25)
  • Petri dish of Navicula pelliculosa diatoms ( protocol 25)
  • Conditioned water (see recipe)
  • Sieves with ~1‐mm and 0.5‐mm pore sizes
  • Spray apparatus (2‐gal deck sprayer pump‐type, typically found in hardware stores)
  • Small spatula
  • Petri dishes (60 × 15 mm)
  • Featherweight forceps (Ward's Biological Supply, #14 V 0520) or small fish net for manipulating snails

Support Protocol 7: Changing Containers of Snails

  Materials
  • Lime (pulverized limestone)
  • Conditioned water
  • Romaine lettuce
  • Petri dish of algae/mud ( protocol 25)
  • Petri dish of diatoms ( protocol 25)
  • Shallow pan or aquarium used for snails
  • Sieves: 1‐ to 2‐mm pore size for Oncomelania hupensis ssp. and juvenile Bulinus and Biomphalaria species, and > 2 mm for adult Bulinus and Biomphalaria species
  • Gauze pads

Support Protocol 8: Infection of Snails with Schistosoma spp. Miracidia

  Materials
  • S. mansoni–infected mice (see protocol 1, protocol 2, or protocol 3), S. japonicum‐infected mice (see protocol 5), or S. haematobium‐infected hamsters (see protocol 7)
  • Sodium pentobarbital with heparin (see recipe)
  • 1.2% (w/v) NaCl
  • Perfusion fluid (see recipe; optional)
  • Conditioned water (see recipe)
  • Juvenile Biomphalaria glabrata snails, 5 to 8 mm diameter, juvenile Oncomelania hupensis ssp. snails, 4 to 6 mm in length, or juvenile Bulinus truncatus truncatus snails, 2 to 3 mm in length (see protocol 9)
  • Waring blender with 300‐ml stainless steel container for blending
  • 50‐ml conical polypropylene centrifuge tubes
  • Tabletop centrifuge
  • 1‐liter side‐arm flask with all but the top few millimeters of the side arm painted black (see Fig. )
  • Light source (stereomicroscope lamp)
  • Hand‐held counter
  • Petri dishes (100 × 25 mm, 60 × 15 mm)
  • 400‐ml beaker
  • Additional reagents and equipment for intraperitoneal injection of mice (unit )
CAUTION: Schistosomes are a biohazard. Workers should wear latex gloves at all times when handling schistosomal suspensions, infected snails, or any material associated with infected snails. Carefully review the discussion of Biohazard Considerations at the beginning of this unit before proceeding.

Basic Protocol 4: In Vitro Transformation of Schistosoma spp. Cercariae to Schistosomules by Vortexing

  Materials
  • Suspension of Schistosoma spp. cercariae (see Support Protocols protocol 41, protocol 62, and protocol 83)
  • Serum‐free Dulbecco's minimum essential medium (DMEM; Life Technologies), or RPMI 1640 (Life Technologies), 4°C
  • Percoll gradient suspension (see recipe), 4°C
  • 50‐ml conical polypropylene centrifuge tubes (e.g., BD Falcon)
  • Refrigerated centrifuge
  • 250‐ml tissue culture flasks
CAUTION: Schistosomes are a biohazard. Workers should wear latex gloves at all times when handling schistosomal suspensions, snails, or any material associated with snails. Carefully review the discussion of Biohazard Considerations at the beginning of this unit before proceeding.

Alternate Protocol 3: In Vitro Transformation of Schistosoma spp. Cercariae to Schistosomules by Needle and Syringe

  Additional Materials (also see protocol 14)
  • 10‐ml plastic syringes with 22‐G disposable hypodermic needles
CAUTION: Schistosomes are a biohazard. Workers should wear latex gloves at all times when handling schistosomal suspensions, infected snails, or any material associated with infected snails. Carefully review the discussion of Biohazard Considerations at the beginning of this unit before proceeding.

