Isolation, Culture and Cryopreservation of Sarcocystis species

S. K. Verma1, D. S. Lindsay2, M. E. Grigg3, J. P. Dubey1

1 United States Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Animal Parasitic Diseases Laboratory, Beltsville, Maryland, 2 Department of Biomedical Science and Pathology, Virginia–Maryland College of Veterinary Medicine, Blacksburg, 3 Molecular Parasitology Section, Laboratory of Parasitic Diseases, National Institutes of Health, National Institutes of Allergy, and Infectious Diseases, Bethesda, Maryland
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 20D.1
DOI:  10.1002/cpmc.32
Online Posting Date:  May, 2017
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Abstract

More than 200 valid Sarcocystis species have been described in the parasitological literature. The developmental life cycle in the intermediate host and definitive host has only been described for a few species. Sarcocystis parasites are common pathogens infecting a wide range of animals, including humans, and this unit reviews the methods used for isolating infective stages of the parasite from both definitive and intermediate host(s), as well as methods used to initiate cultures from sporocysts and merozoites and for cryopreservation of various Sarcocystis spp. These methods are based on published reports and our experience with Sarcocystis species in cell culture over many years. The information presented is suitable for the efficient culture of many Sarcocystis species; however, some minor modifications may be needed based on the unique developmental patterns of some species. © 2017 by John Wiley & Sons, Inc.

Keywords: cryopreservation; culture; host; parasite; isolation; Sarcocystis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Sarcocystis Sporocysts from the Intestine of Definitive Hosts
  • Basic Protocol 2: Isolation of Sarcocystis Sporocysts from the Feces of Definitive Hosts
  • Basic Protocol 3: Isolation of Sarcocystis Sporocysts from Water Samples
  • Basic Protocol 4: Sporocyst Excystation and In Vitro Culture of Merozoites
  • Basic Protocol 5: Examination of Muscles for Sarcocysts
  • Basic Protocol 6: Tissue Digestion to Detect Sarcocystis Infection by Releasing Bradyzoites from Sarcocysts
  • Basic Protocol 7: Isolation, Purification, and Cryopreservation of Infectious Bradyzoites
  • Basic Protocol 8: In Vitro Cultivation of Sarcocystis spp.
  • Alternate Protocol 1: Cell Culture in A 6‐ OR 24‐Well Tissue‐Culture‐Grade Plate
  • Basic Protocol 9: Cryopreservation Of Sarcocytis spp
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Sarcocystis Sporocysts from the Intestine of Definitive Hosts

  Materials
  • Definitive host (e.g., typically a carnivore or an omnivore)
  • Hanks’ balanced salt solution (HBSS; appendix 2A)
  • 5.25% sodium hypochlorite (bleach, e.g., Chlorox)
  • HBSS‐antibiotic mixture (see recipe)
  • Dissecting instruments:
    • surgical scissors
    • scalpels
    • forceps
  • Glass microscope slides
  • Plastic containers with lids
  • Commercial blender or meat grinder
  • Centrifuge
  • 250‐ml centrifuge bottles or 50‐ml conical centrifuge tubes
  • 22‐mm2 coverslips
  • Cheesecloth or 90‐µm wire‐mesh sieve
  • Hemacytometer
  • Microscope
  • Additional reagents and equipment for counting using a hemacytometer ( appendix 4A; Stevenson, )

Basic Protocol 2: Isolation of Sarcocystis Sporocysts from the Feces of Definitive Hosts

  Materials
  • Stool sample
  • 2 M sucrose solution (see recipe)
  • HBSS‐antibiotic mixture (see recipe)
  • 500‐ml plastic containers with lids
  • Tongue depressors
  • Shaker
  • Cheesecloth or 90‐µm wire‐mesh sieve
  • 250‐ml centrifuge bottles
  • Centrifuge
  • 50‐ml conical centrifuge tubes (e.g., Corning Falcon)
  • Microscope slides
  • 22‐mm2 coverslips
  • Microscope
  • Hemacytometer
  • 100‐, 90‐, and 45‐µm wire mesh sieves
  • Additional reagents and equipment for counting using a hemacytometer ( appendix 4A; Stevenson, )

Basic Protocol 3: Isolation of Sarcocystis Sporocysts from Water Samples

  Materials
  • Water sample
  • 2 M sucrose solution (see recipe)
  • HBSS‐antibiotic mixture (see recipe)
  • Centrifuge
  • 250‐ml centrifuge bottles
  • 50‐ml conical centrifuge tubes
  • Glass microscope slides
  • 22‐mm2 coverslips
  • Microscope
CAUTION: Water samples collected from the environment may contain zoonotic oocysts and sporocysts, so these preparations should be handled with extra care; wear gloves, glasses or eye protection, lab coat, and a face mask at all times.

