Laboratory Maintenance of Anaplasma phagocytophilum

Jason A. Carlyon1

1 University of Kentucky College of Medicine, Lexington, Kentucky
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 3A.2
DOI:  10.1002/9780471729259.mc03a02s00
Online Posting Date:  July, 2005
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Abstract

Anaplasma phagocytophilum is the etiologic agent of human granulocytic anaplasmosis (formerly human granulocytic ehrlichiosis), an emerging and potentially deadly disease in the United States, Europe, and Asia. A. phagocytophilum is an obligate intracellular bacterium that displays a unique tropism for neutrophils. Studying this fascinating organism not only provides insight into microbial invasion and intracellular survival strategies, but also offers a unique approach to understanding neutrophil biology and host defense mechanisms. This unit describes the inoculation and maintenance of A. phagocytophilum from an infected blood sample into eukaryotic cell culture or laboratory mice. Cytological staining and immunofluorescent methods for assessing A. phagocytophilum infection are also presented. In addition, this unit describes isolation of viable, host cell–free bacterial preparations from infected cells, as well as the cryopreservation of infected cultures. Lastly, fluorescent labeling of live A. phagocytophilum for the purpose of tracking infection is provided.

Keywords: anaplasmosis; ehrlichiosis; pathogen; leukemic cell line HL‐60; neutrophils

     
 
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Table of Contents

  • Basic Protocol 1: Inoculation of A. phagocytophilum into Laboratory Mice
  • Support Protocol 1: Preparation of Blood Smear for Identifying A. phagocytophilum–Infected Neutrophils
  • Basic Protocol 2: Inoculation and Passaging of A. phagocytophilum Infection in HL‐60 Cell Culture
  • Support Protocol 2: Examination of Tissue Culture Cells for A. phagocytophilum Infection
  • Support Protocol 3: Cytological Staining of A. phagocytophilum–Infected Cells
  • Support Protocol 4: Immunofluorescent Detection of A. phagocytophilum in Infected Cells
  • Support Protocol 5: Cryopreservation of A. phagocytophilum–Infected HL‐60 Cells
  • Basic Protocol 3: Obtaining Host Cell–Free A. phagocytophilum by Differential Centrifugation
  • Support Protocol 6: Density Gradient Purification of Host Cell–Free A. phagocytophilum
  • Support Protocol 7: Fluorescent Labeling of Live A. phagocytophilum
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Inoculation of A. phagocytophilum into Laboratory Mice

  Materials
  • 5% (v/v) Lysol
  • 70% (v/v) ethanol
  • Mouse: C3H/Smn.CicrHsd/scid (SCID; 3 to 5 weeks old; Harlan Sprague‐Dawley) or C3H/HeN (3 to 5 weeks old; National Cancer Institute Animal Production Program, Frederick Cancer Research Center)
  • Ketamine/xylazine anesthetic cocktail (see recipe) or other anesthetic agent as outlined in approved institutional protocol
  • Anticoagulant‐treated, A. phagocytophilum–infected peripheral blood (either from an A. phagocytophilum–infected mouse or a HGA patient that has not yet received antibiotic therapy)
  • Class II, Type B2, vertical flow biological safety cabinet
  • 1‐cc syringes with 28‐G, ½‐in. (12.7‐mm) needles
  • 1‐cc tuberculin syringes with 26‐G, ½‐in. (12.7‐mm) needles
  • Gauze
  • Additional reagents and equipment for anesthesia (Donovan and Brown, ) and intraperitoneal injection (Donovan and Brown, 1995a) of mice
NOTE: The total blood volume of a mouse is ∼5.5 ml per 100 g body weight. Do not collect more than 20% of total volume at one time. Do not collect more than this volume weekly. There is no limit to the number of times a mouse may be bled, provided that the aforementioned parameters are observed. For repeated blood collection via the retro‐orbital route, alternate eyes should be used.

Support Protocol 1: Preparation of Blood Smear for Identifying A. phagocytophilum–Infected Neutrophils

  Materials
  • A. phagocytophilum–infected mice (see protocol 1)
  • Glass tubes coated with anticoagulant (e.g., EDTA, heparin)
  • Glass microscope slides with frosted ends
  • Additional reagents and equipment for blood collection from mice (Donovan and Brown, 1995b)
NOTE: A blood sample from a human patient suspected of having HGA who has yet to receive antibiotic therapy is processed in a manner identical to that described in steps through .