Alternate Protocol 4: Collection of Schistosoma Mansoni and Schistosoma Japonicum Schistosomules from Mouse Lungs

  Materials
  • Schistosoma mansoni‐infected mice, exposed 5 to 7 days prior to procedure (see protocol 1, protocol 2, or protocol 3) or S. japonicum‐infected mice, exposed 3 to 5 days prior to procedure (see protocol 5)
  • Sodium pentobarbital with heparin (see recipe)
  • RPMI 1640 medium (Life Technologies) containing 10 U heparin/ml
  • RPMI 1640 medium (Life Technologies)
  • RPMI 1640 medium (Life Technologies) containing 100 U/ml penicillin and 100 mM streptomycin
  • Dissecting instruments (iris scissors and forceps)
  • 10‐ml syringe and 22‐G needles
  • 60 × 15–mm Petri dishes
  • 30‐ml beaker
  • 50‐ml plastic centrifuge tubes
  • 30‐mesh stainless steel screen (∼500‐µm openings) or cheesecloth
  • Refrigerated centrifuge
  • Dissecting microscope
  • Additional reagents and equipment for intraperitoneal injection of mice (unit )
CAUTION: Schistosomes are a biohazard. Workers should wear latex gloves at all times when handling schistosomal suspensions, snails, or any material associated with snails. Carefully review the discussion of Biohazard Considerations at the beginning of this unit before proceeding.

Basic Protocol 5: Culturing Schistosomules

  Materials
  • Schistosomules (see protocol 14 and Alternate Protocols protocol 153 and protocol 164)
  • Schistosomule Wash + Tween (SWAT; see recipe)
  • Schistosomulum Medium (SM; see recipe)
  • Packed heparinized mouse red blood cells [blood is collected into a heparinized tube; cells are washed twice with schistosomule wash buffer (SW; see recipe) and then stored at 4°C in SW containing 5 U/ml heparin; packed cells can be used for ∼1 week or until red blood cell lysis occurs]
  • Schistosomule Wash (SW; see recipe)
  • 6‐well tissue culture plates
  • 0.45‐µm syringe filter
  • 15‐ml conical centrifuge tube
  • Tabletop centrifuge

Basic Protocol 6: Cryoperserving and Thawing Schistosomules

  Materials
  • Schistosoma spp. cercariae (see Support Protocols protocol 41, protocol 62, and protocol 83)
  • Serum‐free Dulbecco's minimum essential medium (DMEM; Life Technologies) or RPMI 1640 medium (Life Technologies), 37°C
  • 20% and 60% ethylene glycol in serum‐free DMEM or RPMI
  • Chromerge cleaning solution (VWR, chromium trioxide dissolved in sulfuric acid)
  • Liquid N 2
  • Refrigerated centrifuge
  • 50‐ml conical polypropylene centrifuge tubes (e.g., BD Falcon)
  • 15‐ml centrifuge tubes or other small tubes with 1‐ml volume graduations
  • Aluminum weighing pans with aluminum foil lids
  • Liquid nitrogen storage vessels
  • Long forceps
  • Dissecting microscope

Basic Protocol 7: Collection of Adult Schistosome Worms from the Portal Venous Systems of Mice

  Materials
  • S. mansoni–infected mice (see protocol 1, protocol 2, or protocol 3) or S. japonicum‐infected mice (see protocol 5)
  • Sodium pentobarbital with heparin (see recipe)
  • Perfusion fluid (see recipe)
  • Dissecting instruments
  • Silicone tubing (e.g., Masterflex, cat. no. 96420‐14) fitted with a 20‐G, 1‐in. needle
  • Peristaltic perfusion pump (e.g., Masterflex Console Drive; Cole‐Parmer)
  • Foot‐pedal for peristaltic pump (e.g., Treadlite II/Linemaster Switch Corp., http://linemaster.com/)
  • Small plastic pans (7″ W × 11″ L × 2.5″ H) for collecting perfusate
  • Fluorescent light box
  • 100 × 25–mm plastic gridded Petri dish
  • Dissecting microscope
  • Additional reagents and equipment for intraperitoneal injection of mice (unit )
CAUTION: Schistosomes are a biohazard. Workers should wear latex gloves at all times when handling schistosomal suspensions, infected snails, or any material associated with infected snails. Carefully review the discussion of Biohazard Considerations at the beginning of this unit before proceeding.

