Infection of Mice with Francisella as an Immunological Model

J. Wayne Conlan1, Wangxue Chen1, Catharine M. Bosio2, Siobhán C. Cowley3, Karen L. Elkins3

1 National Research Council of Canada, Institute for Biological Sciences, Ottawa, Ontario, Canada, 2 Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, NIH, Hamilton, Montana, 3 Laboratory of Mycobacterial Diseases and Cellular Immunology, Division of Bacterial, Parasitic, and Allergenic Products, OVRR/CBER/U.S. FDA, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 19.14
DOI:  10.1002/0471142735.im1914s93
Online Posting Date:  April, 2011
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Abstract

This unit describes the utility of various mouse models of infection for studying pathogenesis and adaptive immune responses to the facultative intracellular bacteria pathogen Francisella tularensis. By judicious use of different combinations of mouse and bacterial strains, as well as different routes of infection, murine tularemia models may be used to explore a complete picture of F. tularensis infection and immunity. Moreover, studies using Francisella, particularly the Live Vaccine Strain (LVS), serve as a convenient and tractable model system that appears to be representative of mammalian host responses to intracellular pathogens in general. Curr. Protoc. Immunol. 93:19.14.1‐19.14.16. © 2011 by John Wiley & Sons, Inc.

Keywords: Francisella tularensis; intracellular bacteria; mouse models; systemic infection; respiratory infection; biosafety issues

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Systemic Infections of Mice with F. tularensis
  • Alternate Protocol 1: Ear Pinna Infection of Mice with F. tularensis LVS
  • Basic Protocol 2: Respiratory Infection of Mice with Francisella VIA Intranasal Instillation
  • Alternate Protocol 2: Respiratory Aerosol Infections of Mice with Francisella
  • Basic Protocol 3: Intragastric Infection of Mice with Francisella
  • Preparation of Francisella Bacterial Stocks
  • Support Protocol 1: Preparation of F. tularensis (e.g., LVS) Working Stocks
  • Support Protocol 2: Preparation of F. novicida Bacterial Stocks
  • Support Protocol 3: Evaluation of Immune Responses to Francisella
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Systemic Infections of Mice with F. tularensis

  Materials
  • F. tularensis (see unit 14.25; also see Support Protocols protocol 61 and protocol 72 in this unit)
  • Phosphate‐buffered saline (PBS), low endotoxin (Lonza, cat. no. 17‐512F), sterile
  • Modified Mueller‐Hinton agar plates (see recipe)
  • Naïve adult BALB/cByJ, C57BL/6J, or other suitable strains of laboratory mice
  • Mouse restraining device (optional)
  • 1‐ml plastic syringes, sterile
  • 27‐G, ½‐in. sterile needles
  • Additional reagents and equipment for preparing F. tularensis frozen stocks (unit 14.25 and Support Protocols protocol 61 and protocol 72 in this unit) and parenteral injections of mice (unit 1.6)
NOTE: All media components and additives should be sterile, free of antibiotics, and selected from sources that rigorously quantitate and report endotoxin levels; purchase those with the lowest possible endotoxin content.

Alternate Protocol 1: Ear Pinna Infection of Mice with F. tularensis LVS

  • 1‐ml plastic sterile syringes and 25‐G needle, sterile
  • 0.3‐ml insulin syringe fitted with 30‐G needle, sterile
  • Flat‐pronged forceps
  • Additional reagents and equipment for (ketamine/xylazine) anesthesia of the mouse (unit 1.4)

Basic Protocol 2: Respiratory Infection of Mice with Francisella VIA Intranasal Instillation

  Materials
  • F. tularensis (see unit 14.25; also see Support Protocols protocol 61 and protocol 72 in this unit)
  • Phosphate‐buffered saline (PBS; appendix 2A), low endotoxin, sterile
  • Modified Mueller‐Hinton agar plates (see recipe)
  • Naïve adult BALB/cByJ, C57BL/6J, or other suitable strains of laboratory mice
  • 1‐ml plastic syringes and 25‐G needles, sterile
  • Pipet with sterile tip for instillation
  • Additional reagents and equipment for (ketamine/xylazine) anesthesia of the mouse (unit 1.4)