Basic Protocol 2: Inoculation and Passaging of A. phagocytophilum Infection in HL‐60 Cell Culture

  Materials
  • 5% (v/v) Lysol
  • 70% (v/v) ethanol
  • HL‐60 cells (ATCC #CCL240) growing in culture
  • IMDM‐10: Iscove's modified Dulbecco's medium (Invitrogen 12440‐053) supplemented with 10% (v/v) heat‐inactivated FBS
  • A. phagocytophilum–infected blood sample(s) collected in glass tubes coated with anticoagulant (e.g., EDTA, heparin)
  • Class II, Type B2, vertical flow biological safety cabinet
  • 15‐ml polypropylene conical centrifuge tubes with screw caps (BD Falcon)
  • 25‐cm2 tissue culture flasks with vented caps (BD Falcon)
  • Additional reagents and equipment for counting cells using a hemacytometer (Strober, ) and determining cell viability by trypan blue exclusion (Strober, )
NOTE: All solutions and equipment coming into contact with living cells must be sterile and aseptic technique should be used accordingly.NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 2: Examination of Tissue Culture Cells for A. phagocytophilum Infection

  Materials
  • 5 × 105 cell/ml suspension of A. phagocytophilum–infected HL‐60 cells in IMDM‐10 (see protocol 3)
  • 5 × 105 cell/ml suspension of uninfected HL‐60 cells in IMDM‐10 (control)
  • 0.5% (v/v) sodium hypochlorite
  • Glass microscope slides with frosted ends
  • Cytocentrifuge setup (Thermo Electron Corp.) including:
    • Shandon Stainless Steel Cytoclip
    • Shandon TPX Filter Cards
    • Shandon TPX Sample Chamber
    • Shandon Cytospin 4 Cytocentrifuge
  • Absorbent bench paper with plastic backing

Support Protocol 3: Cytological Staining of A. phagocytophilum–Infected Cells

  Materials
  • Protocol Hema‐3 staining solutions (Fisher):
    • Protocol Hema‐3 fixative
    • Protocol Hema‐3 solution I
    • Protocol Hema‐3 solution II
  • Microscope slides with blood smear (see protocol 2 or protocol 42) or HL‐60 cells (see protocol 4)
  • Four glass staining dishes with removable racks (Wheaton model 900200)
  • Absorbent bench paper with plastic backing
  • Light microscope (unit 2.1)

Support Protocol 4: Immunofluorescent Detection of A. phagocytophilum in Infected Cells

  Materials
  • 5% (v/v) Lysol
  • 70% (v/v) ethanol
  • A. phagocytophilum–infected HL‐60 cells (see protocol 3)
  • Uninfected HL‐60 cells (control)
  • Phosphate‐buffered saline, pH 7.4 (PBS; appendix 2A)
  • Acetone
  • PBS/1% and 5% BSA: phosphate‐buffered saline, pH 7.4 (PBS; appendix 2A) containing 1% or 5% (v/v) bovine serum albumin (BSA)
  • Polyclonal A. phagocytophilum antiserum (rabbit or murine; see Cooper and Patterson, )
  • Fluorescein isothiocyanate (FITC)–conjugated secondary antibody: either goat anti‐rabbit IgG (Sigma cat. no. F‐6005) or goat anti‐mouse IgG (Sigma cat. no. F‐8264)
  • PBS‐T: phosphate‐buffered saline, pH 7.4 (PBS; appendix 2A) containing 0.05% (v/v) Tween 20
  • 20 µg/ml propidium iodide (light‐sensitive; store up to 6 months at 4°C; optional)
  • Antifade gel‐mounting medium (Biomeda Gelmount BMM‐01)
  • Clear nail polish
  • 12‐well (5‐mm diameter) Teflon‐coated immunofluorescence slides (Carlson Scientific, cat. no. 101205) with coverslips
  • Class II, Type B2, vertical flow biological safety cabinet
  • Plastic, light‐impermeable container large enough to accommodate slides
  • 1‐ml serological pipets
  • Forceps
  • Fluorescence microscope with filters for detecting FITC and rhodamine emission
  • Additional reagents and equipment for counting cells using a hemacytometer (Strober, )
NOTE: Propidium iodide staining (steps to ) is optional. Propidium iodide binds DNA, which results in the host cell nuclei staining red. This can facilitate easier identification of host cells though it is not necessary.