Support Protocol 9: Perfusion of Adult Schistosoma Haematobium Worms from Hamsters

  Materials
  • S. haematobium–infected hamsters (see protocol 7)
  • Sodium pentobarbital with heparin (see recipe)
  • 70% ethanol
  • Perfusion fluid (see recipe)
  • Physiological saline: 0.85% (w/v) NaCl, sterile
  • Dissecting instruments including fine‐tipped dissecting forceps
  • Screen with clamps for anchoring hamster that can be placed over collecting tray
  • Silicone tubing (e.g., Masterflex 96420‐14) fitted with a 21‐G, 2‐in. needle
  • Peristaltic perfusion pump (e.g., Masterflex Console Drive; Cole‐Parmer)
  • Foot‐pedal for peristaltic pump (e.g., Treadlite II; Linemaster Switch Corp., http://linemaster.com/)
  • Small plastic pan or tray for collecting perfusate
  • Vacuum flask with tubing connected to a suction device
  • Suction device (double‐sided PVC tubing and outlet tubing connected to a sieve (PVC tubing measuring 10 mm in diameter × 20 mm high glued to a stainless steel wire mesh of 45‐µm size; Newark Wire, http://www.newarkwire.com/)

Basic Protocol 8: Enumeration of Schistosome Eggs from Mouse Tissues

  Materials
  • S. mansoni–infected mice (see protocol 1, protocol 2, or protocol 3), S. japonicum‐infected mice ( protocol 5), or S. haematobium–infected hamsters ( protocol 7)
  • Sodium pentobarbital with heparin (see recipe)
  • 1.2% (w/v) NaCl
  • 4% (w/v) KOH
  • Dissecting instruments
  • Scale to weigh mouse tissues
  • 400‐ml glass beakers
  • Dissecting microscope or compound microscope with 3× to 4× objective
  • Additional reagents and equipment for intraperitoneal injection of mice (unit )
CAUTION: Schistosomes are a biohazard. Workers should wear latex gloves at all times when handling schistosomal suspensions, infected snails, or any material associated with infected snails. Carefully review the discussion of Biohazard Considerations at the beginning of this unit before proceeding.

Basic Protocol 9: Collection of Adult Schistosome Eggs and Injection into Mice to Induce Pulmonary Granuloma Formation

  Materials
  • Schistosoma mansoni–infected mice (see protocol 1, protocol 2, or protocol 3), S. japonicum‐infected mice ( protocol 5), or S. haematobium‐infected hamsters ( protocol 7)
  • Sodium pentobarbital with heparin (see recipe)
  • 1.2% (w/v) NaCl, 4°C
  • Physiological saline: 0.85% (w/v) NaCl, sterile
  • Uninfected mice
  • Alcohol swabs
  • Dissecting instruments
  • Waring blender, with variable speed control and 300‐ml stainless steel container for blending
  • Stainless steel sieves of decreasing pore sizes (Newark Wire Cloth; mesh openings of 420 µm, 180 µm, 105 µm, and 45 µm‐ see Fig. )
  • Spray apparatus (2‐gal deck sprayer pump‐type, typically found in hardware stores)
  • Glass Petri dishes (100‐mm diameter) with flat bottoms
  • Light box
  • Falcon #2340 cell strainer, 40 µm nylon (see Fig. ), or sieve (made from PVC tubing measuring 10 mm in diameter × 20 mm high glued to a stainless steel wire mesh of 45‐µm size, Newark Wire, http://www.newarkwire.com/)
  • 15‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Mouse restrainer
  • 1‐ml disposable syringe with 23‐ or 25‐G needle
  • Gauze pads
  • Additional reagents and equipment for enumeration of eggs (see protocol 21), mouse handling and restraint (unit ), injection of mice (unit ), and immunohistochemistry (unit )
CAUTION: Schistosomes are a biohazard. Workers should wear latex gloves at all times when handling schistosomal suspensions, infected snails, or any material associated with infected snails or infected animals. Carefully review the discussion of Biohazard Considerations at the beginning of this unit before proceeding.

