JOURNAL OF CLINICAL MICROBIOLOGY, July 1993, p. 1936-19390095-1137/93/071936-04$02.00/0Copyright 1993, American Society for Microbiology

Use of Immunoelectron Microscopy to DemonstrateFrancisella tularensis

T. W. GEISBERT,l* P. B. JAHRLING,l AND J. W. EZZELL, JR.2

Virology' and Bacteriology2 Divisions, United States Army Medical Research Instituteof Infectious Diseases, Fort Detrick, Frederick Maryland 21702-5011

Received 19 January 1993/Accepted 2 April 1993

Three immunoelectron microscopy (IEM) methods were employed to show laboratory-cultivated Francisellatularensis. By the IEM assays, F. tularensis was distinguished from four antigenically distinct gram-negativebacteria. IEM should be a valuable tool for confirming presumptive isolates ofF. tularensis and may potentiallybe useful for demonstrating other medically important bacteria.

Francisella tularensis, the causative agent of tularemia, isa small gram-negative coccobacillus that can survive for longperiods in reticuloendothelial and circulating macrophagesof animals and humans (9). Laboratory diagnosis of tulare-mia is usually performed by employing assays that detectantibodies against F. tularensis. These assays include thebacterial microagglutination test (1, 13, 14), which may beperformed 8 to 10 days after initial clinical symptoms oftularemia appear, and the enzyme-linked immunosorbentassay (ELISA) (1, 2, 4, 14), which may show class-specificantibodies slightly earlier after the onset of illness thanagglutination assays. However, because human tularemia iseffectively treated with antibiotics (12), methods that showF. tularensis earlier in the disease course may facilitateeffective treatment.The first convincing evidence of F. tularensis antigen was

shown in tissue biopsies utilizing fluorescent antibodies (11).More recent work suggested that it may be possible todemonstrate F. tularensis by 16S rRNA sequence analysis(5), and antigen capture ELISA employed in conjunctionwith an immunoblot assay identified a monoclonal antibodythat also may be useful for detection of F. tularensis (6).Demonstration of F. tularensis, whether by immunofluores-cence tests, rRNA sequence analysis, or antigen captureELISA, should provide an earlier and more specific diagno-sis of tularemia than evidence of antibody formation. Whileimmunoelectron microscopy (IEM) procedures frequentlyrequire more time to perform than rRNA sequence analysisor antigen capture ELISA, we successfully employed IEMto demonstrate a viral antigen in less than 6 h (8). BecauseIEM enables visualization of antibody-antigen reactions onthe surface of morphologically identifiable bacterial cells,IEM may ensure a more definitive diagnosis of tularemiathan immunofluorescence tests, rRNA sequence analysis, or

antigen capture ELISA. We report the application of threeIEM techniques to show F. tularensis in an effort to providea reliable method for confirming isolates reputed to be F.tularensis, for corroborating newly developed assays fordemonstrating F. tularensis, and for assisting studies de-signed to elucidate mechanisms of the pathogenesis of F.tularensis.The live vaccine strain of F. tularensis was obtained as a

frozen suspension from the culture collection of the UnitedStates Army Medical Research Institute of Infectious Dis-

* Corresponding author.

eases (USAMRIID), Fort Detrick, Md. Approximately 1,000CFU of the isolate was plated on 5% sheep blood agar,

supplemented with cysteine, and incubated at 37°C for 48 h.In addition, Brucella suis, Salmonella typhimurium, Pseudo-monas aeruginosa, and Escherichia coli were obtained fromthe culture collection at USAMRIID, grown at 37°C for 48 h,and employed as negative controls. Bacterial growth fromplates was harvested, suspended in phosphate-buffered sa-

line, and centrifuged in 1.5-cm3 Eppendorf microcentrifugetubes at 12,000 x g for 30 s to form loose pellets. Superna-tant was removed, and 2% paraformaldehyde-0.1% glutar-aldehyde in 0.1 M Millonig's phosphate buffer (MPB) (pH7.4) was added to each pellet. After fixation for 2 h, thepellets were rinsed three times with MPB and processedindependently for either negative-contrast IEM (nc-IEM),pre-embedding IEM (pre-IEM), or post-embedding IEM(post-IEM).Nc-IEM was performed as described previously for Re-