Support Protocol 5: Cryopreservation of A. phagocytophilum–Infected HL‐60 Cells

  Materials
  • 5% (v/v) Lysol
  • 70% (v/v) ethanol
  • A. phagocytophilum–infected HL‐60 cells growing in culture (see protocol 3)
  • 100% isopropanol
  • IMDM‐10 with and without 12% (v/v) dimethylsulfoxide (DMSO)
  • 0.5% (v/v) sodium hypochlorite
  • Class II, Type B2, vertical flow biological safety cabinet
  • 10‐ and 1‐ml serological pipets
  • 15‐ml polypropylene conical centrifuge tubes with screw caps (BD Falcon)
  • Refrigerated tabletop centrifuge
  • Alcohol‐resistant pen (VWR)
  • 250‐ml beaker for waste
  • 2‐ml screw‐cap cryovials
  • Nalgene Cryo 1°C freezing container
  • −80°C freezer
  • Cryobiological storage vessel containing liquid nitrogen
  • 25‐cm2 tissue culture flasks with vented caps (BD Falcon)
NOTE: All solutions and equipment coming into contact with living cells must be sterile and aseptic technique should be used accordingly.NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 3: Obtaining Host Cell–Free A. phagocytophilum by Differential Centrifugation

  Materials
  • 5% (v/v) Lysol
  • 70% (v/v) ethanol
  • A. phagocytophilum–infected HL‐60 cells growing in culture (see protocol 3)
  • Uninfected HL‐60 cells (control)
  • Phosphate‐buffered saline, pH 7.4 (PBS; appendix 2A), prechilled
  • 0.5% (v/v) sodium hypochlorite
  • Class II, Type B2, vertical flow biological safety cabinet
  • 25‐, 10‐, and 5‐ml serological pipets
  • 15‐ and 50‐ml polypropylene conical centrifuge tubes with screw caps (BD Falcon)
  • Refrigerated tabletop centrifuge
  • 500‐ml beaker for waste
  • 5‐cc tuberculin syringes
  • 27‐G, ½‐in. (12.7‐mm) needles
  • Additional reagents and equipment for counting cells (Strober, ) and cytological (see protocol 5) or immunofluorescence staining (see protocol 6) of A. phagocytophilum cultures
NOTE: All solutions and equipment coming into contact with living cells must be sterile and aseptic technique should be used accordingly.

Support Protocol 6: Density Gradient Purification of Host Cell–Free A. phagocytophilum

  Materials
  • 5% (v/v) Lysol
  • 70% (v/v) ethanol
  • A. phagocytophilum–infected HL‐60 cells growing in culture (see protocol 3)
  • PBS/glucose (see recipe), ice cold
  • Complete Protease Inhibitor Cocktail Tablets (Roche, cat. no. 1 697 498)
  • DNase I (from bovine pancreas; Roche cat. no. 776785)
  • RNase (from bovine pancreas; Roche cat. no. 1 119 915)
  • 30% Renografin (see recipe)
  • 42% Renografin (see recipe)
  • SPGN buffer (see recipe)
  • 0.5% (v/v) sodium hypochlorite
  • Class II, Type B2, vertical flow biological safety cabinet
  • 50‐ml polypropylene conical centrifuge tubes with screw caps (BD Falcon)
  • Refrigerated tabletop centrifuge
  • 1‐liter beaker for waste
  • 10‐cc tuberculin syringe
  • Beckman L7‐55 ultracentrifuge and Beckman SW 28 swinging‐bucket rotor (or equivalent)
  • 38.5‐ml ultracentrifuge tubes (Beckman Ultra‐Clear)
  • Harvard trip balance
  • 2.0‐ml polypropylene microcentrifuge tubes
  • Additional reagents and equipment for counting cells (Strober, ), cytological (see protocol 5) or immunofluorescence staining (see protocol 6) of A. phagocytophilum cultures, preparing a semipure preparation of A. phagocytophilum (see protocol 8), and protein assay ( appendix 3A)
NOTE: All solutions and equipment coming into contact with living cells must be sterile and aseptic technique should be used accordingly.

