The Ferret Model for Influenza

Yumiko Matsuoka1, Elaine W. Lamirande1, Kanta Subbarao1

1 Laboratory of Infectious Diseases, NIAID, NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 15G.2
DOI:  10.1002/9780471729259.mc15g02s13
Online Posting Date:  May, 2009
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Abstract

A major challenge in influenza research is the selection of an appropriate animal model that accurately reflects the disease and protective immune response to influenza infection in humans. Ferrets are exquisitely susceptible to infection with human influenza viruses and are widely believed to be the ideal small animal model for influenza research. Mice have also been used for influenza vaccine research for decades. Ferrets are used as an animal model for the study of influenza because they are susceptible to human influenza viruses and develop some of the symptoms of influenza that are seen in humans. Although they are not discussed in detail in this unit, hamsters, guinea pigs, and both cotton rats (Sigmodon) and rats (Rattus) have also been used for influenza research. Curr. Protoc. Microbiol. 13:15G.2.1-15G.2.29. © 2009 by John Wiley & Sons, Inc.

Keywords: influenza; animal models; ferrets; virus titration

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Isoflurane Anesthesia of Ferrets
  • Basic Protocol 2: Injectable Anesthesia of Ferrets
  • Basic Protocol 3: Intranasal Inoculation of Ferrets with Influenza Virus
  • Basic Protocol 4: Microchipping of Ferrets
  • Basic Protocol 5: Performing Temperature Reading
  • Basic Protocol 6: Bleeding Ferrets Via Anterior Vena Cava
  • Basic Protocol 7: Euthanasia of Ferrets
  • Basic Protocol 8: Ferret Tissue Harvest
  • Basic Protocol 9: Tissue Homogenization
  • Basic Protocol 10: Titration of Tissue Homogenates on MDCK Cells
  • Support Protocol: Preparation of MDCK Cells
  • Basic Protocol 11: Titration of Tissue Homogenates in Embryonated Eggs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isoflurane Anesthesia of Ferrets

 Materials
  • Isoflurane (1-chloro-2,2,2-trifluoroethyl difluoromethyl ether)
  • 8- to 10-week-old ferrets
  • Isoflurane induction chamber setup (Viking Medical; see Fig. 15G.2.3)
  • Chain mail or leather gloves to handle the ferrets (chain mail gloves are easier to disinfect)
     FigureFigure 15G.2.3 Induction chamber in the BSC is connected to an isoflurane vaporizer through tubing.

Basic Protocol 2: Injectable Anesthesia of Ferrets

 Materials
  • Anesthetics (see Table 15G.2.2) including:
    • Ketamine
    • Xylazine
    • Atropine
  • 8- to 10-week-old ferrets
  • 70% ethanol spray/squirt bottle
  • Transport cages
  • Scale
  • 1-ml syringes
  • 25-G needles
  • Microcentrifuge tubes (e.g., Eppendorf)
  • Microcentrifuge tube rack

NOTE: Ketamine is a controlled substance that must be obtained in accordance with federal regulations. These drugs need to be obtained through the facility veterinarian.
 
Table 15G.2.2 Drug Dosesa,b
DrugDosage (mg drug/kg animal body weight)
Ketamine25 mg/kg
Xylazine2 mg/kg
Atropine0.05 mg/kg
 aThe three drugs will be mixed in a microcentrifuge tube and then injected into the ferret.
 bDrug concentrations can vary; check the bottle each time.

Basic Protocol 3: Intranasal Inoculation of Ferrets with Influenza Virus

 Materials
  • Virus inoculum at appropriate dilution
  • 96-well plate of Madin Darby canine kidney (MDCK) cells (optional for immediate titration)
  • Ice packs or wet ice
  • 8- to 10-week-old ferrets
  • Tubes to hold inoculum
  • Rack for tubes
  • 200-µl pipettor
  • 200-µl pipet tips
  • Additional reagents and equipment for anesthetizing the ferret with inhaled isoflurane (Basic Protocol 1) and injectable drugs (Basic Protocol 2)

Basic Protocol 4: Microchipping of Ferrets

 Materials
  • 8- to 10-week-old ferrets
  • 70% ethanol
  • Transponders IPTT-300 (BioMedic Data Systems; http://www.bmds.com)
  • Wand reader system (Fig. 15G.2.5; BioMedic Data Systems; http://www.bmds.com)
  • Additional reagents and equipment for anesthetizing the ferret with inhaled isoflurane (Basic Protocol 1) and injectable drugs (Basic Protocol 2)
     FigureFigure 15G.2.5 Implantable temperature and ID transponder and wand reader system.

