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Extracellular Bartonella henselae and artifactual intraerythrocytic pseudoinclusions in experimentally infected cats
Veterinary Microbiology 76 (2000) 283±290
Extracellular Bartonella henselae and artifactual intraerythrocytic pseudoinclusions in experimentally infected cats L. Guptilla,*, C-C. Wub, L. Glickmanb, J. Turekc, L. Slaterd, H. HogenEschb a
Department of Veterinary Clinical Sciences, Purdue University, W. Lafayette, IN 47907, USA b Department of Veterinary Pathobiology, Purdue University, W. Lafayette, IN 47907, USA c Department of Basic Medical Sciences, Purdue University, W. Lafayette, IN 47907, USA d Department of Medicine, Infectious Diseases Section, VA Medical Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA Received 10 September 1999; received in revised form 2 May 2000; accepted 2 May 2000
Abstract Blood, spleen and liver of speci®c pathogen-free (SPF) cats and SPF cats experimentally infected with Bartonella henselae were examined. Using immunohistochemical labeling, no intracellular B. henselae were observed in tissues of any cats, but extracellular B. henselae were detected in tissues of infected cats. Pseudoinclusions were detected in erythrocytes of all cats using electron microscopy. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Bartonella henselae; Cat; Erythrocyte; Immunohistochemistry; Artifact
1. Introduction Bartonella henselae causes cat scratch disease, bacillary angiomatosis and other clinical conditions in human beings (Relman et al., 1990; Slater et al., 1992; Koehler et al., 1994). High rates of persistent asymptomatic bacteremia occur in young cats, the apparent major reservoir of B. henselae (Koehler et al., 1994; Breitschwerdt and Kordick, 1995; Chomel et al., 1995). The site of localization of B. henselae in infected cats has not been identi®ed. There is one report of intraerythrocytic B. henselae in two naturally infected cats (Kordick and Breitschwerdt, 1995) based on electron microscopic *
Corresponding author. Tel.: 1-765-496-1107; fax: 1-765-494-9830. E-mail address: [email protected] (L. Guptill). 0378-1135/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 0 0 ) 0 0 2 4 0 - 6
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observations. Published transmission electron photomicrographs (Kordick and Breitschwerdt, 1995) show intraerythrocytic objects with what appear to be single lipid bilayer membranes identical to the erythrocyte membrane. While it is possible that these intraerythrocytic objects are B. henselae with altered cell walls, electron micrographs of B. henselae within other cell types clearly show a trilaminate wall (Batterman et al., 1995) typical of Gram negative bacteria (Stanier et al., 1976). Bartonella bacilliformis is a related organism that is intraerythrocytic and causes hemolytic anemia in human beings (Cuadra and Takano, 1969; Kreier et al., 1992; Ihler, 1996). Within human erythrocytes, B. bacilliformis maintains a well-de®ned trilaminate wall and internal structures typical of Gram negative bacteria (Cuadra and Takano, 1969). The purpose of the study reported here was to investigate whether B. henselae is intraerythrocytic in experimentally infected cats. 2. Materials and methods Spleen and liver were collected from speci®c pathogen-free (SPF) cats infected by intravenous inoculation. The microbiological, immunological and pathological data for these cats was previously reported (Guptill et al., 1997). The B. henselae isolate used was provided by Dr. David F. Welch, University of Oklahoma. This isolate was originally isolated from the blood of a cat owned by a patient with cat scratch disease. It was characterized by standard culture techniques including gas liquid chromatography and immuno¯uorescence, which together are speci®c for identi®cation of Bartonella to the species level (Welch et al., 1993). In addition, blood was examined from four speci®c pathogen-free cats infected by intradermal inoculation using the same isolate after one passage onto chocolate agar after re-isolation from a cat. Fifty microliters of a suspension of 108 colony-forming units (CFU) B. henselae/ml were injected intradermally at each of two sites. Tissues from SPF cats inoculated intradermally with saline were used as controls. Blood from intradermally inoculated cats bacteremic for 6 and 8 weeks (103±104 CFU B. henselae/ml blood) and from a control cat was collected into sodium heparin. An aliquot was washed in phosphate buffered saline (PBS) then ®xed with 3% glutaraldehyde at 48C for 24±48 h; processing for electron microscopy is described below. Heparinized blood diluted 1:2 with PBS was layered onto Ficoll±Hypaque (Sigma, St. Louis, MO), speci®c gravity (SG) 1.077, and centrifuged. The mononuclear cell band was resuspended in Percoll (Sigma), SG 1.03, layered onto a discontinuous Percoll gradient (layers of 1.05 and 1.07 SG) and centrifuged (Bishop, 1995). Plasma was centrifuged at 13,600g at 48C for 15 min, and the pellet resuspended in PBS. Individual bands from the Percoll gradient were collected and resuspended in PBS. Cytocentrifugation slides were prepared from each white blood cell fraction, the platelet fraction, and the plasma pellet. Cytocentrifugation slides were labeled with polyclonal goat anti-B. henselae serum as previously described (Guptill et al., 1997). Spleen and liver specimens from two cats inoculated intravenously with B. henselae and from one uninfected cat (Guptill et al., 1997) were snap frozen at the time of euthanasia 4 or 32 weeks after inoculation. Frozen sections mounted on positively charged glass slides were ®xed with acetone at ÿ208C.
