This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rolain, J. M.
Right arrow Articles by Raoult, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rolain, J. M.
Right arrow Articles by Raoult, D.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, August 2001, p. 2978-2980, Vol. 39, No. 8
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.8.2978-2980.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Immunofluorescent Detection of Intraerythrocytic Bartonella henselae in Naturally Infected Cats

J. M. Rolain,1 B. La Scola,1 Z. Liang,1 B. Davoust,2 and D. Raoult1,*

Unité des Rickettsies, CNRS UMR 6020, Faculté de Médecine, 13385 Marseille Cedex 05,1 and Conseiller Vétérinaire Régional Interarmées, 69998 Lyon Armées,2 France

Received 21 March 2001/Returned for modification 18 April 2001/Accepted 8 May 2001


    ABSTRACT
Top
Abstract
Text
References

To determine the presence of Bartonella henselae bacteremia in six cats, we compared isolation using blood culture with direct immunofluorescence on blood smears. Three cats that were positive by blood culture were also positive by direct immunofluorescence, and laser confocal microscopy confirmed the intraerythrocytic location of B. henselae.


    TEXT
Top
Abstract
Text
References

Bartonella henselae is an agent of cat scratch disease and can be responsible for other human diseases such as bacillary angiomatosis, peliosis hepatitis, and endocarditis (12). The natural host of B. henselae is the domestic cat, and its vector is the cat flea. The organism can be isolated from the blood of 4 to 70% of apparently healthy cats depending on the cat population concerned and its geographic location (2, 5, 7). In infected cats, the target cells of B. henselae seem to be the erythrocytes (8, 14), although this has been refuted in a recent study (4). The detection of B. henselae in feline erythrocytes using immunological techniques has not been described, and the sensitivity of such methods is thus unknown. In our study we compared the use of blood cultures and a monoclonal antibody (MAb) against B. henselae in direct immunofluorescence tests to detect the organism in cat blood. Also, we used laser confocal microscopy to determine the position of B. henselae in blood smears that were positive by immunofluorescence.

The study was performed in June 2000 during an exercise to eradicate stray cats from a French military base. Six cats were captured using oral Dexeutanol (Instituto DEX, Seville, Spain) in fresh meat according to the manufacturer's instructions. Once sedated, the cats were anesthetized with Imalgène (Merial, Lyon, France), and 3 ml of blood was collected aseptically using intracardiac puncture. Cats were euthanized by intracardiac inoculation of Dolethal (Vétoquinol SA, Lure, France) according to the manufacturer's directions. Thin blood smears were made from the fresh blood and stored at room temperature until the direct immunofluorescence assays were carried out. The remaining fresh blood was stored at -20°C until it was thawed at room temperature and 1-ml aliquots were inoculated onto Columbia 5% sheep blood agar plates (BioMerieux, Marcy l'Etoile, France). The plates were placed in polyethylene bags to prevent desiccation and incubated at 37°C under 5% CO2 (Genbag CO2 system; BioMerieux). For 3 months the plates were examined weekly for evidence of bacterial growth. When Bartonella-like colonies were observed, they were characterized using the sequences of their 16S rRNA genes and the BLAST 2.0 program (National Center for Biotechnology Information) as described previously (6). The blood smears were examined for evidence of B. henselae infection using two specific MAbs prepared in our laboratory: the first (B3D4) was Bartonella genus specific (11) and the second (H2A10) (10) was B. henselae specific using standard immunofluorescence techniques (9). MAb H2A10 was of the immunoglobulin G2a (IgG2a) subclass and reacted with a 43-kDa epitope present only in B. henselae strains. This MAb does not cross-react with other Bartonella species, including B. quintana, B. elizabethae, B. clarridgeiae, B. bacilliformis, B. taylori, B. doshiae, and B. vinsonii, or with any of the bacteria already tested for MAb B3D4 (11).

Positive smears were examined under a laser scanning confocal microscope with an excitation wavelength of 488 nm and an emission wavelength of 617 nm to determine the location of the organisms in the erythrocytes. Sections of the erythrocytes were made in increments of 0.5 µm as previously described (13).

