Rat Model of Chronic Leptospirosis

Rubén Bonilla‐Santiago1, Jarlath E. Nally1

1 Veterinary Sciences Centre, UCD School of Agriculture Food Science & Veterinary Medicine, UCD Conway Institute of Biomolecular and Biomedical Research, College of Life Sciences, University College Dublin, Belfield, Dublin, Republic of Ireland
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 12E.3
DOI:  10.1002/9780471729259.mc12e03s20
Online Posting Date:  February, 2011
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Abstract

Reservoir hosts of pathogenic Leptospira species, including wild and domestic animal species, persistently excrete leptospires from infected renal tubules via urine into the environment. Reservoir hosts are typically asymptomatic. In contrast to the hamster model of leptospirosis (described in UNIT 12E.2), which emulates the severe form of human leptospirosis, the rat model of chronic leptospirosis emulates the asymptomatic infection observed in reservoir hosts. Rats are the primary reservoir host for pathogenic Leptospira species associated with the severe forms of human leptospirosis. Curr. Protoc. Microbiol. 20:12E.3.1‐12E.3.8. © 2011 by John Wiley & Sons, Inc.

Keywords: Leptospira; Rattus norvegicus; leptospirosis; reservoir hosts

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Intraperitoneal Challenge of Rats with Leptospira
  • Basic Protocol 2: Collection of Urine from Rats for Assessment of Leptospira Infection
  • Basic Protocol 3: Collection of Blood from Tail Vein for Assessment of Leptospira Infection
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Intraperitoneal Challenge of Rats with Leptospira

  Materials
  • 6–week‐old Male Wistar rats (Rattus norvegicus)
  • Low‐passage, log‐phase cultures (∼1 × 108 leptospires/ml of medium) of suitable strain of Leptospira, e.g., L. interrogans serovar Copenhageni strain RJ16441 (available from the authors as a courtesy)
  • Uninfected (negative‐control) Leptospira medium (EMJH medium; see recipe)
  • 70% ethanol
  • Autoclavable rat cages and bedding
  • Dark‐field microscope (unit 2.1)
  • Petroff‐Hauser ( appendix 4A) or similar counting chamber
  • Microscope slides and coverslips (22 mm × 22 mm)
  • Biocontainment cabinet
  • 1‐ml syringes
  • 25‐G needles
  • Sterile gauze
  • Rat restraint apparatus (e.g., Harvard Apparatus Rodent Restrainer; also see Donovan and Brown, )
  • Additional reagents and equipment for culture and maintenance of Leptospira (unit 12.1), dark‐field microscopy (unit 2.1), rat restraint (Donovan and Brown, ), and intraperitoneal injection of the rat (Donovan and Brown, )
NOTE: The items above represent a list of the minimum equipment necessary. Additional equipment or reagents should be discussed and acquired as per the Principal Investigator and University policies.

Basic Protocol 2: Collection of Urine from Rats for Assessment of Leptospira Infection

  Materials
  • Male Wistar rats (Rattus norvegicus)
  • Metabolic cages to separate urine and fecal samples (e.g., Harvard Apparatus, Tecniplast)
  • Dark‐field microscope (unit 2.1)
  • Petroff‐Hauser ( appendix 4A) or similar counting chamber
  • Microscope slides and coverslips (22 mm × 22 mm)
  • Biocontainment cabinet
  • Additional reagents and equipment for dark‐field microscopy (unit 2.1), counting cells ( protocol 1, step 2), and fluorescent antibody test (unit 12.1)

Basic Protocol 3: Collection of Blood from Tail Vein for Assessment of Leptospira Infection

  Materials
  • Male Wistar rats (Rattus norvegicus) experimentally infected with Leptospira
  • 70% ethanol
  • Rat restraint apparatus (e.g., Harvard Apparatus Rodent Restrainer; also see Donovan and Brown, )
  • Heat lamp
  • 25‐G needles
  • Microcentrifuge tubes
  • Capillary tubes or suitable pipet
  • Sterile gauze
  • 70% ethanol
  • Additional reagents and equipment for rat restraint (Donovan and Brown, )
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Figures

Videos

Literature Cited

Literature Cited
   Athanazio, D.A., Silva, E.F., Santos, C.S., Rocha, G.M., Vannier‐Santos, M.A., McBride, A.J., Ko, A.I., and Reis, M.G. 2008. Rattus norvegicus as a model for persistent renal colonization by pathogenic Leptospira interrogans. Acta Tropica 105:176‐180.
   Bharti, A.R., Nally, J.E., Ricaldi, J.N., Matthias, M.A., Diaz, M.M., Lovett, M.A., Levett, P.N., Gilman, R.H., Willig, M.R., Gotuzzo, E., and Vinetz, J.M. 2003. Leptospirosis: A zoonotic disease of global importance. Lancet Infect. Dis. 3:757‐771.
   Bolin, C.A. and Alt, D.P. 2001. Use of a monovalent Leptospiral vaccine to prevent renal colonization and urinary shedding in cattle exposed to Leptospira borgpetersenii serovar hardjo. Am. J. Vet. Res. 62:995‐1000.
   Donovan, J. and Brown, P. 2006a. Handling and restraint. Curr. Protoc. Immunol. 73:1.3.1‐1.3.5.
   Donovan, J. and Brown, P. 2006b. Parenteral injections. Curr. Protoc. Immunol. 73:1.6.1‐1.6.10.
   Ganoza, C.A., Matthias, M.A., Saito, M., Cespedes, M., Gotuzzo, E., and Vinetz, J.M. 2010. Asymptomatic renal colonization of humans in the peruvian Amazon by Leptospira. PLoS Neglect. Trop. Dis. 4:e612.
   Ido, Y., Hoki, R., Ito, H., and Wani, H. 1917. The rat as a carrier of Spirocheta icterohaemorrhagiae, the causative agent of Weil's disease (Spirochaetosis icterohaemorrhagica). J. Exp. Med. 26:341‐353.
   Ko, A.I., Goarant, C., and Picardeau, M. 2009. Leptospira: the dawn of the molecular genetics era for an emerging zoonotic pathogen. Nature Rev. 7:736‐747.
   Miller, J.N. 1971. Spirochetes in Body Fluids and Tissues. Charles C. Thomas, Springfield, Illinois.
   Monahan, A.M., Callanan, J.J., and Nally, J.E. 2008. Proteomic analysis of Leptospira interrogans shed in urine of chronically infected hosts. Infect. Immun. 76:4952‐4958.
   Monahan, A.M., Callanan, J.J., and Nally, J.E. 2009. Review paper: Host‐pathogen interactions in the kidney during chronic leptospirosis. Vet. Pathol. 46:792‐799.
   Nally, J.E., Chow, E., Fishbein, M.C., Blanco, D.R., and Lovett, M.A. 2005. Changes in lipopolysaccharide O antigen distinguish acute versus chronic Leptospira interrogans infections. Infect. Immun. 73:3251‐3260.
   Rojas, P., Monahan, A.M., Schuller, S., Miller, I.S., Markey, B.K., and Nally, J.E. 2010. Detection and quantification of leptospires in urine of dogs: A maintenance host for the zoonotic disease leptospirosis. Eur. J. Clin. Microbiol. Infect. Dis. 29:1305‐1309.
   Thiermann, A.B. 1977. Incidence of leptospirosis in the Detroit rat population. Am. J. Trop. Med. Hyg. 26:970‐974.
   Thiermann, A.B. 1981. The Norway rat as a selective chronic carrier of Leptospira icterohaemorrhagiae. J. Wildlife Dis. 17:39‐43.
   Wolfensohn, S. and Lloyd, M. 2003. Handbook of Laboratory Animal Management and Welfare, 3rd edition. Blackwell Publishing Ltd., Oxford.
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