Alternate Protocol 2: Respiratory Aerosol Infections of Mice with Francisella

  Materials
  • Naïve adult BALB/cByJ, C57BL/6J, or other suitable strains of laboratory mice
  • F. tularensis LVS bacteria (see unit 14.25; also see Support Protocols protocol 61 and protocol 72 in this unit)
  • Phosphate‐buffered saline (PBS; appendix 2A), low endotoxin, sterile
  • Alcohol wipes
  • Modified Mueller‐Hinton agar plates (see recipe)
  • Sterile stainless steel 18‐G, 2‐in. (5.1 cm) curved gavage needle (Harvard Apparatus or equivalent); note that if gavage is performed on young mice, a smaller‐sized gavage needle or feeding tube will be required
  • 1‐ml tuberculin syringe
  • Additional reagents and equipment for evaluation of immune responses to Francisella ( protocol 8)
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Figures

  •   FigureFigure 19.14.1 Relative virulence of Francisella subspecies for mice by different routes of infection. The subspecies name is placed at the intersection of the route and the approximate corresponding LD50

Videos

Literature Cited

   Anthony, L.S.D., Skamene, E., and Knogshavn, P.A.L. 1988. Influence of genetic background on host resistance to experimental murine tularemia. Infect. Immun. 56:2089‐2093.
   Baker, C.N., Hollis, D.G., and Thornsberry, C. 1985. Anti‐microbial susceptibility testing of Francisella tularensis with a modified Mueller‐Hinton broth. J. Clin. Micro. 22:212‐215.
   Collazo, C.M., Meierovics, A.I., De Pascalis, R., Wu, T.H., Lyons, C.R., and Elkins, K.L. 2009. T cells from lungs and livers of Francisella tularensis‐immune mice control the growth of intracellular bacteria. Infect. Immun. 77:2010‐2021.
   Cong, Y., Yu, J.J., Guentzel, M.N., Berton, M.T., Seshu, J., Klose, K.E., and Arulanandam, B.P. 2009. Vaccination with a defined Francisella tularensis subsp. novicida pathogenicity island mutant (DeltaiglB) induces protective immunity against homotypic and heterotypic challenge. Vaccine 27:5554‐5561.
   Dennis, D.T., Inglesby, T.V., Henderson, D.A., Bartlett, J.G., Ascher, M.S., Eitzen, E., Fine, A.D., Friedlander, A.M., Hauer, J., Layton, M., Lillibridge, S.R., McDade, J.E., Osterholm, M.T., O'Toole, T., Parker, G., Perl, T.M., Russell, P.K., and Tonat, K. 2001. Tularemia as a biological weapon: Medical and public health management. JAMA 285:2763‐2773.
   Elkins, K.L., Cowley, S.C., and Bosio, C.M. 2003. Innate and adaptive immune responses to an intracellular bacterium, Francisella tularensis live vaccine strain. Microbes Infect. 5:132‐142.
   Elkins, K.L., Cowley, S.C., and Bosio, C.M. 2007. Innate and adaptive immunity to Francisella. Ann. N.Y. Acad. Sci. 1105:284‐324.
   Elkins, K.L., Colombini, S.M., Meierovics, A.I., Chu, M.C., Chou, A.Y., and Cowley, S.C. 2010. Survival of secondary lethal systemic Francisella LVS challenge depends largely on interferon gamma. Microbes Infect. 12:28‐36.
   Hornick, R.B., Dawkins, A.T., Eigelsbach, H.T., and Tulis, J.J. 1966. Oral tularemia vaccine in man. Antimicrob. Agents Chemother. 6:11‐14.
   Klimpel, G.R., Eaves‐Pyles, T, Moen, S.T., Taormina, J., Peterson, J.W., Chopra, A.K., Niesel, D.W., Carness, P., Haithcoat, J.L., Kirtley, M., and Nasr, A.B. 2008. Levofloxacin rescues mice from lethal intra‐nasal infections with virulent Francisella tularensis and induces immunity and production of protective antibody. Vaccine 26:6874‐6882.