ston virus (8). Primary antibodies utilized in the nc-IEMassay were dilutions (1 in 75) of either normal rabbit serum(NRS) or rabbit antiserum raised against F. tularensis livevaccine strain (anti-FT). The anti-FT was collected andpooled 90 days after intravenous inoculation of rabbits with107 F. tularensis live vaccine strain organisms; employmentof a microagglutination assay (1, 3) showed that the micro-agglutinin titer of the pooled rabbit antisera was 1 in 320. Thesecondary antibody employed for nc-IEM was a dilution (1in 10) of goat anti-rabbit immunoglobulin G labeled with10-nm gold spheres (GARaG10) (Ted Pella, Inc., Redding,Calif.). After the antibody incubations, nc-IEM specimenswere negatively contrasted by the addition of 1% phospho-tungstic acid (pH 6.6) and examined at 80 kV with a JEOL100CX electron microscope (JEOL Ltd., Peabody, Mass.).Pre-IEM was performed in 1.5-cm3 Eppendorf microcentri-fuge tubes. Bacterial pellets were successively resuspendedtwice in BTT, once in 4% normal goat serum (AmershamCorp., Arlington Heights, Ill.) (to reduce nonspecific immu-nolabeling potentially caused by employment of the second-ary antibody) for 15 min at 23°C, and once again in BTT;they were then incubated for 16 h at 4°C or for 2 h at 23°C indilutions (1 in 75) of either NRS or anti-FT in BTT. Speci-mens were then microcentrifuged, resuspended three timesin BTT, and incubated in GARaGlO diluted 1 in 10 in BTTfor 1 h at 23°C. Specimens were resuspended twice in BTTand twice in MPB, pelleted by microcentrifugation, and fixedfor 20 min in 2% glutaraldehyde in MPB to fix the antibody-antigen complex. After fixation, pellets were rinsed in MPB

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FIG. 1. Electron micrograph showing attachment of gold spheresto surface of an F. tularensis bacterium incubated with a polyclonal(rabbit) antiserum and examined by nc-IEM. Bar = 250 nm.

and processed for transmission electron microscopy (7). Forpost-IEM, fixed-bacterium pellets were dehydrated in etha-nol, infiltrated with either LR White resin (Polysciences) orPOLY/BED 812 resin (Polysciences), and cured for 18 h at490C (LR White) or 60°C (POLY/BED 812). Post-IEM wasperformed as previously described (7) with the immunereagents employed as described above for nc-IEM andpre-IEM.The nc-IEM procedure demonstrated F. tularensis less

than 7 h after sampling. Figure 1 shows positive immunogoldstaining of an F. tularensis bacterium incubated with theanti-FT. The bacteria readily adsorbed the negative-contraststain employed and appeared to be exceedingly electrondense. This positive-contrast staining of bacteria treatedwith a negative-contrast reagent was previously addressedby Hayat and Miller (10). While positive-contrast staining ofbacteria hindered the visualization of gold probes and thephotography of specimens, the diagnosis was not affected.No reaction was observed when F. tularensis was incubatedwith NRS or when B. suis, S. typhimunum, P. aeruginosa,or E. coli was incubated with anti-FT.Pre-IEM successfully demonstrated antigens along the

outer membrane and a layer of extracellular material (floc-culent layer) encircling the outer membrane of F. tularensis(Fig. 2 and 3). The flocculent layer, which comprised lipids,proteins, and carbohydrates, was shown to be nontoxic forguinea pigs and was considered to represent a capsule (13).Differences in the pre-IEM reaction product due to primaryantibody incubation time were difficult to assess. The 16-hincubation period appeared to reveal somewhat more intensegold sphere labeling of F. tularensis than the 2-h incubationperiod, but the stronger immunolabeling may be due to thelower temperature employed in the 16-h procedure. Whilepre-IEM required more time to perform than nc-IEM, theantibody-antigen complex was more easily visualized be-cause pre-IEM provided better resolution of bacterial mem-branes and internal structures than results demonstrated by

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FIG. 2. Electron micrograph illustrating adhesion of gold spheres (arrows) to extracellular antigens of F. tularensis by pre-IEM. Bar = 500nm.

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FIG. 3. Electron micrograph showing attachment of gold spheres (large arrows) to outer membrane (arrowheads) and flocculent layer(small arrows) of F. tularensis bacteria (F) by pre-IEM. Bar = 250 nm.

nc-IEM. As with nc-IEM, pre-IEM showed no reactionwhen F. tularensis was incubated with NRS or when thenegative-control bacteria were incubated with anti-FT. Al-though the gold spheres employed in the pre-IEM procedurewere 10 nm in diameter, they did not penetrate the outermembrane of F. tularensis and were not capable of labelinginternal antigens.Post-IEM, performed within 48 h after bacterial growth

was harvested, showed positive gold sphere labeling of F.tularensis when either LR White or POLY/BED 812 (Fig. 4)sections were incubated with anti-FT. Because no significantdifference in the intensity or specificity of labeling wasdetected between the two resins, POLY/BED 812 appearedto be the more appropriate choice for IEM as it showedbetter ultrastructural detail and contrast of bacteria than LRWhite. No reaction was seen when LR White or POLY/BED812 sections were incubated with NRS or when thin sectionsof the four antigenically distinct gram-negative bacteria wereincubated with anti-FT.By employing anti-FT in three easily performed IEM

