JOURNAL OF CLINICAL MICROBIOLOGY, June 1995, p. 1655–1656 Vol. 33, No. 6 0095-1137/95/$04.00ϩ0 Copyright ