Infant Rabbit Model for Diarrheal Diseases

Sören Abel1, Matthew K. Waldor2

1 Department of Pharmacy, Faculty of Health Sciences, UiT, The Arctic University of Norway, Tromsø, 2 Howard Hughes Medical Institute, Boston, Massachusetts
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 6A.6
DOI:  10.1002/9780471729259.mc06a06s38
Online Posting Date:  August, 2015
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Abstract

Vibrio cholerae is the agent of cholera, a potentially lethal diarrheal disease that remains a significant threat to populations in developing nations. The infant rabbit model of cholera is the only non‐surgical small animal model system that closely mimics human cholera. Following orogastric inoculation, V. cholerae colonizes the intestines of infant rabbits, and the animals develop severe cholera‐like diarrhea. In this unit, we provide a detailed description of the preparation of the V. cholerae inoculum, the inoculation process and the collection and processing of tissue samples. This infection model is useful for studies of V. cholerae factors and mechanisms that promote its intestinal colonization and enterotoxicity, as well as the host response to infection. The infant rabbit model of cholera enables investigations that will further our understanding of the pathophysiology of cholera and provides a platform for testing new therapeutics. © 2015 by John Wiley & Sons, Inc.

Keywords: Vibrio cholerae; cholera; enteric disease; animal infection model; infant rabbits

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Infectious Inoculum
  • Basic Protocol 2: Orogastric Infection of Infant Rabbits
  • Basic Protocol 3: Euthanization of Infected Infant Rabbits
  • Basic Protocol 4: Necropsy, Dissection of the Gut and Harvesting Tissue
  • Basic Protocol 5: Tissue Homogenization and Colony‐Forming Unit (CFU) Determination
  • Alternate Protocol 1: Tissue Homogenization with Glass Plates
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Infectious Inoculum

  Materials
  • Vibrio cholerae strains of interest with selectable marker (e.g., chromosomal streptomycin resistance)
  • LB‐agar culture plate with appropriate selective agent (e.g., 200 μg/ml streptomycin)
  • LB medium with appropriate selective agent (e.g., 200 μg/ml streptomycin)
  • 300 mM sodium bicarbonate (NaHCO 3) solution, pH 9.0, sterilized by filtration through a 0.22‐μm filter
  • Culture tube, sterile
  • Incubator, 37°C
  • Toothpicks, sterile
  • Shaking incubator, 30°C or 37°C
  • Erlenmeyer flask, sterile
  • 50 ml centrifugation tube, sterile
  • Centrifuge with appropriate rotor for centrifugation tube, room temperature
  • Spectrophotometer, 600 nm
  • Cuvette
  • Eppendorf tubes, sterile

Basic Protocol 2: Orogastric Infection of Infant Rabbits

  Materials
  • New Zealand white rabbit adult mother with 2‐ to 3‐day‐old litter (e.g., from Charles River Laboratories)
  • 70% (v/v) ethanol
  • 25 mg/ml Zantac (ranitidine hydrochloride), injection grade, USP
  • Sterile water for injection, USP (preservative‐free)
  • Infectious inoculum ( protocol 1)
  • Water, sterile
  • Spray bottle (for ethanol)
  • Permanent marker with soft tip
  • Cardboard animal transfer boxes
  • Balance
  • Disposable exam gloves
  • Eppendorf tubes, sterile
  • 1‐ml and 10‐ml syringe, sterile
  • 30‐G × ½ (0.3 mm × 13 mm) hypodermic needle, sterile
  • Size 4 French peripherally inserted central catheter (PICC) with flexible tip (Arrow International), sterile, with a permanent marker mark ∼4 cm behind its tip

Basic Protocol 3: Euthanization of Infected Infant Rabbits

  Materials
  • Isoflurane, USP
  • 2 M potassium chloride (KCl), injection grade, USP
  • Fume hood
  • 1‐liter beaker or metal container with airtight lid
  • Metal grid that inserts into container and covers the surface completely
  • Gauze
  • Forceps
  • 3‐ml syringe

Basic Protocol 4: Necropsy, Dissection of the Gut and Harvesting Tissue

  Materials
  • PBS, sterile
  • 70% (v/v) ethanol
  • 1.5‐ml screw‐cap microvials, sterile
  • 3.2‐mm stainless steel tissue disruption beads, sterile (Biospec)
  • Dissection board (cork, Styrofoam or plastic)
  • Medium dissection scissors
  • Medium tissue forceps
  • Small surgical scissors
  • Small angled forceps
  • 1‐ml syringe, sterile
  • 26‐G × 5/8 (0.45 mm × 16 mm) hypodermic needle
  • Body disposal bag

Basic Protocol 5: Tissue Homogenization and Colony‐Forming Unit (CFU) Determination

  Materials
  • Gut tissue samples ( protocol 4)
  • Cecal fluid ( protocol 4)
  • PBS, sterile
  • LB‐agar culture plate with appropriate selective agent (e.g., 200 μg/ml streptomycin)
  • Thiosulfate‐citrate‐bile salts‐sucrose (TCBS) agar plates (e.g., Becton Dickinson)
  • Bead‐beater‐type tissue homogenizers (e.g., Mini‐Beadbeater‐16; Biospec)
  • Eppendorf tubes, sterile
  • Vortexer

Alternate Protocol 1: Tissue Homogenization with Glass Plates

  Materials
  • Gut tissue samples ( protocol 4)
  • PBS, sterile
  • Standard microscope slides with frosted labeling area, sterile
  • 90‐mm bacteriological Petri dish, sterile
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Figures

Videos

Literature Cited

Literature Cited
  Abel, S., Abel zur Wiesch, P., Chang, H.‐H., Davis, B.M., Lipsitch, M., and Waldor, M.K. 2015. Sequence tag‐based analysis of microbial population dynamics. Nat. Methods 12:223‐226.
  Blachman, U., Goss, S.J., and Pickett, M.J. 1974. Experimental cholera in the chinchilla. J. Infect. Dis. 129:376‐384.
  Blow, N.S., Salomon, R.N., Garrity, K., Reveillaud, I., Kopin, A., Jackson, F.R., and Watnick, P.I. 2005. Vibrio cholerae infection of Drosophila melanogaster mimics the human disease cholera. PLoS Pathog. 1:e8.
  Burnett, L.C., Lunn, G., and Coico, R. 2009. Biosafety: Guidelines for working with pathogenic and infectious microorganisms. Curr. Protoc. Microbiol. 13:1A.1.1‐1A.1.14.
  Chao, M.C., Pritchard, J.R., Zhang, Y.J., Rubin, E.J., Livny, J., Davis, B.M., and Waldor, M.K. 2013. High‐resolution definition of the Vibrio cholerae essential gene set with hidden Markov model‐based analyses of transposon‐insertion sequencing data. Nucleic Acids Res. 41:9033‐9048.
  De, S.N. 1959. Enterotoxicity of bacteria‐free culture‐filtrate of Vibrio cholerae. Nature 183:1533‐1534.
  De, S.N. and Chatterje, D.N. 1953. An experimental study of the mechanism of action of Vibrio cholerae on the intestinal mucous membrane. J. Pathol. Bacteriol. 66:559‐562.
  Dutta, N.K. and Habbu, M.K. 1955. Experimental cholera in infant rabbits: A method for chemotherapeutic investigation. Br. J. Pharmacol. Chemother. 10:153‐159.
  Finkelstein, R.A., Boesman‐Finkelstein, M., Chang, Y., and Häse, C.C. 1992. Vibrio cholerae hemagglutinin/protease, colonial variation, virulence, and detachment. Infect. Immun. 60:472‐478.
  Freter, R. 1956. Experimental enteric Shigella and Vibrio infections in mice and guinea pigs. J. Exp. Med. 104:411‐418.
  Fu, Y., Waldor, M.K., and Mekalanos, J.J. 2013. Tn‐Seq analysis of Vibrio cholerae intestinal colonization reveals a role for T6SS‐mediated antibacterial activity in the host. Cell Host Microbe 14:652‐663.
  Guentzel, M.N. and Berry, L.J. 1974. Protection of suckling mice from experimental cholera by maternal immunization: Comparison of the efficacy of whole‐cell, ribosomal‐derived, and enterotoxin immunogens. Infect. Immun. 10:167‐172.
  Kamp, H.D., Patimalla‐Dipali, B., Lazinski, D.W., Wallace‐Gadsden, F., and Camilli, A. 2013. Gene fitness landscapes of Vibrio cholerae at important stages of its life cycle. PLoS Pathog. 9:e1003800.
  Kaper, J.B., Morris, J.G. Jr, and Levine, M.M. 1995. Cholera. Clin. Microbiol. Rev. 8:48‐86.
  Leitch, G.J. 1988. Cholera enterotoxin‐induced mucus secretion and increase in the mucus blanket of the rabbit ileum in vivo. Infect. Immun. 56:2871‐2875.
  Madden, J.M., Nematollahi, W.P., Hill, W.E., McCardell, B.A., and Twedt, R.M. 1981. Virulence of three clinical isolates of Vibrio cholerae non‐O‐1 serogroup in experimental enteric infections in rabbits. Infect. Immun. 33:616‐619.