Basic Protocol 10: Preparation of Schistosoma spp. Crude Soluble Egg Antigen

  Materials
  • Purified Schistosoma spp. eggs (see protocol 22, steps 1 to 7)
  • Phosphate‐buffered saline, pH 7.4 (PBS; ), 4°C
  • Protein assay kit
  • Hand‐held Potter‐Elvehjem glass homogenizer (15‐ml capacity) with tight pestle, prechilled
  • Dissecting microscope
  • 15‐ or 50‐ml centrifuge tubes
  • Refrigerated centrifuge
  • Ultracentrifuge
  • 10‐ml disposable syringe with 0.2‐µm pore‐size syringe filter
CAUTION: Schistosomes are a biohazard. Workers should wear latex gloves at all times when handling schistosomal suspensions, snails, or any material associated with snails. Carefully review the discussion of Biohazard Considerations at the beginning of this unit before proceeding.

Support Protocol 10: Preparation of Snail Food

  Materials
  • 8 g barley grass powder (available at health food stores)
  • 2.0 g wheat germ (available at grocery stores)
  • 2.0 g fish food (Tetramin large tropical flakes, available at pet stores)
  • 1.0 g powdered milk (available at grocery stores)
  • 2.0 g sodium alginate (alginic acid sodium salt, medium viscosity, sold as a fine powder; Sigma, cat. no. A‐2033)
  • 2% (w/v) calcium chloride solution (Sigma, cat. no. C‐4901, anhydrous)
  • 1‐liter beaker
  • Mortar and pestle
  • 7‐in. × 9‐in. × 6‐in. plastic pans
  • Glass plates

Support Protocol 11: Preparation of Mud Base Medium for Growth of Nostoc sp. Algae and Navicula Pelliculosa Diatoms