Basic Protocol 4: Sporocyst Excystation and In Vitro Culture of Merozoites

  Materials
  • Sporocyst pellet ( protocol 1, 2, or 3)
  • 2.6% (v/v) NaOCl [1:1 bleach (Chlorox or Purox):water]
  • Normal saline (0.85% NaCl; see recipe) or phosphate‐buffered saline (PBS; appendix 2A)
  • Cell culture growth medium with 10% FBS (see recipe)
  • Excysting fluid (2×): HBSS, pH 7.2 to 7.4 ( appendix 2A) containing 0.5% (w/v) trypsin or chymotrypsin (activity of 1:250 or 1:300) and 1.5% (w/v) bile salt (sodium taurocholate obtained from the appropriate intermediate host such as bovine, ovine, etc.)
  • Cell culture maintenance medium with 2% FBS (see recipe)
  • Cell line to be tested (see annotation to step 6, below; also see Tables 20.1.1 and 20.1.2), growing in appropriate‐sized flasks
  • 1× saline‐antibiotic solution (see recipe for 2×)
  • 15‐ and 50‐ml conical centrifuge tubes
  • Centrifuge
  • Microscope
  • Hemacytometer
  • Cell culture flasks
  • 20‐, 23‐, and 27‐G needles
  • Sterile 5‐ or 10‐ml syringe
  • 5‐μm cell strainer
  • Additional reagents and equipment for counting using a hemacytometer ( appendix 4A; Stevenson, )
Table 0.0.1   MaterialsIn Vitro Culture of Sarcocystis spp. Gamonts aSpecies of Sarcocystis Schizonts Cultivated In Vitro

Species Host Type of cells successful Type of cells unsuccessful References
S. muris Mouse MDCK, FE, MF Not stated (Entzeroth, ; Entzeroth & Chobotar, )
S. muris Mouse CL, DK HF, SK (Becker, Mehlhorn, & Heydorn, )
S. suihominis Pig HF CL, DK, SK
S. tenella Sheep DK HF, SK, CL
S. capracanis Goat DK HF, SK, CL
Sarcocystis sp. Grackle MDCK, EBK, EBTr, ETK, ECM ECK (Fayer, ; Fayer, )
S. suihominis Pig HEI, HSF (Mehlhorn & Heydorn, )
Host Species Type of cells successful Type of cells unsuccessful Schizonts days References
Ox S. cruzi BM, CPA Mouse macrophages 18‐1, 320 (Andrews, Fayer, & Dubey, ; Speer & Burgess, ; Speer, Whitmire, Reduker, & Dubey, )
Ox S. hirsuta BPE BM 14‐62 (Cawthorn, Markham, Hitt, & Despres, )
Rat S. singaporensis Rat brain endothelial cells, rat pneumonocytes Hepatoma, fibroblats, myoblasts 3‐18 (Jäkel et al., ; Jäkel, Henke, Weingarten, Kliemt, & Seidinger, )
Sheep S. tenella BM, CPA MDBK, OM 14‐50 (Speer, Cawthorn, & Dubey, )
Goat S. capracanis BM MDBK, OM, CPA 60‐100 (Speer et al., )
Budgerigar Didelphis virginiana S. falcatula BT 3‐4 (Lindsay, Dubey, Horton, & Bowman, )
Budgerigar Didelphis albiventris S. falcatula BM 7 (Dubey, Venturini, Venturini, Basso, & Unzaga, )
Budgerigar Didelphis marsupialis S. falcatula CV‐1, BT EK 4 (Dubey et al., )
Budgerigar Didelphis albiventris S. falcatula BT, Vero ED, Hep‐2 4‐6 (Elsheikha, Saeed, Fitzgerald, Murphy, & Mansfield, ) (510)
Budgerigar Didelphis albiventris S. lindsayi EK, BT, CV‐1 2‐3 (Dubey, Lindsay, Rezende, & Costa, )
Mouse Didelphis virginiana S. speeri BM, EK CV‐1 3 (Dubey, Speer, & Lindsay, )
Mouse Didelphis marsupialis S. speeri BT 15 (Dubey, Kerber, Lindsay, Kasai, & Pena, )