  Mandlik, A., Livny, J., Robins, W.P., Ritchie, J.M., Mekalanos, J.J., and Waldor, M.K. 2011. RNA‐Seq‐based monitoring of infection‐linked changes in Vibrio cholerae gene expression. Cell Host Microbe 10:165‐174.
  Metchnikoff, E. 1894. Recherches sur le cholera et les vibrions. Receptivite des jeunes lapins pour le cholera intestinal. Ann. Inst. Pasteur 8:557.
  Miller, C.E., Wong, K.H., Feeley, J.C., and Forlines, M.E. 1972. Immunological conversion of Vibrio cholerae in gnotobiotic mice. Infect. Immun. 6:739‐742.
  Munera, D., Ritchie, J.M., Hatzios, S.K., Bronson, R., Fang, G., Schadt, E.E., Davis, B.M., and Waldor, M.K. 2014. Autotransporters but not pAA are critical for rabbit colonization by Shiga toxin‐producing Escherichia coli O104:H4. Nat. Commun. 5:3080. doi:10.1038/ncomms4080
  Nygren, E., Li, B.‐L., Holmgren, J., and Attridge, S.R. 2009. Establishment of an adult mouse model for direct evaluation of the efficacy of vaccines against Vibrio cholerae. Infect. Immun. 77:3475‐3484.
  Pritchard, J.R., Chao, M.C., Abel, S., Davis, B.M., Baranowski, C., Zhang, Y.J., Rubin, E.J., and Waldor, M.K. 2014. ARTIST: High‐resolution genome‐wide assessment of fitness using transposon‐insertion sequencing. PLoS Genet. 10:e1004782.
  Ritchie, J.M. and Waldor, M.K. 2005. The locus of enterocyte effacement‐encoded effector proteins all promote enterohemorrhagic Escherichia coli pathogenicity in infant rabbits. Infect. Immun. 73:1466‐1474.
  Ritchie, J.M. and Waldor, M.K. 2009. Vibrio cholerae interactions with the gastrointestinal tract: Lessons from animal studies. Curr. Topics Microbiol. Immunol. 337:37‐59.
  Ritchie, J.M., Thorpe, C.M., Rogers, A.B., and Waldor, M.K. 2003. Critical roles for stx2, eae, and tir in enterohemorrhagic Escherichia coli‐induced diarrhea and intestinal inflammation in infant rabbits. Infect. Immun. 71:7129‐7139.
  Ritchie, J.M., Rui, H., Bronson, R.T., and Waldor, M.K. 2010. Back to the future: Studying cholera pathogenesis using infant rabbits. mBio 1:e00047‐10. doi:10.1128/mBio.00047‐10
  Ritchie, J.M., Rui, H., Zhou, X., Iida, T., Kodoma, T., Ito, S., Davis, B.M., Bronson, R.T., and Waldor, M.K. 2012. Inflammation and disintegration of intestinal villi in an experimental model for Vibrio parahaemolyticus‐induced diarrhea. PLoS Pathog. 8:e1002593.
  Rui, H., Ritchie, J.M., Bronson, R.T., Mekalanos, J.J., Zhang, Y., and Waldor, M.K. 2010. Reactogenicity of live‐attenuated Vibrio cholerae vaccines is dependent on flagellins. Proc. Natl. Acad. Sci. U.S.A. 107:4359‐4364.
  Runft, D.L., Mitchell, K.C., Abuaita, B.H., Allen, J.P., Bajer, S., Ginsburg, K., Neely, M.N., and Withey, J.H. 2014. Zebrafish as a natural host model for Vibrio cholerae colonization and transmission. Appl. Environ. Microbiol. 80:1710‐1717.
  Sack, R.B., Carpenter, C.C., Steenburg, R.W., and Pierce, N.F. 1966. Experimental cholera. A canine model. Lancet 2:206‐207.
  Sears, C.L. and Kaper, J.B. 1996. Enteric bacterial toxins: Mechanisms of action and linkage to intestinal secretion. Microbiol. Rev. 60:167‐215.
  Taylor, R.K., Miller, V.L., Furlong, D.B., and Mekalanos, J.J. 1987. Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. Proc. Natl. Acad. Sci. U.S.A. 84:2833‐2837.
  Zhou, X., Gewurz, B.E., Ritchie, J.M., Takasaki, K., Greenfeld, H., Kieff, E., Davis, B.M., and Waldor, M.K. 2013. A Vibrio parahaemolyticus T3SS effector mediates pathogenesis by independently enabling intestinal colonization and inhibiting TAK1 activation. Cell Rep. 3:1690‐1702.
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