Support Protocol 7: Fluorescent Labeling of Live A. phagocytophilum

  Materials
  • Semipure preparation of host cell–free A. phagocytophilum (see protocol 8)
  • Phosphate‐buffered saline, pH, pH 7.4 (PBS; appendix 2A), chilled
  • 10 µM CellTracker Green CMFDA (see recipe)
  • 250‐ml beaker for waste
  • Fluorescence microscope with filters for detecting FITC emission
NOTE: All solutions and equipment coming into contact with living cells must be sterile and aseptic technique should be used accordingly.
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Figures

Videos

Literature Cited

   Borjesson, D.L. and Barthold, S.W. 2002. The mouse as a model for investigation of human granulocytic ehrlichiosis: Current knowledge and future directions. Comp. Med. 52:403‐413.
   Carlyon, J.A. and Fikrig, E. 2003. Invasion and survival strategies of Anaplasma phagocytophilum. Cell Microbiol. 5:743‐754.
   Carlyon, J.A. and Fikrig, E. 2004. Pathogenic strategies of Anaplasma phagocytophilum, a unique bacterium that colonizes neutrophils. In Sixty‐third Symposium of the Society for General Microbiology (M. Scourfield, ed.) pp. 301‐329. Cambridge University Press, Cambridge, U.K.
   Carlyon, J.A., Akkoyunlu, M., Xia, L., Yago, T., Wang, T., Cummings, R.D., McEver, R.P., and Fikrig, E. 2003. Murine neutrophils require alpha 1,3‐fucosylation but not PSGL‐1 for productive infection with Anaplasma phagocytophilum. Blood 102:3387‐3395.
   Chen, S.M., Dumler, J.S., Bakken, J.S. and Walker, D.H. 1994a. Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease. J. Clin. Microbiol. 32:589‐595.
   Chen, S.M., Dumler, J.S., Feng, H.M. and Walker, D.H. 1994b. Identification of the antigenic constituents of Ehrlichia chaffeensis. Am. J. Trop. Med. Hyg. 50:52‐58.
   Cooper, H.M. and Patterson, Y. Production of polyclonal antisera. 1995. In Current Protocols in Immunology (S.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and Warren Strober, eds.) pp. 2.4.1‐2.4.9. John Wiley and Sons, Hoboken, N.J.
   Donovan, J. and Brown, P. 1995. Parenteral injections. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 1.6.1‐1.6.10. John Wiley & Sons, Hoboken, N.J.
   Donovan, J. and Brown, P. 1998. Anesthesia. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 1.4.1‐1.4.5. John Wiley & Sons, Hoboken, N.J.
   Dumler, J.S. and Bakken, J.S. 1998. Human ehrlichioses: Newly recognized infections transmitted by ticks. Annu. Rev. Med. 49:201‐213.
   Dumler, J.S. and Brouqui, P. 2004. Molecular diagnosis of human granulocytic anaplasmosis. Expert Rev. Mol. Diagn. 4:559‐569.
   Dumler, J.S., Barbet, A.F., Bekker, C.P., Dasch, G.A., Palmer, G.H., Ray, S.C., Rikihisa, Y., and Rurangirwa, F.R. 2001. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: Unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and ‘HGE agent” as subjective synonyms of Ehrlichia phagocytophila. Int. J. Syst. Evol. Micr. 51:2145‐2165.
   Goodman, J.L., Nelson, C.M., Klein, M.B., Hayes, S.F. and Weston, B.W. 1999. Leukocyte infection by the granulocytic ehrlichiosis agent is linked to expression of a selectin ligand. J. Clin. Invest. 103:407‐412.
   Goodman, J.L., Nelson, C., Vitale, B., Madigan, J.E., Dumler, J.S., Kurtti, T.J. and Munderloh, U.G. 1996. Direct cultivation of the causative agent of human granulocytic ehrlichiosis. New Engl. J. Med. 334:209‐215.