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The tissue sections were labeled with polyclonal goat anti-B. henselae serum as previously described (Guptill et al., 1997). Erythrocytes collected after Ficoll centrifugation were washed twice in PBS, resuspended in 3% glutaraldehyde and held at 48C for 24±48 h. Fixed erythrocytes or ®xed whole blood cells were washed twice in PBS, post-®xed in 1% osmium tetroxide (OsO4) for 1 h at 258C, and pelleted in 2% agarose. Pellets were diced into 1 mm pieces, dehydrated in ethanol solutions, propylene dioxide, and embedded in epoxy resin (Poly/ Bed 8121, Polysciences, Warrinton, PA). Thin sections were stained with uranyl acetate and lead citrate and examined on a JEOL 100 CX electron microscope. Erythrocyte pellets not post-®xed in OsO4 were dehydrated in acetone solutions and processed using indium trichloride to label bacterial DNA (Knight, 1977), embedded, and examined. B. henselae grown on chocolate agar were also prepared and stained for electron microscopy using these techniques. 3. Results and discussion Extracellular B. henselae, labeled immunohistochemically, were seen both individually and in clusters in plasma and platelet fractions of infected cats (Fig. 1). Extracellular B. henselae were also seen in frozen sections of spleen (Fig. 2) and liver, and on occasional blood smears of infected cats. No B. henselae were observed in mononuclear cell or granulocyte fractions or in any preparations from uninfected cats. Pseudoinclusions (Fig. 3) but not bacteria were observed in every grid of erythrocytes examined from infected and uninfected SPF cats using electron microscopy. In contrast,
Fig. 1. Extracellular Bartonella henselae (arrow) in the platelet fraction of a cat 8 weeks after infection, labeled using Fast Red as substrate for alkaline phosphatase. Magni®cation 1000. Bar7.5 mm.
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Fig. 2. Extracellular Bartonella henselae in the spleen of a cat 8 weeks after infection, labeled using ¯uorescein isothiocyanate. Magni®cation 1000. Bar7.5 mm.
no labeling with indium trichloride was seen within erythrocytes of infected or uninfected cats. Indium trichloride labeling of nucleic acids of B. henselae grown on chocolate agar was clearly positive (data not shown) and B. henselae grown on chocolate agar had typical Gram negative cell wall structure (Fig. 4). Pseudoinclusions result when ®xed erythrocytes are sectioned through an irregular border and viewed in two dimensions. These artifacts of preparation are pleomorphic and may resemble bacteria. Pseudoinclusions are often surrounded by a single lipid bilayer and contain debris that resembles bacterial DNA but is not labeled with indium trichloride. Intraerythrocytic objects were previously reported in 2.9% of 1220 erythrocytes and 6.2% of 645 erythrocytes examined in two bacteremic cats (Kordick and Breitschwerdt, 1995). The magnitude of bacteremia in these cats was not reported (Kordick and Breitschwerdt, 1995). In a normal adult cat with 6106 erythrocytes per microliter of blood the above values would equate to a bacteremia of 1.74108 and 3.72108 CFU/ml blood. This value is greater than published values for naturally and experimentally infected cats by 2±3 orders of magnitude. While current culture methods may not detect all bacteria present in blood samples, it seems unlikely that only 1 per 100 to 1 per 1000 of bacteria present are detected. Recovery of B. henselae from SPF cat blood inoculated in vitro with B. henselae was excellent (unpublished data). Colony counts from saline dilutions of bacteria used to inoculate blood and from inoculated blood of the same dilution were always within one order of magnitude. Pseudoinclusions in the current study were detected in 5.9% of 1947 erythrocytes of an uninfected SPF cat and in 5.2% of 2000 erythrocytes of a cat with a bacteremia of 2.4103 CFU/ml blood. Infected cats in this study, in several previous studies of experimentally infected cats (Chomel et al., 1996; Guptill et al., 1997; Abbott et al., 1997; Guptill et al., 1998), and in other
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Fig. 3. (a) Pseudoinclusions in erythrocytes of a cat infected with Bartonella henselae (magni®cation 47,850 bar0.2 mm), (b) Pseudoinclusions in erythrocytes of an uninfected speci®c pathogen free cat (magni®cation 72,500, bar0.1 mm).
