Animal Model of Nippostrongylus brasiliensis and Heligmosomoides polygyrus

Mali Camberis1, Graham Le Gros1, Joseph Urban2

1 Malaghan Institute of Medical Research, Wellington, 2 Nutrient Requirements and Functions Laboratory, Beltsville Human Nutrition Research Center, U.S. Department of Agriculture, Beltsville, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 19.12
DOI:  10.1002/0471142735.im1912s55
Online Posting Date:  August, 2003
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Abstract

Animal models of Nippostrongylus brasiliensis and Heligmosomoides polygyrus infection are powerful tools for the investigation of the basic biology of immune responses and protective immunity. In particular, they model the induction and maintenance of Th2 type immune responses and exhibit all the requisite hallmarks of CD4 T cell–dependent IgE production, eosinophilia, mastocytosis, and mucus production. This chapter describes simple, cost‐effective techniques for using and maintaining these easy‐to‐work‐with parasites in the context of a modern laboratory.

Keywords: immunoparasitology; nematodes; Nippostrongylus brasiliensis; Heligmosomoides polygyrus; Th2 immune responses; cytokines; mucosal immunity; allergic responses; IL4; IL5; IL9; IL13; IgE; eosinophils; mastocytosis; protective immunity; worm migration; parasitic diseases; inflammatory responses; nematode biology

     
 
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Table of Contents

  • Maintenance and Propagation of Nippostrongylus brasiliensis
  • Basic Protocol 1: Inoculation of Rats with Nippostrongylus brasiliensis Third‐Stage (L3) Infective Larvae
  • Basic Protocol 2: Collection, Counting, and Culturing of Nippostrongylus brasiliensis Eggs and Larvae from Infected Rats
  • Basic Protocol 3: Harvesting of Nippostrongylus brasiliensis Third‐Stage (L3) Infective Larvae from Fecal Cultures
  • Alternate Protocol 1: Modified Baermann Methods for Harvesting Nippostrongylus brasiliensis Third‐Stage (L3) Infective Larvae from Fecal Cultures
  • Basic Protocol 4: Storage of Nippostrongylus brasiliensis Third‐Stage (L3) Infective Larvae
  • Basic Protocol 5: Mouse Model of Nippostrongylus brasiliensis Infection: Inoculation of Mice with Nippostrongylus brasiliensis Third‐Stage (L3) Infective Larvae
  • Basic Protocol 6: Enumeration of Nippostrongylus brasiliensis Egg Production in Infected Mice
  • Alternate Protocol 2: Quick Method for Determining Egg Counts in Individual Mice
  • Basic Protocol 7: Collection of Nippostrongylus brasiliensis Fourth‐Stage (L4) Lung Larvae
  • Alternate Protocol 3: Broncheoalveolar Lavage as an Alternative Method for the Recovery of Nippostrongylus brasiliensis Fourth‐Stage (L4) Lung Larvae
  • Basic Protocol 8: Collection of Nippostrongylus brasiliensis Adult Worms (L5)
  • Basic Protocol 9: Recovery of Nippostrongylus brasiliensis Third‐Stage (L3) Infective Larvae from the Site of Infection
  • Support Protocol 1: Preparation of Nippostrongylus brasiliensis Antigen (NES)
  • Maintenance and Propagation of Heligmosomoides polygyrus
  • Basic Protocol 10: Inoculation of Mice with Heligmosomoides polygyrus Infective Larvae
  • Basic Protocol 11: Collection, Counting, and Culturing of Heligmosomoides polygyrus Eggs from Fecal Pellets of Infected Mice
  • Basic Protocol 12: Harvesting and Storage of Heligmosomoides polygyrus Infective L3 from Fecal Cultures
  • Basic Protocol 13: Obtaining Heligmosomoides polygyrus Adult Worms and Antigen
  • Support Protocol 2: Measurement of CD4+ T Cell–Dependent Primary and Secondary Immune Responses to Heligmosomoides polygyrus
  • Support Protocol 3: Immunological Assays of Cytokines, Parasite‐Specific Antibody Production, Eosinophilia, Mucosal Mastocytosis, Goblet Cell Hyperplasia, and Mucus Production
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Inoculation of Rats with Nippostrongylus brasiliensis Third‐Stage (L3) Infective Larvae

  Materials
  • Nippostrongylus brasiliensis third‐stage (L3) infective larvae (see protocol 3 and protocol 4)
  • PBS ( appendix 2A), sterile
  • Female Lewis rats, 4 months old, or female Sprague‐Dawley rats with a minimum weight of 150 g
  • 5‐ml sterile plastic bottles
  • 1‐ml disposable syringes and 21‐ or 23‐G needles
  • Additional reagents and equipment for rat restraint (unit 1.3) and injection (unit 1.6)