  Materials
  • Mud, or soil source (see above)
  • Lime (pulverized limestone)
  • Dried chicken manure
  • Clay
  • Conditioned water (see recipe), autoclaved
  • 0.06% (w/v) sodium nitrate solution prepared with conditioned water (only needed if the soil is collected from nutrient‐poor locations), sterile
  • Nostoc sp. (stock cultures can be obtained from Ward's Biological Supply, Rochester, N.Y.)
  • Series of crude screens (7‐mm and 2‐mm pore sizes)
  • Stainless steel baking pan (10 × 15 × 3 in.)
  • Plastic bucket
  • Photography developing pans measuring 62 cm × 44 cm × 8 cm
  • Plastic Petri dishes (100 × 25 mm)
  • Spatulas
  • 40‐W, cool‐white fluorescent light
  • Diatoms (Navicula pelliculosa purchased from Ward's Scientific)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Basch, P.F. 1981. Cultivation of Schistosoma mansoni in vitro. I. Establishment of cultures from cercariae and development until pairing. J. Parasitol. 67:179‐185.
   Boros, D.L. and Warren, K.S. 1970. Delayed hypersensitivity granuloma formation and dermal reaction induced and elicited by a soluble factor isolated from Schistosoma mansoni eggs. J. Exp. Med. 132:488‐507.
   Bruce, J.I. and Liang, Y.‐S. 1992. Cultivation of schistosomes and snails for researchers in the United States of America and other countries. J. Med. Appl. Malacol. 4:13‐30.
   Bruce, J.I. , Radke, M.G. , and Davis, G.M. 1971. Culturing Biomphalaria and Oncomelania (Gastropoda) for large‐scale studies of schistosomiasis. Biomedical Report No. 19, 406th Medical Laboratory, U.S. Army.
   Cai, Y. , Langley, J.G. , Smith, D.I. , and Boros, D.L. 1996. A cloned major Schistosoma mansoni egg antigen with homologies to small heat shock proteins elicits Th1 responsiveness. Infect. Immun. 64:1750‐1755.
   Campbell, W.C. and Cuckler, A.C. 1961. The prophylactic effect of topically applied cedarwood oil on infection with Schistosoma mansoni in mice. Am. J. Trop. Med. Hyg. 10:712‐715.
   Cheever, A.W. 1970. Relative resistance of the eggs of human schistosomes to digestion in potassium hydroxide. Bull. WHO 43:601‐603.
   Cheever, A.W. 1987. Comparison of pathologic changes in mammalian hosts infected with Schistosoma mansoni, S. japonicum and S. haematobium . Mem. Inst. Oswaldo Cruz 82:39‐45.
   Cheever, A.W. and Yap, G.S. 1997. Immunologic basis of disease and disease regulation in schistosomiasis. In Immunopathogenetic Aspects of Disease Induced by Helminth Parasites ( D.O. Freedman , ed.) pp. 159‐176. S. Karger, Basel, Switzerland.
   Chernin, E. and Michelson, E.H. 1957. Studies on the biological control of schistosome‐bearing snails. III. The effects of population density on the growth and fecundity in Australorbis glabratus . Am. J. Hyg. 65:57‐70.
   Cohen, L.M. , Neimark, H. , and Eveland, L.K. 1980. Schistosoma mansoni: Response of cercariae to a thermal gradient. J. Parasitol. 66:363‐364.
   Colley, D.G. and Wikel, S.K. 1974. Schistosoma mansoni: Simplified method for the production of schistosomules. Exp. Parasitol. 35:44‐51.
   Cooper, L.A. , Lewis, F.A. , and File‐Emperador, S. 1989. Re‐establishing a life cycle of Schistosoma mansoni from cryopreserved larvae. J. Parasitol. 75:353‐356.
   Cousin, C.E. , Stirewalt, M.S. , and Dorsey, C.H. 1981. Schistosoma mansoni: Ultrastructure of early transformation of skin‐ and shear‐pressure‐derived schistosomules. Exp. Parasitol. 51:341‐365.
   Damian, R.T. 1984. Immunity in schistosomiasis: A holistic view. In Contemporary Topics in Immunobiology ( J.J. Marchalonis , ed.) pp. 359‐420. Plenum, New York.
   deWalick, S. , Tielens, A.G. , and van Hellemond, J.J. 2012. Schistosoma mansoni: The egg, biosynthesis of the shell and interaction with the host. Exp. Parasitol. 132:7‐13.
   Duvall, R.H. and DeWitt, W.B. 1967. An improved perfusion technique for recovering adult schistosomes from laboratory animals. Am. J. Trop. Med. Hyg. 16:483‐486.
   Edungbola, L.D. and Schiller, E.L. 1979. Histopathology of hepatic and pulmonary granulomata experimentally induced with eggs of Schistosoma mansoni . J. Parasitol. 65:253‐261.