experimental infections from our laboratory (unpublished data) were not anemic at any time. Reports of naturally infected cats state that infected cats are clinically healthy, but hematological data are not provided (Koehler et al., 1994; Kordick et al., 1995). Transient (2±3 weeks) anemia occurred in seven of eight experimentally infected cats in one study, although intraerythrocytic B. henselae were not speci®cally reported in those cats (Kordick and Breitschwerdt, 1997).
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Fig. 4. Electron photomicrograph of Bartonella henselae grown on chocolate agar. Gram-negative cell wall structures including outer membrane, peptidoglycan layer and the inner cell wall are visible. Magni®cation 66,000. Bar0.25 mm.
There was no evidence in the current study that the isolate of B. henselae used, either plate-grown after a small number of laboratory passages or after re-isolation from a cat following intradermal inoculation, infects erythrocytes. No erythrocytes of infected or uninfected cats were labeled with indium trichloride, whereas nucleic acids of B. henselae grown on chocolate agar were clearly labeled with indium trichloride. This indicates that B. henselae were not intraerythrocytic or they would have been detected using this procedure. The documented mechanism of human erythrocyte invasion by B. bacilliformis includes a combination of pili for attachment, elaboration of a protein, deformin, that alters the cytoskeleton of erythrocytes in the area of Bartonella entry, and spiral motion of a polar ¯agellum to propel the bacteria into erythrocytes (Benson et al., 1986; Mernaugh and Ihler, 1992; Mitchell and Minnick, 1995; Minnick et al., 1996). A locus from B. henselae that has sequence homology to the invasion locus of B. bacilliformis was isolated recently (Murakawa et al., 1996). When inserted into the genome of E. coli and expressed, this locus greatly enhanced the ability of E. coli to enter human erythrocytes. However, as has been demonstrated for toxin genes in Clostridium perfringens (Meer and Songer, 1997), the presence of a gene locus does not necessarily mean that the genes are transcribed or expressed. One study indicates that B. henselae invade feline erythrocytes in vitro (Mehock et al., 1998). Further work is necessary to determine whether some or most isolates of B. henselae carry and express a functional invasion locus, and whether this results in an intraerythrocytic location of B. henselae in infected cats.
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The results of the study reported here suggest that the isolate of B. henselae used does not infect erythrocytes in cats. The intraerythrocytic objects observed by us as reported here, and by others (Kordick and Breitschwerdt, 1995) are probably pseudoinclusions and not B. henselae. The presence of such structures alone does not suggest that B. henselae is intraerythrocytic in cats. In conclusion, there is currently insuf®cient evidence to conclude that B. henselae infect feline erythrocytes in vivo. Unless the presence of intraerythrocytic B. henselae can be con®rmed by immunological labeling techniques, the status of these bacteria as intraerythrocytic in cats should remain provisional. Acknowledgements This work was presented in part as poster #1 at the 77th Annual Meeting of the Conference of Research Workers in Animal Diseases, Chicago, IL, November 1996. We thank Deborah A.H. Van Horn for the electron photomicrographs. This work was supported in part by Intervet, Inc. and in part by an Andrews Fellowship, Purdue University (LG). References Abbott, R.C., Chomel, B.B., Kasten, R.W., Floyd-Hawkins, K.A., Kikuchi, Y., Koehler, J.E., Pedersen, N.C., 1997. Experimental and natural infection with Bartonella henselae in cats. Comp. Immunol. Microbiol. Infect. Dis. 20, 41±57. Batterman, H.J., Peek, J.A., Loutit, J.S., Falkow, S., Tompkins, L.S., 1995. Bartonella henselae and Bartonella quintana adherence to and entry into cultured human epithelial cells. Infect. Immun. 