 aMDCK: Madin‐Darby canine kidney; FE: feline embryo; CL: cat lung; DK: dog kidney; SK: swine kidney; MF: mouse fibroblasts; HF: human fibroblasts; EBK: embryonic bovine kidney; EBTr: embryonic bovine trachea; ECK: embryonic chicken kidney; ECM: embryonic chicken muscle; ETK: embryonic turkey kidney; HEI: human embryonic intestine; HSF: human skin fibroblasts.
Table 0.0.2   MaterialsIn Vitro Culture of Sarcocystis spp. Gamonts aSpecies of Sarcocystis Schizonts Cultivated In Vitro

Species Host Type of cells successful Type of cells unsuccessful References
S. muris Mouse MDCK, FE, MF Not stated (Entzeroth, ; Entzeroth & Chobotar, )
S. muris Mouse CL, DK HF, SK (Becker, Mehlhorn, & Heydorn, )
S. suihominis Pig HF CL, DK, SK
S. tenella Sheep DK HF, SK, CL
S. capracanis Goat DK HF, SK, CL
Sarcocystis sp. Grackle MDCK, EBK, EBTr, ETK, ECM ECK (Fayer, ; Fayer, )
S. suihominis Pig HEI, HSF (Mehlhorn & Heydorn, )
Host Species Type of cells successful Type of cells unsuccessful Schizonts days References
Ox S. cruzi BM, CPA Mouse macrophages 18‐1, 320 (Andrews, Fayer, & Dubey, ; Speer & Burgess, ; Speer, Whitmire, Reduker, & Dubey, )
Ox S. hirsuta BPE BM 14‐62 (Cawthorn, Markham, Hitt, & Despres, )
Rat S. singaporensis Rat brain endothelial cells, rat pneumonocytes Hepatoma, fibroblats, myoblasts 3‐18 (Jäkel et al., ; Jäkel, Henke, Weingarten, Kliemt, & Seidinger, )
Sheep S. tenella BM, CPA MDBK, OM 14‐50 (Speer, Cawthorn, & Dubey, )
Goat S. capracanis BM MDBK, OM, CPA 60‐100 (Speer et al., )
Budgerigar Didelphis virginiana S. falcatula BT 3‐4 (Lindsay, Dubey, Horton, & Bowman, )
Budgerigar Didelphis albiventris S. falcatula BM 7 (Dubey, Venturini, Venturini, Basso, & Unzaga, )
Budgerigar Didelphis marsupialis S. falcatula CV‐1, BT EK 4 (Dubey et al., )
Budgerigar Didelphis albiventris S. falcatula BT, Vero ED, Hep‐2 4‐6 (Elsheikha, Saeed, Fitzgerald, Murphy, & Mansfield, ) (510)
Budgerigar Didelphis albiventris S. lindsayi EK, BT, CV‐1 2‐3 (Dubey, Lindsay, Rezende, & Costa, )
Mouse Didelphis virginiana S. speeri BM, EK CV‐1 3 (Dubey, Speer, & Lindsay, )
Mouse Didelphis marsupialis S. speeri BT 15 (Dubey, Kerber, Lindsay, Kasai, & Pena, )

 aBM: bovine monocytes; BPE: bovine pulmonary endothelial; BT: bovine turbinates; CPA: cardiopulmonary endothelial cells; Viro and CV‐1: African green monkey kidney; ED: equine dermal; EK: equine kidney; Hep‐2: human hepatocytes; MDBK: Madin‐Darby bovine kidney; OM: ovine monocytes.
 bUnlike these species, S. neurona schizonts have been cultivated in many cell lines and from many hosts (Reviewed in Dubey et al., ).