   Everts, B. , Perona‐Wright, G. , Smits, H. H. , Hokke, C.H. , van der Ham, A.J. , Fitzsimmons, C.M. , Doenhoff, M.J. , van der Bosch, J. , Mohrs, K. , Haas, H. , Mohrs, M. , Yazdanbakhsh, M. , and Schramm, G. 2009. Omega‐1, a glycoprotein secreted by Schistosoma mansoni eggs, drives Th2 responses. J. Exp. Med. 206:1673‐1680.
   Gobert, G.N. , Chai, M. , McManus, D.P. 2007. Biology of the schistosome lung‐stage schistosomulum. Parasitology 134:453‐460.
   Gui, M. , Kusel, J.R. , Shi, Y.E. , and Ruppel, A. 1995. Schistosoma japonicum and S. mansoni: Comparison of larval migration patterns in mice. J. Helminthol. 69:19‐25.
   James, E.R. 1981. Schistosoma mansoni: Cryopreservation of schistomules by two‐step addition of ethanediol and rapid cooling. Exp. Parasitol. 52:105‐116.
   James, E.R. and Farrant, J. 1977. Recovery of infective Schistosoma mansoni schistosomula from liquid nitrogen. A step towards storage of a live schistosomiasis vaccine. Trans. R Soc. Trop. Med. Hyg. 71:498‐500.
   Lazdins, J.K. , Stein, M.J. , David, J.R. , and Sher, A. 1982. Schistosoma mansoni: Rapid isolation and purification of schistosomula of different developmental stages by centrifugation on discontinuous density gradients of percoll. Exp Parasitol. 53:39‐44.
   Lewis, F.A. 1998. Schistosomiasis. Curr. Protoc. Immunol. 28:19.1.1‐19.1.28. Available at http://onlinelibrary.wiley.com/doi/10.1002/0471142735.im1901s28/full.
   Lewis, F.A. and Colley, D.G. 1977. Modification of the lung recovery assay for schistosomula and correlations with worm burdens in mice infected with Schistosoma mansoni . J. Parasitol. 63:413‐417.
   Lewis, F.A. , Stirewalt, M.A. , Souza, C.P. , and Gazzinelli, G. 1986. Large‐scale laboratory maintenance of Schistosoma mansoni, with observations on three schistosome/snail host combinations. J. Parasitol. 72:813‐829.
   Lewis, F.A. , White‐Ziegler, C.A. , Ball, J.E. , and Niemann, G.M. 1990. Schistosoma mansoni larvacidal activity of murine bronchoalveolar lavage cells. Infect. Immun. 58:3903‐3908.
   Liang, Y.‐S. 1974. Cultivation of Bulinus (Physopsis) globosus (Morelet) and Biomphalaria pfeifferi pfeifferi (Krauss), snail hosts of schistosomiasis. Sterkiana 54:7‐73.
   Liang, Y.‐S. , van der Schalie, H. , and Berry, E.G. , 1973. Transmission of ostracods in snails. Malacol. Rev. 6:66.
   Liang, Y.‐S. , Bruce, J.I. , and Boyd, D.A. 1987. Laboratory cultivation of schistosome vector snails and maintenance of schistosome life cycles. Proc. First Sino‐Am. Sym. 1:34‐48.
   Lo, C.T. 1967. The inhibiting action of ostracods on snail cultures. Trans. Am. Microsc. Soc. 86:402‐405.
   Lukacs, N.W. and Boros, D.L. 1991. Splenic and granuloma T‐lymphocyte responses to fractionated soluble egg antigens of Schistosoma mansoni–infected mice. Infect. Immun. 59:941‐948.
   Lustigman, S. , Mahmoud, A.A.F. , and Hamburger, J. 1985. Glycoproteins in soluble egg antigen of Schistosoma mansoni: Isolation, characterization, and elucidation of their immunochemical and immunopathological relation to the major egg glycoprotein (MEG). J. Immunol. 134:1961‐1971.
   Michaelson, E.H. 1964. The protective action of Chaetogaster limnaei on snails exposed to Schistosoma mansoni . J. Parasitol. 50:441‐444.
   Meevissen, M.H. , Yazdanbakhsh, M. , and Hokke, C.H. 2012. Schistosoma mansoni egg glycoproteins and C‐type lectins of host immune cells: Molecular partners that shape immune responses. Exp Parasitol. 132:14‐21.
   Moloney, N.A. , Hinchcliffe, P. , and Webbe, G. 1987. The simple laboratory maintenance of a highly productive Schistosoma japonicum life cycle. Trans. R Soc. Trop. Med. Hyg. 81:67‐68.
   Moore, D.V. and Meleney, H.E. 1954. Comparative susceptibility of common laboratory animals to experimental infection with Schistosoma haematobium . J. Parasitol. 40:392‐397.
   Moore, D.V. , Thillet, C.J. , Carney, D.M. , and Meleney, H.E. 1953. Experimental infection of Bulinus truncatus with Schistosoma haematobium . J. Parasitol. 39:215‐221.
   Najarian, H.H. 1961. Biological studies on the snail, Bulinus truncatus, in central Iraq. Bull. World Health Organ. 25:435‐446.