63, 4553±4556. Benson, L.A., Kar, S., McLaughlin, G., Ihler, G.M., 1986. Entry of Bartonella bacilliformis into erythrocytes. Infect. Immun. 54, 347±353. Bishop, S.A., 1995. Functional abnormalities in the immune system of FIV-infected cats. In: Willett, B.J., Jarrett, O. (Eds.), Feline Immunology and Immunode®ciency. Oxford Science Publications, Oxford, 150 pp. Breitschwerdt, E.B., Kordick, D.L., 1995. Bartonellosis. J. Am. Vet. Med. Assoc. 206, 1928±1931. Chomel, B.B., Abbott, R.C., Kasten, R.W., Floyd-Hawkins, K.A., Kass, P.H., Glaser, C.A., Pedersen, N.C., Koehler, J.E., 1995. Bartonella henselae prevalence in domestic cats in California: risk factors and association between bacteremia and antibody titers. J. Clin. Microbiol. 33, 2445±2450. Chomel, B.B., Kasten, R.W., Floyd-Hawkins, K.A., Chi, B., Yamamoto, K., Roberts-Wilson, J., Gur®eld, A.N., Abbott, R.C., Pedersen, N.C., Koehler, J., 1996. Experimental transmission of Bartonella henselae by the cat ¯ea. J. Clin. Microbiol. 34, 1952±1956. Cuadra, M., Takano, J., 1969. The relationship of Bartonella bacilliformis to the red blood cell as revealed by electron microscopy. Blood 33, 708±716. Guptill, L., Slater, L., Wu, C-C., Lin, T-L., Glickman, L.T., Welch, D.F., HogenEsch, H., 1997. Experimental infection of young speci®c pathogen-free cats with Bartonella henselae. J. Infect. Dis. 176, 206±216. Guptill, L., Slater, L.N., Wu, C-C., Lin, T-L., Glickman, L.T., Welch, D.F., Tobolski, J., HogenEsch, H., 1998. Evidence of reproductive failure and lack of perinatal transmission of Bartonella henselae in experimentally infected cats. Vet. Immunol. Immunopathol. 65, 177±189. Ihler, G.M., 1996. Bartonella bacilliformis: dangerous pathogen slowly emerging from deep background. FEMS Microbiol. Lett. 144, 1±11. Knight, D.P., 1977. Cytological staining methods in electron microscopy. In: Lewis, P.R., Knight, D.P. (Eds.), Practical Methods in Electron Microscopy. North-Holland, Amsterdam, 25 pp.
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Koehler, J.E., Glaser, C.A., Tappero, J.W., 1994. Rochalimaea henselae infection: a new zoonosis with the domestic cat as reservoir. JAMA 271, 531±535. Kordick, D.L., Breitschwerdt, E.B., 1995. Intraerythrocytic presence of Bartonella henselae. J. Clin. Microbiol. 33, 1655±1656. Kordick, D.L., Breitschwerdt, E.B., 1997. Relapsing bacteremia after blood transfusion of Bartonella henselae to cats. Am. J. Vet. Res. 58, 492±497. Kordick, D.L., Wilson, K.H., Sexton, D.J., Had®eld, T.L., Berkhoff, H.A., Breitschwerdt, E.B., 1995. Prolonged Bartonella bacteremia in cats associated with cat-scratch disease patients. J. Clin. Microbiol. 33, 3245±3251. Kreier, J.P., Gothe, R., Ihler, G.M., Krampitz, H.E., Mernaugh, G., Palmer, G.H., 1992. The hemotrophic bacteria: the families Bartonellaceae and Anaplasmataceae. In: Balows, A., Truper, H.G., Dworkin, M., Harder, W., Schleifer, K-H. (Eds.), The Prokaryotes. A handbook on the biology of bacteria: ecophysiology, isolation, identi®cation, applications. Vol. 2, Springer, New York, 3994 pp. Meer, R.R., Songer, J.G., 1997. Multiplex polymerase chain reaction assay for genotyping Clostridium perfringens. Am. J. Vet. Res. 58, 702±705. Mehock, J.R., Greene, C., Gherardini, F.C., Hahn, T-W., Krause, D.C., 1998. Bartonella henselae invasion of feline erythrocytes in vitro. Infect. Immun. 66, 3462±3466. Mernaugh, G., Ihler, G.M., 1992. Deformation factor: an extracellular protein synthesized by Bartonella bacilliformis that deforms erythrocyte membranes. Infect. Immun. 60, 937±943. Minnick, M.F., Mitchell, S.J., McAllister, S.J., 1996. Cell entry and the pathogenesis of Bartonella infections. Trends Microbiol. 4, 343±347. Mitchell, S.J., Minnick, M.F., 1995. Characterization of a two-gene locus from Bartonella bacilliformis associated with the ability to invade human erythrocytes. Infect. Immun. 