Basic Protocol 2: Collection, Counting, and Culturing of Nippostrongylus brasiliensis Eggs and Larvae from Infected Rats

  Materials
  • Nippostrongylus brasiliensis–infected rats (see protocol 1)
  • Saturated NaCl solution
  • Coarsely granulated charcoal, autoclaved
  • Sphagnum moss (available from garden supply stores), cut into small pieces and autoclaved
  • Cage with wire‐grid floor (Tecniplast USA)
  • Paper towels
  • 100‐ and 250‐ml sterile containers
  • Spoon or spatula
  • McMaster worm egg counting chamber (J.A. Whitlock & Co.)
  • Gridded coverslip or glass sheet scored with a diamond pen
  • Stereoscopic microscope
  • 140‐mm sterile petri dishes
  • Sponge‐cut into 4 × 4–cm pieces (dishwashing cloth or cotton wool can also be used), autoclaved
  • 110‐mm Whatman filter paper circles
  • Plastic lidded container large enough to hold culture plates, lined with moistened paper towels or dishcloth
  • Incubator set at 26°C, without CO 2 (room temperature benchtop incubation is adequate)

Basic Protocol 3: Harvesting of Nippostrongylus brasiliensis Third‐Stage (L3) Infective Larvae from Fecal Cultures

  Materials
  • Nippostrongylus brasiliensis fecal culture dish (see protocol 2)
  • 25‐ml sterile LabServ plastic container with conical bottom or other sterile universal container
  • Gridded petri dish or glass sheet scored with a diamond pen
  • Stereoscopic microscope

Alternate Protocol 1: Modified Baermann Methods for Harvesting Nippostrongylus brasiliensis Third‐Stage (L3) Infective Larvae from Fecal Cultures

  Materials
  • Phosphate‐buffered saline (PBS; appendix 2A; optional)
  • Nippostrongylus brasiliensis fecal culture dishes (see protocol 2)
  • 10 × 13–cm glass filter funnel
  • 1.5 × 10–cm plastic tubing
  • 5 ml (1.2 × 7.5–cm) plastic collection tube
  • Stand and clamp
  • Cheesecloth cut into 8 × 8–cm squares
  • Additional reagents and equipment for counting larvae (see protocol 3)

Basic Protocol 4: Storage of Nippostrongylus brasiliensis Third‐Stage (L3) Infective Larvae

  Materials
  • Nippostrongylus brasiliensis L3 larvae harvested in 5 ml water (see protocol 3)
  • 100‐mm Whatman filter paper circles
  • 25‐ml sterile LabServ plastic container with conical bottom or other universal container with lid

Basic Protocol 5: Mouse Model of Nippostrongylus brasiliensis Infection: Inoculation of Mice with Nippostrongylus brasiliensis Third‐Stage (L3) Infective Larvae

  Materials
  • Nippostrongylus brasiliensis infective L3 larvae (see protocol 3)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 6‐ to 8‐week‐old C57Bl/6 or Balb/c mice
  • 25‐ml sterile LabServ plastic containers with conical bottoms or other universal plastic containers
  • 1‐ml syringes with 21‐ or 23‐G needles
  • Additional reagents and equipment for counting larvae (see protocol 3), mouse restraint (unit 1.3), and injection of mice (unit 1.6)

Basic Protocol 6: Enumeration of Nippostrongylus brasiliensis Egg Production in Infected Mice

  Materials
  • N. brasiliensis–infected mice (See protocol 6)
  • 1.5‐ml microcentrifuge tubes
  • Analytical balance
Additional reagents and equipment for mouse restraint (unit 1.3) and enumeration of worm eggs (see protocol 2)

Alternate Protocol 2: Quick Method for Determining Egg Counts in Individual Mice

  Materials
  • N. brasiliensis–infected mice (see protocol 6)
  • 70% ethanol
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 1% (w/v) agarose
  • Dissecting instruments including iris scissors and forceps
  • 6‐well plastic tissue culture dishes
  • Cheesecloth cut into 8 × 8–cm squares
  • 8 × 8–cm plastic disposable weigh boats
  • 50‐ml centrifuge tubes and corresponding racks
  • 40‐mm petri dishes
  • Dissecting microscope
  • Additional reagents and equipment for euthanasia of the mouse (unit 1.8)

Basic Protocol 7: Collection of Nippostrongylus brasiliensis Fourth‐Stage (L4) Lung Larvae