   Oswald, I.P. , Caspar, P. , Jankovic, D. , Wynn, T.A. , Pearce, E.J. , and Sher, A. 1994. IL‐12 inhibits Th2 cytokine responses induced by eggs of Schistosoma mansoni . J. Immunol. 153:1707‐1713.
   Pearce, E.J. 2005. Priming of the immune response by schistosome eggs. Parasite Immunol. 27:265‐270.
   Pearce, E.J. and James, S.L. 1986. Post lung‐stage schistosomula of Schistosoma mansoni exhibit transient susceptibility to macrophage‐mediated cytotoxicity in vitro that may relate to late phase killing in vivo . Parasite Immunol. 8:513‐527.
   Perez, H. , Clegg, J.A. , and Smithers, S.R. 1974. Acquired immunity to Schistosoma mansoni in the rat: Measurement of immunity by the lung recovery technique. Parasitology 69:99‐116.
   Ramalho‐Pinto, F.J. , Gazzinelli, G. , Howells, R.E. , Monta‐Santos, T.A. , Figueiredo, E.A. , and Pellegrino, J. 1974. Schistosoma mansoni: A defined system for the stepwise transformation of a cercaria to schistosomule in vitro . Exp. Parasitol. 36:360‐372.
   Richards, C.S. and Shade, P.C. 1987. The genetic variation of compatibility in Biomphalaria glabrata and Schistosoma mansoni . J. Parasitol. 73:1146‐1151.
   Rowan, W.B. 1958. Mass cultivation of Australorbis glabratus, intermediate host of Schistosoma mansoni in Puerto Rico. J. Parasitol. 44:247.
   Sher, F.A. , MacKenzie, P. , and Smithers, S.R. 1974. Decreased recovery of invading parasites from the lungs as a parameter of acquired immunity to schistosomiasis in the laboratory mouse. J. Infect. Dis. 130:626‐634.
   Sher, A. , Jankovic, D. , Cheever, A. , and Wynn, T. 1996. An IL‐12 based vaccine approach for preventing immunopathology in schistosomiasis. Interleukin 12:288‐301.
   Smithers, S.R. and Terry, R.J. 1965. The infection of laboratory hosts with cercariae of Schistosoma mansoni and the recovery of the adult worms. Parasitology 55:695‐700.
   Sodeman, W.A. Jr. and Dowda, M. 1973. Laboratory maintenance of Bulinus vectors of Schistosoma haematobium . Am. J. Trop. Med. Hyg. 22:678‐681.
   Standen, O.D. 1951. Some observations upon the maintenance of Australorbis glabratus in the laboratory. Ann. Trop. Med. Parasitol. 45:80‐83.
   Steinfelder, S. , Andersen, J.F. , Cannons, J.L. , Feng, C.G. , Joshi, M. , Dwyer, D. , Caspar, P. , Schwartzberg, P.L. , Sher, A. , and Jankovic, D. 2009. The major component in schistosome eggs responsible for conditioning dendritic cells for Th2 polarization is a T2 ribonuclease (omega‐1). J. Exp. Med. 206:1681‐1690.
   Stirewalt, M.A. 1954. Effect of snail maintenance temperatures on development of Schistosoma mansoni . Exp. Parasitol. 3:504‐516.
   Stirewalt, M.A. 1974. Schistosoma mansoni: Cercaria to schistosomule. In Advances in Parasitology ( B. Dawes , ed.) pp. 115‐182. Academic Press, London.
   Stirewalt, M.A. and Lewis, F.A. 1981. Schistosoma mansoni: Effect of rotifers on cercarial output, motility and infectivity. Int. J. Parasitol. 11:301‐308.
   Stirewalt, M.A. , Shepperson, J.R. , and Lincicome, D.R. 1965. Comparison of penetration and maturation of Schistosoma mansoni in four strains of mice. Parasitology 55:227‐235.
   Stirewalt, M.A. , Cousin, C.E. , and Dorsey, C.H. 1983. Schistosoma mansoni: Stimulus and transformation of cercariae into schistosomules. Exp. Parasitol. 56:358‐368.
   Stirewalt, M. , Lewis, F.A. , Cousin, C.E. , and Leef, J.L. 1984. Cryopreservation of schistosomules of Schistosoma mansoni in quantity. Am. J. Trop. Med. Hyg. 33:116‐124.
   Warren, K.S. , Boros, D.L. , Hang, L.M. , and Mahmoud, A.A. 1975. The Schistosoma japonicum egg granuloma. Am. J. Pathol. 80:279‐294.
   Weiss, J.B. , Magnani, J.L. , and Strand, M. 1986. Identification of Schistosoma mansoni glycolipids that share immunogenic carbohydrate epitopes with glycoproteins. J. Immunol. 136:4275‐4282.
   Wilson, R.A. and Coulson, P.S. 1989. Lung‐phase immunity to schistosomes: A new perspective on an old problem. Parasitol. Today 5:274‐278.
   Wright, C.A. 1960. The crowding phenomenon in laboratory colonies of freshwater snails. Ann. Trop. Med. Parasitol. 54:224‐232.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library