63, 1552±1562. Murakawa, J., Peek, J.A., Tompkins, L.S., Falkow, S., 1996. Sequence and characterization of an invasion locus in Bartonella henselae. Abstracts of the 12th Sesqui-Annual meeting of the American Society for Rickettsiology and Rickettsial Diseases 50 (Abstr.). Relman, D.A., Loutit, J.S., Schmidt, T.M., Falkow, S., Tompkins, L.S., 1990. The agent of bacillary angiomatosis: an approach to the identi®cation of uncultured pathogens. N. Engl. J. Med. 323, 1573±1580. Slater, L.N., Welch, D.F., Min, K-W., 1992. Rochalimaea henselae causes bacillary angiomatosis and peliosis hepatis. Arch. Intern. Med. 152, 602±606. Stanier, R.Y., Adelberg, E.A., Ingraham, J.L., 1976. The prokaryotes: an introductory survey. In: Stanier, R.Y., Adelbert, E.A., Ingraham, J.L. (Eds.), The Microbial World. Prentice-Hall, Englewood Cliffs, 119 pp. Welch, D.F., Hensel, D.M., Pickett, D.A., San Joaquin, V.H., Robinson, A., Slater, L.N., 1993. Bacteremia due to Rochalimaea henselae in a child: practical identi®cation of isolates in the clinical laboratory. J. Clin. Microbiol. 31, 2381±2386.
Extracellular Bartonella henselae and artifactual intraerythrocytic pseudoinclusions in experimentally infected cats L. Guptilla,*, C-C. Wub, L. Glickmanb, J. Turekc, L. Slaterd, H. HogenEschb a
Department of Veterinary Clinical Sciences, Purdue University, W. Lafayette, IN 47907, USA b Department of Veterinary Pathobiology, Purdue University, W. Lafayette, IN 47907, USA c Department of Basic Medical Sciences, Purdue University, W. Lafayette, IN 47907, USA d Department of Medicine, Infectious Diseases Section, VA Medical Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA Received 10 September 1999; received in revised form 2 May 2000; accepted 2 May 2000
Abstract Blood, spleen and liver of speci®c pathogen-free (SPF) cats and SPF cats experimentally infected with Bartonella henselae were examined. Using immunohistochemical labeling, no intracellular B. henselae were observed in tissues of any cats, but extracellular B. henselae were detected in tissues of infected cats. Pseudoinclusions were detected in erythrocytes of all cats using electron microscopy. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Bartonella henselae; Cat; Erythrocyte; Immunohistochemistry; Artifact
1. Introduction Bartonella henselae causes cat scratch disease, bacillary angiomatosis and other clinical conditions in human beings (Relman et al., 1990; Slater et al., 1992; Koehler et al., 1994). High rates of persistent asymptomatic bacteremia occur in young cats, the apparent major reservoir of B. henselae (Koehler et al., 1994; Breitschwerdt and Kordick, 1995; Chomel et al., 1995). The site of localization of B. henselae in infected cats has not been identi®ed. There is one report of intraerythrocytic B. henselae in two naturally infected cats (Kordick and Breitschwerdt, 1995) based on electron microscopic *
Corresponding author. Tel.: 1-765-496-1107; fax: 1-765-494-9830. E-mail address: [email protected] (L. Guptill). 0378-1135/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 3 5 ( 0 0 ) 0 0 2 4 0 - 6
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observations. Published transmission electron photomicrographs (Kordick and Breitschwerdt, 1995) show intraerythrocytic objects with what appear to be single lipid bilayer membranes identical to the erythrocyte membrane. While it is possible that these intraerythrocytic objects are B. henselae with altered cell walls, electron micrographs of B. henselae within other cell types clearly show a trilaminate wall (Batterman et al., 1995) typical of Gram negative bacteria (Stanier et al., 1976). Bartonella bacilliformis is a related organism that is intraerythrocytic and causes hemolytic anemia in human beings (Cuadra and Takano, 1969; Kreier et al., 1992; Ihler, 1996). Within human erythrocytes, B. bacilliformis maintains a well-de®ned trilaminate wall and internal structures typical of Gram negative bacteria (Cuadra and Takano, 1969). The purpose of the study reported here was to investigate whether B. henselae is intraerythrocytic in experimentally infected cats. 2. Materials and methods Spleen and liver were collected from speci®c pathogen-free (SPF) cats infected by intravenous inoculation. The microbiological, immunological and pathological data for these cats was previously