  Materials
  • N. brasiliensis–infected mice (see protocol 6)
  • 70% ethanol
  • Dissecting instruments including iris scissors and forceps
  • 18‐G Insyte catheter (Becton Dickinson)
  • 1‐ml syringes
  • 90‐mm petri dish
  • Dissecting microscope
  • Additional reagents and equipment for euthanasia of the mouse (unit 1.8)

Alternate Protocol 3: Broncheoalveolar Lavage as an Alternative Method for the Recovery of Nippostrongylus brasiliensis Fourth‐Stage (L4) Lung Larvae

  Materials
  • N. brasiliensis–infected mice (see protocol 6)
  • 70% ethanol
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Dissecting instruments including iris scissors and forceps
  • 50‐ml centrifuge tubes and corresponding racks
  • Cheesecloth cut into 8 × 8–cm squares
  • 40‐mm petri dishes
  • Dissecting microscope
  • Additional reagents and equipment for euthanasia of the mouse (unit 1.8)

Basic Protocol 8: Collection of Nippostrongylus brasiliensis Adult Worms (L5)

  Materials
  • N. brasiliensis infected mice (see protocol 6)
  • 70% ethanol
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 1% (w/v) agarose
  • Dissecting instruments including iris scissors and forceps
  • 6‐well plastic tissue culture dishes
  • Cheesecloth cut into 8 × 8–cm squares
  • 8 × 8–cm plastic disposable weigh boats
  • 50‐ml centrifuge tubes and corresponding racks
  • 40‐mm petri dishes
  • Dissecting microscope
  • Additional reagents and equipment for euthanasia of the mouse (unit 1.8)

Basic Protocol 9: Recovery of Nippostrongylus brasiliensis Third‐Stage (L3) Infective Larvae from the Site of Infection

  Materials
  • N. brasiliensis: L3 larvae from 4 culture plates (see protocol 3), L4 larvae from 10 mice (see protocol 9 or protocol 10), or L5 adult worms from 10 mice (see protocol 11)
  • NES wash solution I (see recipe)
  • NES wash solution II (see recipe)
  • NES culturing solution (see recipe)
  • 15% SDS‐PAGE gel (unit 8.4)
  • 50‐ml sterile glass test tubes
  • 50 ml sterile tissue culture flasks
  • 37°C, 5% CO 2 incubator
  • 0.2‐µm filter units
  • Ultrafiltration concentrator unit, 5000 MWCO ( appendix 3H)
  • Additional reagents and equipment for counting worms (see protocol 3), concentration via ultrafiltration ( appendix 3H), protein assay (e.g., unit 2.11), and SDS‐PAGE (unit 8.4)

Support Protocol 1: Preparation of Nippostrongylus brasiliensis Antigen (NES)

  Materials
  • Heligmosomoides polygyrus infective L3 at 2000 L3/ml (see protocol 1)
  • Swiss Webster mice or relevant inbred strains
  • 1‐ml plastic syringe fitted with 18‐G rounded‐end feeding needle
  • Additional reagents and equipment for mouse restraint (unit 1.3)

Basic Protocol 10: Inoculation of Mice with Heligmosomoides polygyrus Infective Larvae

  Materials
  • H. polygyrus infected mice (see protocol 15)
  • Granulated charcoal/sphagnum moss
  • Spoon or spatula
  • 90‐mm sterile petri dishes
  • Incubator set at 26°C, without CO 2 (room temperature benchtop incubation is adequate)
  • Additional reagents and equipment for collection and counting of worm eggs from rodent fecal pellets (as for N. brasiliensis; see protocol 2)

Basic Protocol 11: Collection, Counting, and Culturing of Heligmosomoides polygyrus Eggs from Fecal Pellets of Infected Mice

  Materials
  • H. polygyrus infective L3 at 2000 L3/ml (see protocol 16)
  • Mice of relevant in‐bred strain
  • Anti‐CD4 monoclonal antibody
  • 50 mg/ ml liquid suspension of pyrantel pamoate (Pfizer)
  • 1‐ml plastic syringe fitted with 18‐G rounded‐end feeding needle
  • Additional reagents and equipment for mouse restraint (unit 1.3) injection of mice (unit 1.6), determining egg counts in individual mice (see protocol 8)
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Figures

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Selected References
  Anderson, R.C. 2000. Nematode Parasites of Vertebrates: Their Development and Transmission. 2nd Ed. CABI Publishing, CAB International, Wallingford Oxon, U.K.
  Thorough review of the basic techniques involved in preparation, handling and use of Nippostrongylus brasiliensis.
  Kassai, T. 1982, Handbook of Nippostrongylus brasiliensis, Akademia Kiado, Budapest.
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