Bartonella organisms at 105 CFU/ml were isolated from the blood of three of six (50%) cats, and their 16S rRNA gene sequences were identical to that of B. henselae URLLY8 strain Marseille (3). The blood smears made from these cats had positive immunofluorescence when assayed either with MAb B3D4, specific for the Bartonella genus, or with MAb H2A10, specific to B. henselae strains (Fig. 1). Three to 8% (mean, 5%) of the erythrocytes were found to be infected, with most of these cells containing only one bacterium (average, 1.08 bacteria/erythrocyte). These results are consistent with previous reports that the prevalence of bacteremia in cats is 22 to 59% (5) and that the percentage of infected red blood cells varies from 1 to 20% (1, 2, 7). The average red cell count in the cat is 5 × 109/ml, and with the 5% level of infection we detected, there were around 2.5 × 108 infected erythrocytes per milliliter of blood. In our blood cultures, however, we detected only 105 CFU/ml, and we conclude that a large number of bacteria were killed during the processing of the blood and/or its storage at -20°C and thus that 1 in 2,500 bacteria was cultivable.


View larger version (58K):
[in this window]
[in a new window]
  		FIG. 1.   Digital sections of feline red blood cells infected with B. henselae as viewed by laser scanning confocal microscopy. Sections were taken in 0.5-µm increments from top to bottom. B. henselae organisms were revealed with a specific MAb. Arrows indicate intracellular B. henselae.

By laser scanning confocal microscopy B. henselae was seen within the erythrocytes, particularly near the cell membrane but also in the center of the cell (Fig. 1). These findings using immunological techniques confirm the intraerythrocytic location of B. henselae in naturally infected cats. Also, our findings show that direct fluorescence with a specific MAb is a sensitive, rapid, and simple technique which may replace blood cultures for detecting Bartonella infections in cats.


    ACKNOWLEDGMENTS

We thank Patrick Kelly for review of the manuscript and Robert Pistoresi for technical assistance.


    FOOTNOTES

* Corresponding author. Mailing address: Unité des Rickettsies, CNRS UMR 6020, Faculté de Médecine, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France. Phone: 33 91 38 55 17. Fax: 33 91 83 03 90. E-mail: Didier.Raoult{at}medecine.univ-mrs.fr.


    REFERENCES
Top
Abstract
Text
References

1. Bergmans, A. M. C., J. F. P. Schellekens, J. D. A. Van Embden, and L. M. Schouls. 1996. Predominance of two Bartonella henselae variants among cat scratch disease patients in the Netherlands. J. Clin. Microbiol. 34:254-260[Abstract].
2. Chomel, B. B., R. C. Abbott, R. W. Kasten, K. A. Floyd-Hawkins, P. H. Kass, C. A. Glaser, N. C. Pedersen, and J. E. Koehler. 1995. Bartonella henselae prevalence in domestic cats in California: risk factors and association between bacteremia and antibody titers. J. Clin. Microbiol. 33:2445-2450[Abstract].
3. Drancourt, M., R. J. Birtles, G. Chaumentin, F. Vandenesch, J. Etienne, and D. Raoult. 1996. New serotype of Bartonella henselae in endocarditis and cat-scratch disease. Lancet 347:441-443[CrossRef][Medline].
4. Guptill, L., C. C. Wu, L. Glickman, J. Turek, L. Slater, and H. HogenEsch. 2000. Extracellular Bartonella henselae and artifactual intraerythrocytic pseudoinclusions in experimentally infected cats. Vet. Microbiol. 76:283-290[CrossRef][Medline].
5. Heller, R., M. Artois, V. Xemar, D. De Briel, H. Gehin, B. Jaulhac, H. Monteil, and Y. Piemont. 1997. Prevalence of Bartonella henselae and Bartonella clarridgeiae in stray cats. J. Clin. Microbiol. 35:1327-1331[Abstract].
6. Joblet, C., V. Roux, M. Drancourt, J. Gouvernet, and D. Raoult. 1995. Identification of Bartonella (Rochalimaea) species among fastidious gram-negative bacteria on the basis of the partial sequence of the citrate synthase gene. J. Clin. Microbiol. 33:1879-1883[Abstract].
7. Koehler, J. E., C. A. Glaser, and J. W. Tappero. 1994. Rochalimaea henselae infection: a new zoonosis with the domestic cat as a reservoir. JAMA 271:531-535[Abstract/Free Full Text].
8. Kordick, D. L., and E. B. Breitschwerdt. 1995. Intraerythrocytic presence of Bartonella henselae. J. Clin. Microbiol. 33:1655-1656[Abstract].
9. La Scola, B., and D. Raoult. 1999. Culture of Bartonella quintana and Bartonella henselae from human samples: a 5-year experience (1993 to 1998). J. Clin. Microbiol. 37:1899-1905[Abstract/Free Full Text].
10. Liang, Z. 2001. Rôle des anticorps monoclonaux dans la classification et l'identification des bactéries intracellularies. Ph. D. thesis. Université de la Méditerranée, Marseille, France.
11. Liang, Z., B. La Scola, and D. Raoult. Production of Bartonella genus-specific monoclonal antibodies. Clin. Diagn. Lab. Immun., in press.
12. Maurin, M., R. J. Birtles, and D. Raoult. 1997. Current knowledge of Bartonella species. Eur. J. Clin. Microbiol. Infect. Dis. 16:487-506[CrossRef][Medline].
13. Meconi, S., V. Jacomo, P. Boquet, D. Raoult, J. L. Mege, and C. Capo. 1998. Coxiella burnetii induces reorganization of the actin cytoskeleton in human monocytes. Infect. Immun. 66:5527-5533[Abstract/Free Full Text].
14. Mehock, J. R., C. E. Greene, F. C. Gherardini, T. W. Hahn, and D. C. Krause. 1998. Bartonella henselae invasion of feline erythrocytes in vitro. Infect. Immun. 66:3462-3466[Abstract/Free Full Text].