reported (Guptill et al., 1997). The B. henselae isolate used was provided by Dr. David F. Welch, University of Oklahoma. This isolate was originally isolated from the blood of a cat owned by a patient with cat scratch disease. It was characterized by standard culture techniques including gas liquid chromatography and immuno¯uorescence, which together are speci®c for identi®cation of Bartonella to the species level (Welch et al., 1993). In addition, blood was examined from four speci®c pathogen-free cats infected by intradermal inoculation using the same isolate after one passage onto chocolate agar after re-isolation from a cat. Fifty microliters of a suspension of 108 colony-forming units (CFU) B. henselae/ml were injected intradermally at each of two sites. Tissues from SPF cats inoculated intradermally with saline were used as controls. Blood from intradermally inoculated cats bacteremic for 6 and 8 weeks (103±104 CFU B. henselae/ml blood) and from a control cat was collected into sodium heparin. An aliquot was washed in phosphate buffered saline (PBS) then ®xed with 3% glutaraldehyde at 48C for 24±48 h; processing for electron microscopy is described below. Heparinized blood diluted 1:2 with PBS was layered onto Ficoll±Hypaque (Sigma, St. Louis, MO), speci®c gravity (SG) 1.077, and centrifuged. The mononuclear cell band was resuspended in Percoll (Sigma), SG 1.03, layered onto a discontinuous Percoll gradient (layers of 1.05 and 1.07 SG) and centrifuged (Bishop, 1995). Plasma was centrifuged at 13,600g at 48C for 15 min, and the pellet resuspended in PBS. Individual bands from the Percoll gradient were collected and resuspended in PBS. Cytocentrifugation slides were prepared from each white blood cell fraction, the platelet fraction, and the plasma pellet. Cytocentrifugation slides were labeled with polyclonal goat anti-B. henselae serum as previously described (Guptill et al., 1997). Spleen and liver specimens from two cats inoculated intravenously with B. henselae and from one uninfected cat (Guptill et al., 1997) were snap frozen at the time of euthanasia 4 or 32 weeks after inoculation. Frozen sections mounted on positively charged glass slides were ®xed with acetone at ÿ208C.
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The tissue sections were labeled with polyclonal goat anti-B. henselae serum as previously described (Guptill et al., 1997). Erythrocytes collected after Ficoll centrifugation were washed twice in PBS, resuspended in 3% glutaraldehyde and held at 48C for 24±48 h. Fixed erythrocytes or ®xed whole blood cells were washed twice in PBS, post-®xed in 1% osmium tetroxide (OsO4) for 1 h at 258C, and pelleted in 2% agarose. Pellets were diced into 1 mm pieces, dehydrated in ethanol solutions, propylene dioxide, and embedded in epoxy resin (Poly/ Bed 8121, Polysciences, Warrinton, PA). Thin sections were stained with uranyl acetate and lead citrate and examined on a JEOL 100 CX electron microscope. Erythrocyte pellets not post-®xed in OsO4 were dehydrated in acetone solutions and processed using indium trichloride to label bacterial DNA (Knight, 1977), embedded, and examined. B. henselae grown on chocolate agar were also prepared and stained for electron microscopy using these techniques. 3. Results and discussion Extracellular B. henselae, labeled immunohistochemically, were seen both individually and in clusters in plasma and platelet fractions of infected cats (Fig. 1). Extracellular B. henselae were also seen in frozen sections of spleen (Fig. 2) and liver, and on occasional blood smears of infected cats. No B. henselae were observed in mononuclear cell or granulocyte fractions or in any preparations from uninfected cats. Pseudoinclusions (Fig. 3) but not bacteria were observed in every grid of erythrocytes examined from infected and uninfected SPF cats using electron microscopy. In contrast,
Fig. 1. Extracellular Bartonella henselae (arrow) in the platelet fraction of a cat 8 weeks after infection, labeled using Fast Red as substrate for alkaline phosphatase. Magni®cation 1000. Bar7.5 mm.