   KuoLee, R., Zhao, X., Austin, J., Harris, G., Conlan, J.W., and Chen, W. 2007a. Mouse model of oral infection with virulent Type A Francisella tularensis. Infect Immun. 75:1651‐1660.
   KuoLee, R., Harris, G., Conlan, J.W., and Chen, W. 2007b. Oral immunization of mice with the live vaccine strain (LVS) of Francisella tularensis protects mice against respiratory challenge with virulent type A F. tularensis. Vaccine 25:3781‐3791.
   Owen, C.R., Buker, E.O., Jellison, W.L., Lackman, D.B., and Bell, J.F. 1964. Comparative studies of Francisella tularensis and Francisella novicida. J. Bacteriol. 87:676–683.
   Ray, H.J., Cong, Y., Murthy, A.K., Selby, D.M., Klose, K.E., Barker, J.R., Guentzel, M.N., and Arulanandam, B.P. 2009. Oral live vaccine strain‐induced protective immunity against pulmonary Francisella tularensis challenge is mediated by CD4+ T cells and antibodies, including immunoglobulin A. Clin. Vaccine Immunol. 16:444‐452.
   Rhinehart‐Jones, T.R., Fortier, A.H., and Elkins, K.L. 1994. Transfer of immunity against lethal murine Francisella infection by specific antibody depends on host gamma interferon and T cells. Infect Immun. 62:3129‐3137.
   Shen, H., Chen, W., and Conlan, J.W. 2004. Susceptibility of various mouse strains to systemically‐ or aerosol‐initiated tularemia by virulent type A Francisella tularensis before and after immunization with the attenuated live vaccine strain of the pathogen. Vaccine 22:2116‐2121.
   Sjostedt, A. 2007. Tularemia: History, epidemiology, pathogen physiology, and clinical manifestations. Ann. N.Y. Acad. Sci. 1105:1‐29.
   Wu, T.H., Hutt, J.A., Garrison, K.A., Berliba, L.S., Zhou, Y., and Lyons, C.R. 2005. Intranasal vaccination induces protective immunity against intranasal infection with virulent Francisella tularensis biovar A. Infect. Immun. 73:2644‐2654.
Key References
   Bosio, C.M. and Elkins, K.L. 2001. Susceptibility to secondary Francisella tularensis LVS infection in B cell deficient mice is associated with neutrophilia but not with defects in specific T cell mediated immunity. Infect. Immun. 69:194‐203.
  These two publications provide detailed accounts, and experimental examples, of an in vitro method for determining the contributions of various lymphocyte subsets to acquired immunity (see also UNIT ).
   Cowley, S.C. and Elkins, K.L. 2003. Multiple T cell subsets control Francisella tularensis LVS intracellular growth without stimulation through macrophage interferon gamma receptors. J. Exp. Med. 198:379‐389.
  Provides detailed descriptions of the course of infection of mice with virulent F. tularensis subspecies holarctica and tularensis.
   Conlan, J.W., Chen, W., Shen, H., Webb, A., and KuoLee, R. 2003. Experimental tularemia in mice challenged by aerosol or intradermally with virulent strains of Francisella tularensis: Bacteriologic and histopathologic studies. Microbial Pathogenesis 34:239‐248.
  A comprehensive review of the use of LVS infection of mice to examine innate and adaptive immunity.
   Elkins et al., 2003. See above.
  These references provide detailed descriptions of an experimental mouse model of oral infection with virulent F. tularensis.
   KuoLee et al., 2007a,b. See above.
  A comprehensive review of the uses and limitations of mouse models of infection with virulent F. tularensis strains.
   Lyons C.R. and Wu, T.H. 2007. Animal models of Francisella tularensis infection. Ann. N.Y. Acad. Sci. 1105:238‐265.
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