Basic Protocol 5: Examination of Muscles for Sarcocysts

  Materials
  • Animal for inspection
  • Normal saline (0.85% NaCl; see recipe)
  • 10% (v/v) neutral buffered formalin solution (see recipe)
  • Glutaraldehyde buffer (for TEM or SEM preparation): 2.5% (v/v) to 3% (v/v) glutaraldehyde/0.05 M sodium cacodylate buffer, 0.05 M CaCl 2, pH 7
  • Dissecting instruments:
    • surgical scissors
    • scalpels
    • forceps
  • Glass slides
  • Containers with lids
  • 22‐mm2 coverslips
  • Microscope
  • Histology cassettes
  • Additional reagents and equipment for paraffin embedding and sectioning (e.g., Hofman & Taylor, )

Basic Protocol 6: Tissue Digestion to Detect Sarcocystis Infection by Releasing Bradyzoites from Sarcocysts

  Materials
  • Animal for inspection
  • Normal saline (0.85% NaCl; see recipe)
  • Digestion fluid: 1% (w/v) trypsin solution, pH 7.4, or HCl‐pepsin solution (see recipe)
  • 1.2% (w/v) sodium bicarbonate, pH 8.3
  • Phenol red
  • 1× saline‐antibiotic solution (see recipe for 2×) or HBSS‐antibiotic mixture (see recipe)
  • Dissecting instruments:
    • surgical scissors
    • scalpels
    • forceps
  • Commercial blender or meat grinder
  • Magnetic stirrer or shaking water bath
  • Cheesecloth
  • Centrifuge
  • 250‐ml centrifuge bottles
  • 50‐ml conical centrifuge tubes
  • Glass slides
  • 22‐mm2 coverslips
  • Microscope

Basic Protocol 7: Isolation, Purification, and Cryopreservation of Infectious Bradyzoites

  Materials
  • Infected tissue
  • Digestion fluid: 1% (w/v) trypsin solution, pH 7.4, or HCl‐pepsin solution (see recipe)
  • Normal saline (0.85% NaCl; see recipe) or Hanks’ balanced salt solution (HBSS; appendix 2A)
  • 1× saline‐antibiotic solution (see recipe for 2×) or HBSS‐antibiotic mixture (see recipe)
  • Isotonic Percoll solution: mix 9 parts of Percoll with 1 part 9% (w/v) NaCl solution
  • Dissecting instruments:
    • surgical scissors
    • scalpels
    • forceps
  • Commercial blender or meat grinder
  • Magnetic stirrer or shaking water bath
  • Cheesecloth
  • Centrifuge
  • Glass slides
  • 22‐mm2 coverslips
  • Microscope
  • Containers with lids
  • 250‐ml centrifuge bottles
  • 50‐ml conical centrifuge tubes

Basic Protocol 8: In Vitro Cultivation of Sarcocystis spp.

  Materials
  • Cell line (See Tables 20.1.1 and 20.1.2)
  • Cell culture growth medium with 10% FBS (see recipe)
  • Cell culture maintenance medium with 2% FBS (see recipe)
  • Hanks’ balanced salt solution (HBSS; appendix 2A)
  • 1× saline‐antibiotic solution (see recipe for 2×) or HBSS‐antibiotic mixture (see recipe)
  • 25‐cm2 culture flasks
  • 37°C humidified, 5% CO 2 incubator
  • 5 or 10 ml syringe
  • 18‐, 20‐, and 23‐G needles
  • Sterile 15‐ and 50‐ml conical screw‐cap centrifuge tubes
  • Sterile 2‐mm glass beads
  • Glass slides
  • 22‐mm2 coverslips
  • Microscope
  • Centrifuge

Alternate Protocol 1: Cell Culture in A 6‐ OR 24‐Well Tissue‐Culture‐Grade Plate

  Additional Materials (also see protocol 8)
  • 10% (v/v) neutral buffered formalin solution (see recipe)
  • Giemsa stain (Sigma‐Aldrich; cat. no. GS)
  • Permount
  • Round glass coverslips
  • 6‐ or 24‐well cell culture plates
  • Small forceps

Basic Protocol 9: Cryopreservation Of Sarcocytis spp

  Materials
  • 2× cryopreservation medium (see recipe)
  • Cell culture growth medium with 10% FBS (see recipe)
  • Parasites (see protocols above)
  • Liquid N 2
  • Hanks’ balanced salt solution (HBSS; appendix 2A)
  • Cell scrapers
  • Sterile 15‐ and 50‐ml conical centrifuge tubes
  • Refrigerated centrifuge
  • Cryovials
  • Mr. Frosty or similar freezing box
  • Liquid nitrogen storage tank
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Figures

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Literature Cited

Literature Cited
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