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Fig. 2. Extracellular Bartonella henselae in the spleen of a cat 8 weeks after infection, labeled using ¯uorescein isothiocyanate. Magni®cation 1000. Bar7.5 mm.
no labeling with indium trichloride was seen within erythrocytes of infected or uninfected cats. Indium trichloride labeling of nucleic acids of B. henselae grown on chocolate agar was clearly positive (data not shown) and B. henselae grown on chocolate agar had typical Gram negative cell wall structure (Fig. 4). Pseudoinclusions result when ®xed erythrocytes are sectioned through an irregular border and viewed in two dimensions. These artifacts of preparation are pleomorphic and may resemble bacteria. Pseudoinclusions are often surrounded by a single lipid bilayer and contain debris that resembles bacterial DNA but is not labeled with indium trichloride. Intraerythrocytic objects were previously reported in 2.9% of 1220 erythrocytes and 6.2% of 645 erythrocytes examined in two bacteremic cats (Kordick and Breitschwerdt, 1995). The magnitude of bacteremia in these cats was not reported (Kordick and Breitschwerdt, 1995). In a normal adult cat with 6106 erythrocytes per microliter of blood the above values would equate to a bacteremia of 1.74108 and 3.72108 CFU/ml blood. This value is greater than published values for naturally and experimentally infected cats by 2±3 orders of magnitude. While current culture methods may not detect all bacteria present in blood samples, it seems unlikely that only 1 per 100 to 1 per 1000 of bacteria present are detected. Recovery of B. henselae from SPF cat blood inoculated in vitro with B. henselae was excellent (unpublished data). Colony counts from saline dilutions of bacteria used to inoculate blood and from inoculated blood of the same dilution were always within one order of magnitude. Pseudoinclusions in the current study were detected in 5.9% of 1947 erythrocytes of an uninfected SPF cat and in 5.2% of 2000 erythrocytes of a cat with a bacteremia of 2.4103 CFU/ml blood. Infected cats in this study, in several previous studies of experimentally infected cats (Chomel et al., 1996; Guptill et al., 1997; Abbott et al., 1997; Guptill et al., 1998), and in other
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Fig. 3. (a) Pseudoinclusions in erythrocytes of a cat infected with Bartonella henselae (magni®cation 47,850 bar0.2 mm), (b) Pseudoinclusions in erythrocytes of an uninfected speci®c pathogen free cat (magni®cation 72,500, bar0.1 mm).
experimental infections from our laboratory (unpublished data) were not anemic at any time. Reports of naturally infected cats state that infected cats are clinically healthy, but hematological data are not provided (Koehler et al., 1994; Kordick et al., 1995). Transient (2±3 weeks) anemia occurred in seven of eight experimentally infected cats in one study, although intraerythrocytic B. henselae were not speci®cally reported in those cats (Kordick and Breitschwerdt, 1997).
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Fig. 4. Electron photomicrograph of Bartonella henselae grown on chocolate agar. Gram-negative cell wall structures including outer membrane, peptidoglycan layer and the inner cell wall are visible. Magni®cation 66,000. Bar0.25 mm.
There was no evidence in the current study that the isolate of B. henselae used, either plate-grown after a small number of laboratory passages or after re-isolation from a cat following intradermal inoculation, infects erythrocytes. No erythrocytes of infected or uninfected cats were labeled with indium trichloride, whereas nucleic acids of B. henselae grown on chocolate agar were clearly labeled with indium trichloride. This indicates that B. henselae were not intraerythrocytic or they would have been detected using this procedure. The documented mechanism of human erythrocyte invasion by B. bacilliformis includes a combination of pili for attachment, elaboration of a protein, deformin, that alters the cytoskeleton of erythrocytes in the area of Bartonella entry, and spiral motion of a polar ¯agellum to propel the bacteria into erythrocytes (Benson et al., 1986; Mernaugh and Ihler, 1992; Mitchell and Minnick, 1995; Minnick et al., 1996). A locus from B. henselae that has sequence homology to the invasion locus of B. bacilliformis was isolated recently (Murakawa et al., 1996). When inserted into the genome of E. coli and expressed, this locus greatly enhanced the ability of E. coli to enter human erythrocytes. However, as has been demonstrated for toxin genes in Clostridium perfringens (Meer and Songer, 1997), the presence of a gene locus does not necessarily mean that the genes are transcribed or expressed. One study indicates that B. henselae invade feline erythrocytes in vitro (Mehock et al., 1998). Further work is necessary to determine whether some or most isolates of B. henselae carry and express a functional invasion locus, and whether this results in an intraerythrocytic location of B. henselae in infected cats.