procedures, we demonstrated laboratory-cultivated F. tula-rensis 7 to 48 h after initiating the assays. In addition, weemployed anti-FT to distinguish F. tularensis from fourantigenically distinct gram-negative bacteria. Pre-IEMshowed better resolution of bacterial membranes and inter-nal structures than nc-IEM or post-IEM, but post-IEM maybe preferred when tissues are used, since this techniqueenables antigens to be more easily accessed by gold sphere-conjugated secondary antibodies. Because nc-IEM demon-strated F. tularensis in less than 7 h, this IEM method maycomplement the recently developed 16S rRNA sequenceanalysis assay (5) and antigen capture ELISA (6) to providean earlier and more specific diagnosis of tularemia thandemonstration of antibodies against the bacteria. Also, IEMmay be safer to perform than other assays demonstratingpathogenic bacteria, considering that most manipulations areperformed with glutaraldehyde-killed specimens. Future ap-plication of our IEM procedures will be directed toward

identifying immune reagents to differentiate species of Fran-cisella and to support studies employing monoclonal anti-bodies to distinguish and compare antigens of medicallyimportant gram-negative bacteria. In addition, these IEM

FIG. 4. Electron micrograph demonstrating association of goldspheres with the outer membrane (arrowheads) of an F. tularensisbacterium by post-IEM. Bar = 125 nm.

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procedures are currently being used to assist pathogenesisstudies by demonstrating F. tularensis in fluids and tissues ofexperimentally infected animals.

Thanks are expressed to Kathy Kuehl and Eric Oldenburg of theelectron microscopy laboratory at USAMRIID for expert technicalassistance. We are grateful to Michael J. Langford and Judith G.Pace for reviewing the manuscript.

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and antibodies to Francisella tularensis outer membrane anti-gens in the early diagnosis of disease during an outbreak oftularemia. J. Clin. Microbiol. 26:433-437.

2. Bevanger, L., J. A. Maeland, and A. I. Naess. 1989. Competitiveenzyme immunoassay for antibodies to a 43,000-molecular-weight Francisella tularensis outer membrane protein for thediagnosis of tularemia. J. Clin. Microbiol. 27:922-926.

3. Brown, S. L., F. T. McKinney, G. C. Klein, and W. L. Jones.1980. Evaluation of a safranin-O-stained antigen microaggluti-nation test for Francisella tularensis antibodies. J. Clin. Micro-biol. 11:146-148.

4. Carlsson, H. E., A. A. Lindberg, G. Lindberg, B. Hederstedt,K.-A. Karisson, and B. 0. Agell. 1979. Enzyme-linked immu-nosorbent assay for immunological diagnosis of human tulare-mia. J. Clin. Microbiol. 10:615-621.

5. Forsman, M., G. Sandstrom, and B. Jaurin. 1990. Identificationof Francisella species and discrimination of type A and type Bstrains of F. tularensis by 16S rRNA analysis. Appl. Environ.Microbiol. 56:949-955.

6. Fulop, M. J., T. Webber, R. J. Manchee, and D. C. Kelly. 1991.Production and characterization of monoclonal antibodies di-

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7. Geisbert, T. W., P. B. Jahrling, M. A. Hanes, and P. M. Zack.1992. Association of Ebola-related Reston virus particles andantigen with tissue lesions of monkeys imported to the UnitedStates. J. Comp. Pathol. 106:137-152.

8. Geisbert, T. W., J. B. Rhoderick, and P. B. Jahrling. 1991.Rapid identification of Ebola virus and related filoviruses in fluidspecimens using indirect immunoelectron microscopy. J. Clin.Pathol. 44:521-522.

9. Gutman, L. T. 1988. Francisella, p. 502-505. In W. K. Joklik,H. P. Willett, D. B. Amos, and C. M. Wilfert (ed.), Zinssermicrobiology, 20th ed. Appleton and Lange, Norwalk, Conn.

10. Hayat, M. A., and S. E. Miller. 1990. Negative staining, p. 1-48.In M. A. Hayat and S. E. Miller (ed.), Negative staining.McGraw-Hill Publishing Co., New York.

11. Karlsson, K.-A., S. Dahlstrand, E. Hanks, and 0. Soderlind.1970. Demonstration of Francisella tularensis (syn. Pasteurellatularensis) in sylvan animals with the aid of fluorescent antibod-ies. Acta Pathol. Microbiol. Scand. Sect. B 78:647-651.

12. Overhold, E. L., W. D. Tigertt, P. J. Kadull, M. K. Ward, N. D.Charkes, R. M. Rene, T. E. Salzman, and M. Stephens. 1961. Ananalysis of 42 cases of laboratory acquired tularemia. Treatmentwith broad spectrum antibiotics. Am. J. Med. 30:785.

13. Swartz, M. N. 1990. Yersinia, Francisella, Pasteurella, andBrucella, p. 601-614. In B. D. Davis, R. Dulbecco, H. N. Eisen,and H. S. Ginsberg (ed.), Microbiology. J. B. Lippincott Co.,Philadelphia.

14. Syrjala, H., P. Koskela, T. Rippatti, A. Salminen, and E. Herva.1986. Agglutination and ELISA methods in the diagnosis oftularemia in different clinical forms and severities of the disease.J. Infect. Dis. 153:142-145.

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