Basic Protocol 6: Bleeding Ferrets Via Anterior Vena Cava

 Materials
  • 8- to 10-week-old ferrets
  • 70% ethanol
  • Electric clippers
  • 1-ml syringe
  • 23-G needles
  • Serum separator tubes (Sarstedt, cat. no. 41.1378.005)
  • Microcentrifuge
  • Microcentrifuge tubes (e.g., Eppendorf)
  • Additional reagents and equipment for anesthetizing the ferrets with injectable anesthetics (Basic Protocol 2)

Basic Protocol 7: Euthanasia of Ferrets

 Materials
  • 8- to 10-week-old ferrets
  • Euthanasia-III (Med-Pharmex, ANADA cat. no. 200-280)
  • 1-ml syringes
  • 23-G needles
  • Additional reagents and equipment for anesthetizing the ferrets with injectable anesthetics (Basic Protocol 2)

Basic Protocol 8: Ferret Tissue Harvest

 Materials
  • 70% ethanol
  • Dry ice (if freezing samples immediately to process later)
  • Wet ice (if processing samples that same day)
  • 10% neutral-buffered formalin
  • 15-ml conical tubes, prelabeled and preweighed
  • 50-m conical tubes, prelabeled and preweighed
  • Tri-pour beakers or other containers to hold 70% ethanol to disinfect instruments
  • Racks to hold tubes
  • Surgical instruments including:
    • 5-in. scissors
    • Rongeurs, 5.5-in.
    • Forceps
    • Small spatula
    • Sterile disposable scalpels
    • Microdissecting curette
    • Cut-resistant gloves (optional)
  • 100-mm sterile petri dishes
  • Containers with a screw-top lid to hold at least 15 vol of 10% formalin and 1 vol of tissue
  • 12-ml syringes
  • 20-G needles
  • Suture material

Basic Protocol 9: Tissue Homogenization

 Materials
  • Wescodyne solution (Steris)
  • Tissue samples in 15-ml or 50-ml conical tubes
  • Wet ice
  • L-15 tissue grinding medium (see recipe)
  • Dry ice
  • Cryotubes (Nunc, cat. no. 375418)
  • Autoclave
  • Hard disposable homogenizer tips in autoclavable bags (OMNI, cat. no. 34750-AC-16)
  • 1-liter beakers for Wescodyne solution
  • Scale
  • 5-, 10-, 25-, and 50-ml pipets
  • Pipettor
  • OMNI tissue homogenizer
  • Absorbent pads (blue pads)
  • Tube racks

Basic Protocol 10: Titration of Tissue Homogenates on MDCK Cells

 Materials
  • 96-well plates of MDCK cells
  • 24-well plates of MDCK cells
  • Homogenized tissue samples (see Basic Protocol 9)
  • Wet ice
  • Wescodyne solution (Steris)
  • Complete medium with TPCK trypsin (see recipe)
  • Dry ice
  • Absorbent pads
  • Multichannel pipettors
  • Pipet tips
  • 1-liter beakers for Wescodyne solution
  • Rocker
  • Additional reagents and equipment for preparing MDCK cells (Support Protocol)

Support Protocol: Preparation of MDCK Cells

 Materials
  • MDCK cells
  • MDCK growth medium (see recipe)
  • MEM medium (Invitrogen, cat. no. 12360-038)
  • Complete medium with TPCK trypsin (see recipe)
  • 96- and 24-well plates
  • Container lined with absorbent pads

Basic Protocol 11: Titration of Tissue Homogenates in Embryonated Eggs

 Materials
  • 10- to 11-day-old embryonated hen's eggs
  • Dilution medium or buffer (L15 medium, PBS, or other appropriate buffer)
  • Virus or samples to be titrated
  • Disinfectant
  • Wet ice
  • 70% ethanol
  • Washed red blood cells (0.5% turkey or chicken RBCS in PBS, or 1% horse RBCs in PBS and 0.5% BSA)
  • Egg candler (Brinsea, Egg Lume Candling lamp, cat. no. USF150)
  • 1.5-ml microtubes (for preparation of dilutions)
  • Vortex
  • Repeat dispenser (with tips of appropriate size for 0.9 ml) or pipettor (with 5- to 10-ml disposable pipets)
  • Egg punch (We use sterile and disposable Lancet capillary blood sampling devices available from diabetic supply stores)
  • 1-ml syringes
  • 22-G, 1-in. needles
  • Incubator for eggs (35°C to 37°C)
  • Glue (Elmers)
  • Forceps
  • 50- to 200-µl multichannel pipettor
  • 50- to 200-µl pipet tips
  • 96-well V-shaped plates
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Figures