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The results of the study reported here suggest that the isolate of B. henselae used does not infect erythrocytes in cats. The intraerythrocytic objects observed by us as reported here, and by others (Kordick and Breitschwerdt, 1995) are probably pseudoinclusions and not B. henselae. The presence of such structures alone does not suggest that B. henselae is intraerythrocytic in cats. In conclusion, there is currently insuf®cient evidence to conclude that B. henselae infect feline erythrocytes in vivo. Unless the presence of intraerythrocytic B. henselae can be con®rmed by immunological labeling techniques, the status of these bacteria as intraerythrocytic in cats should remain provisional. Acknowledgements This work was presented in part as poster #1 at the 77th Annual Meeting of the Conference of Research Workers in Animal Diseases, Chicago, IL, November 1996. We thank Deborah A.H. Van Horn for the electron photomicrographs. This work was supported in part by Intervet, Inc. and in part by an Andrews Fellowship, Purdue University (LG). References Abbott, R.C., Chomel, B.B., Kasten, R.W., Floyd-Hawkins, K.A., Kikuchi, Y., Koehler, J.E., Pedersen, N.C., 1997. Experimental and natural infection with Bartonella henselae in cats. Comp. Immunol. Microbiol. Infect. Dis. 20, 41±57. Batterman, H.J., Peek, J.A., Loutit, J.S., Falkow, S., Tompkins, L.S., 1995. Bartonella henselae and Bartonella quintana adherence to and entry into cultured human epithelial cells. Infect. Immun. 63, 4553±4556. Benson, L.A., Kar, S., McLaughlin, G., Ihler, G.M., 1986. Entry of Bartonella bacilliformis into erythrocytes. Infect. Immun. 54, 347±353. Bishop, S.A., 1995. Functional abnormalities in the immune system of FIV-infected cats. In: Willett, B.J., Jarrett, O. (Eds.), Feline Immunology and Immunode®ciency. Oxford Science Publications, Oxford, 150 pp. Breitschwerdt, E.B., Kordick, D.L., 1995. Bartonellosis. J. Am. Vet. Med. Assoc. 206, 1928±1931. Chomel, B.B., Abbott, R.C., Kasten, R.W., Floyd-Hawkins, K.A., Kass, P.H., Glaser, C.A., Pedersen, N.C., Koehler, J.E., 1995. Bartonella henselae prevalence in domestic cats in California: risk factors and association between bacteremia and antibody titers. J. Clin. Microbiol. 33, 2445±2450. Chomel, B.B., Kasten, R.W., Floyd-Hawkins, K.A., Chi, B., Yamamoto, K., Roberts-Wilson, J., Gur®eld, A.N., Abbott, R.C., Pedersen, N.C., Koehler, J., 1996. Experimental transmission of Bartonella henselae by the cat ¯ea. J. Clin. Microbiol. 34, 1952±1956. Cuadra, M., Takano, J., 1969. The relationship of Bartonella bacilliformis to the red blood cell as revealed by electron microscopy. Blood 33, 708±716. Guptill, L., Slater, L., Wu, C-C., Lin, T-L., Glickman, L.T., Welch, D.F., HogenEsch, H., 1997. Experimental infection of young speci®c pathogen-free cats with Bartonella henselae. J. Infect. Dis. 176, 206±216. Guptill, L., Slater, L.N., Wu, C-C., Lin, T-L., Glickman, L.T., Welch, D.F., Tobolski, J., HogenEsch, H., 1998. Evidence of reproductive failure and lack of perinatal transmission of Bartonella henselae in experimentally infected cats. Vet. Immunol. Immunopathol. 65, 177±189. Ihler, G.M., 1996. Bartonella bacilliformis: dangerous pathogen slowly emerging from deep background. FEMS Microbiol. Lett. 144, 1±11. Knight, D.P., 1977. Cytological staining methods in electron microscopy. In: Lewis, P.R., Knight, D.P. (Eds.), Practical Methods in Electron Microscopy. North-Holland, Amsterdam, 25 pp.
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