Journal of Clinical Microbiology, August 2001, p. 2978-2980, Vol. 39, No. 8
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.8.2978-2980.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Kunz, S., Oberle, K., Sander, A., Bogdan, C., Schleicher, U. (2008). Lymphadenopathy in a Novel Mouse Model of Bartonella-Induced Cat Scratch Disease Results from Lymphocyte Immigration and Proliferation and Is Regulated by Interferon-{alpha}/{beta}. Am. J. Pathol. 172: 1005-1018 [Abstract] [Full Text]  
  • Koehler, J. E., Duncan, L. M. (2005). Case records of the Massachusetts General Hospital. Case 30-2005. A 56-year-old man with Fever and axillary lymphadenopathy.. NEJM 353: 1387-1394 [Full Text]  
  • Mandle, T., Einsele, H., Schaller, M., Neumann, D., Vogel, W., Autenrieth, I. B., Kempf, V. A. J. (2005). Infection of human CD34+ progenitor cells with Bartonella henselae results in intraerythrocytic presence of B henselae. Blood 106: 1215-1222 [Abstract] [Full Text]  
  • Rolain, J. M., Brouqui, P., Koehler, J. E., Maguina, C., Dolan, M. J., Raoult, D. (2004). Recommendations for Treatment of Human Infections Caused by Bartonella Species. Antimicrob. Agents Chemother. 48: 1921-1933 [Full Text]  
  • Woestyn, S., Olive, N., Bigaignon, G., Avesani, V., Delmee, M. (2004). Study of Genotypes and virB4 Secretion Gene of Bartonella henselae Strains from Patients with Clinically Defined Cat Scratch Disease. J. Clin. Microbiol. 42: 1420-1427 [Abstract] [Full Text]  
  • Rolain, J.-M., Locatelli, C., Chabanne, L., Davoust, B., Raoult, D. (2004). Prevalence of Bartonella clarridgeiae and Bartonella henselae in Domestic Cats from France and Detection of the Organisms in Erythrocytes by Immunofluorescence. CVI 11: 423-425 [Abstract] [Full Text]  
  • Rolain, J.-M., Fournier, P. E., Raoult, D., Bonerandi, J. J. (2003). First Isolation and Detection by Immunofluorescence Assay of Bartonella koehlerae in Erythrocytes from a French Cat. J. Clin. Microbiol. 41: 4001-4002 [Full Text]  
  • Rolain, J. M., Gouriet, F., Enea, M., Aboud, M., Raoult, D. (2003). Detection by Immunofluorescence Assay of Bartonella henselae in Lymph Nodes from Patients with Cat Scratch Disease. CVI 10: 686-691 [Abstract] [Full Text]  
  • Zimmermann, R., Kempf, V. A. J., Schiltz, E., Oberle, K., Sander, A. (2003). Hemin Binding, Functional Expression, and Complementation Analysis of Pap 31 from Bartonella henselae. J. Bacteriol. 185: 1739-1744 [Abstract] [Full Text]  
  • Rolain, J.-M., Maurin, M., Mallet, M.-N., Parzy, D., Raoult, D. (2003). Culture and Antibiotic Susceptibility of Bartonella quintana in Human Erythrocytes. Antimicrob. Agents Chemother. 47: 614-619 [Abstract] [Full Text]  
  • GREUB, G., RAOULT, D. (2002). Bartonella: new explanations for old diseases. J Med Microbiol 51: 915-923 [Full Text]  
  • Jacomo, V., Kelly, P. J., Raoult, D. (2002). Natural History of Bartonella Infections (an Exception to Koch's Postulate). CVI 9: 8-18 [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rolain, J. M.
Right arrow Articles by Raoult, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rolain, J. M.
Right arrow Articles by Raoult, D.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»