  •  FigureFigure 15G.2.1 A typical study design for a one (A) or two dose (B) immunization study.
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  •  FigureFigure 15G.2.2 Hold ferrets firmly by the scruff of the neck.
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  •  FigureFigure 15G.2.3 Induction chamber in the BSC is connected to an isoflurane vaporizer through tubing.
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  •  FigureFigure 15G.2.4 A mouse cage can be used to hold small ferrets for weighing.
    View Image
  •  FigureFigure 15G.2.5 Implantable temperature and ID transponder and wand reader system.
    View Image
  •  FigureFigure 15G.2.6 Needle is inserted under the sternum into the vena cava.
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  •  FigureFigure 15G.2.7 Blood is seen in the syringe when the plunger is drawn back if the needle is properly placed in the heart.
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  •  FigureFigure 15G.2.8 Use forceps and scissors to remove skin from the skull.
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  •  FigureFigure 15G.2.9 Carefully cut through the skull with rongeurs.
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  •  FigureFigure 15G.2.10 Gently tease brain from the skull with a spatula.
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  •  FigureFigure 15G.2.11 Cut the ribcage with scissors and lift away with forceps.
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  •  FigureFigure 15G.2.12 Remove nasal turbinates with a curette.
    View Image
  •  FigureFigure 15G.2.13 Samples are diluted in 96-well plates containing a MDCK monolayer. The sample is inoculated into four wells and serial 10-fold dilutions are made in successive rows of the plate.
    View Image