   Herron, M.J., Nelson, C.M., Larson, J., Snapp, K.R., Kansas, G.S. and Goodman, J.L. 2000. Intracellular parasitism by the human granulocytic ehrlichiosis bacterium through the P‐selectin ligand, PSGL‐1. Science 288:1653‐1656.
   Hodzic, E., Ijdo, J.W., Feng, S., Katavolos, P., Sun, W., Maretzki, C.H., Fish, D., Fikrig, E., Telford, S.R. 3rd, and Barthold, S.W. 1998. Granulocytic ehrlichiosis in the laboratory mouse. J. Infect. Dis. 177:737‐745.
   McCaul, T.F. and Williams, J.C. 1981. Developmental cycle of Coxiella burnetii: Structure and morphogenesis of vegetative and sporogenic differentiations. J. Bacteriol. 147:1063‐1076.
   Munderloh, U.G., Madigan, J.E., Dumler, J.S., Goodman, J.L., Hayes, S.F., Barlough, J.E., Nelson, C.M., and Kurtii, T.J. 1996. Isolation of the equine granulocytic ehrlichiosis agent, Ehrlichia equi, in tick cell culture. J. Clin. Microbiol. 34:664‐670.
   Munderloh, U.G., Jauron, S.D., Fingerle, V., Leitritz, L., Hayes, S.F., Hautman, J.M., Nelson, C.M., Huberty, B.W., Kurtii, T.J., Ahlstrand, G.G., Greig, B., Mellencamp, M.A., and Goodman, J.L. 1999. Invasion and intracellular development of the human granulocytic ehrlichiosis agent in tick cell culture. J. Clin. Microbiol. 37:2518‐2524.
   Plano, G.V. and Winkler, H.H. 1989. Solubilization and reconstitution of the Rickettsia prowazekii ATP/ADP translocase. J. Membrane Biol. 110:227‐233.
   Rikihisa, Y. 2003. Mechanisms to create a safe haven by members of the family Anaplasmataceae. Ann. N.Y. Acad. Sci. 990:548‐555.
   Strober, W. 1997a. Monitoring cell growth. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. A.3A.1‐A.3A.2. John Wiley & Sons, Hoboken, N.J.
   Strober, W. 1997b. Trypan blue exclusion test of cell viability. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. A.3B.1‐A.3B.2. John Wiley & Sons, Hoboken, N.J.
   Weiss, E., Williams, J.C., Dasch, G.A. and Kang, Y.H. 1989. Energy metabolism of monocytic Ehrlichia. Proc. Natl. Acad. Sci. U.S.A. 86:1674‐1678.
   Yago, T., Leppanen, A., Carlyon, J.A., Akkoyunlu, M., Karmakar, S., Fikrig, E., Cummings, R.D., and McEver, R.P. 2003. Structurally distinct requirements for binding of P‐selectin glycoprotein ligand‐1 and sialyl Lewis x to Anaplasma phagocytophilum and P‐selectin. J. Biol. Chem. 278:37987‐37997.
Key References
   Borjesson and Barthold 2002. See above.
  An excellent review of the use of laboratory mice for in vivo maintenance and studies of A. phagocytophilum.
   Carlyon and Fikrig 2004. See above.
  This reference provides a comprehensive review of the epidemiology, and clinical symptoms of HGA, as well as of the strategies used by A. phagocytophilum for invasion and intracellular survival.
   Goodman et al., 1996. See above.
  Presents the first demonstration of in vitro cultivation of A. phagocytophilum.
   Herron et al., 2000. See above.
  This seminal paper identified PSGL‐1 as a ligand required for A. phagocytophilum binding and invasion of human neutrophils and HL‐60 cells, and at least partially explains the bacterium's tropism for neutrophils. It also includes the first documentation of the use of CellTracker Green CMFDA for labeling A. phagocytophilum and tracing the bacterium's interactions with host cells.
   Hodzic et al., 1998. See above.
  This presents the first demonstration of the use of the laboratory mouse as an in vivo model for maintaining and studying A. phagocytophilum.
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