Videos

Literature Cited

Literature Cited
    Abou-Donia, H., Jennings, R., and Potter, C.W. 1980. Growth of influenza A viruses in hamsters. Arch. Virol. 65:99-107.
    Brown, S.A. 2004. "Basic anatomy, physiology, and husbandry". In Ferrets, Rabbits, and Rodents Clinical Medicine and Surgery, 2nd edition (K.E. Queensbury and J.W. Carpenter, eds.) pp. 2-12. Saunders, St. Louis, Mo.
    Carson, F. 2007. Histotechnology: A Self-Instructional Text, 2nd edition. p. 4. American Society for Clinical Pathology Press, Chicago.
    Heath, A.W., Addison, C., Ali, M., Teale, D., and Potter, C.W. 1983. In vivo and in vitro hamster models in the assessment of virulence of recombinant influenza viruses. Antiviral. Res. 3:241-252.
    Jin, H., Manetz, S., Leininger, J., Luke, C., Subbarao, K., Murphy, B., Kemble, G., and Coelingh, K.L. 2007. Toxicological evaluation of live attenuated, cold-adapted H5N1 vaccines in ferrets. Vaccine 25:8664-8672.
    Lowen, A.C., Mubareka, S., Tumpey, T.M., Garcia-Sastre, A., and Palese, P. 2006. The guinea pig as a transmission model for human influenza viruses. Proc. Natl. Acad. Sci. U.S.A. 103:9988-9992.
    Luke, C.J. and Subbarao, K. 2008. "The role of animal models in influenza vaccine research". In Influenza Vaccines for the Future Series: Advances in Infectious Diseases (R. Rappuoli and G. Del Giudice, eds.) pp. 161-202. Birkhäuser Verlag, Basel, Switzerland.
    Niewiesk, S. and Prince, G. 2002. Diversifying animal models: The use of hispid cotton rats (Sigmodon hispidus) in infectious diseases. Lab. Anim. 36:357-372.
    Ottolini, M.G., Blanco, J.C., Eichelberger, M.C., Porter, D.D., Pletneva, L., Richardson, J.Y., and Prince, G.A. 2005. The cotton rat provides a useful small-animal model for the study of influenza virus pathogenesis. J. Gen. Virol. 86:2823-2830.
    Phair, J.P., Kauffman, C.A., Jennings, R., and Potter, C.W. 1979. Influenza virus infection of the guinea pig: Immune response and resistance. Med. Microbiol. Immunol. 165:241-254.
    Reed, L.J. and Muench, H. 1938. Simple method of estimating fifty percent endpoints. Amer. J. Hygiene 27:493-497.
    Reuman, P.D., Keely, S., and Schiff, G.M. 1989. Assessment of signs of influenza illness in the ferret model. J. Virol. Methods 24:27-34.
    Rosenthal, K.L. 2004. "Respiratory diseases". In Ferrets, Rabbits, and Rodents Clinical Medicine and Surgery, 2nd edition (K.E. Queensbury and J.W. Carpenter, eds.) pp. 72-78. Saunders, St. Louis, Mo.
    Smith, W., Andrewes, C.H., and Laidlaw, P.P. 1933. A virus obtained from influenza patients. Lancet 2:66-68.
    Spackman, E. and Suarez, D.L. 2008. Type A influenza virus detection and quantitation by real-time RT-PCR. Methods Molec. Biol. 436:19-26.
    Subbarao, E.K., Kawaoka, Y., and Murphy, B.R. 1993. Rescue of an influenza A virus wild-type PB2 gene and a mutant derivative bearing a site-specific temperature-sensitive and attenuating mutation. J. Virol. 67:7223-7228.
    Subbarao, K., McAuliffe, J., Vogel, L., Fahle, G., Fischer, S., Tatti, K., Packard, M., Shieh, W. J., Zaki, S., and Murphy, B. 2004. Prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice. J. Virol. 78:3572-3577.
    Takiguchi, K., Sugawara, K., Hongo, S., Nishimura, H., Kitame, F., and Nakamura, K. 1992. Protective effect of serum antibody on respiratory infection of influenza C virus in rats. Arch. Virol. 122:1-11.
    Teh, C., Jennings, R., and Potter, C.W. 1980. Influenza virus infection of newborn rats: Virulence of recombinant strains prepared from influenza virus strain A/Okuda/57. J. Med. Microbiol. 13:297-306.
    van der Laan, J.W., Herberts, C., Lambkin-Williams, R., Boyers, A., Mann, A.J., and Oxford, J. 2008. Animal models in influenza vaccine testing. Expert Rev. Vaccines 7:783-793.
    Van Riel, D., Munster, V.J., de Wit, E., Rimmelzwaan, G.F., Fouchier, R.A.M., Osterhaus, A.D.M.E., and Kuiken, T. 2006. H5N1 virus attachment to lower respiratory tract. Science 312:399.
    Zitzow, L.A., Rowe, T., Morken, T., Shieh, W., Zaki, S., and Katz, J.M. 2002. Pathogenesis of avian influenza A (H5N1) viruses in ferrets. J. Virol. 76:4420-4429.
 Key References
    Queensbury, K.E. and Carpenter, J.W., eds. 2004. Ferrets, Rabbits, and Rodents Clinical Medicine and Surgery, 2nd edition. Saunders, St. Louis, Mo.

This book discusses the anatomy, husbandry, physiology, and diseases of small mammals including ferrets, rabbits, guinea pigs, and other small rodents.

    van der Laan et al., 2008. See above.

This review article discusses various animal species that can be experimentally infected with influenza.

    Luke and Subbarao, 2008. See above.

This chapter discusses the evaluation of influenza virus vaccines in animal models.

 Internet Resources
    http://www.cdc.gov/od/sap

CDC Select Agent program.

    http://www.bmds.com

Use of ferret microchips.

    http://www.avma.org/resources/euthanasia.pdf

Euthanasia guidelines from American Veterinary Medical Association.

    http://www.searo.who.int/LinkFiles/CDS_CDS-Guidelines-Laboratory.pdf

WHO Guidelines on Laboratory Diagnosis of Avian Influenza (2007).

    http://www.wpro.who.int/NR/rdonlyres/EFD2B9A7-2265-4AD0-BC98-97937B4FA83C/0/manualonanimalaidiagnosisandsurveillance.pdf

WHO Manual on Animal Influenza Diagnosis and Surveillance.

    http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm

5th edition of Biosafety in Microbiological and Biomedical Laboratories.

    http://www.cdc.gov/flu/pdf/h2n2bsl3/pdf

Biosafety guidelines for noncontemporary or nonhuman influenza viruses from the 5th edition of Biosafety in Microbiological and Biomedical Laboratories.

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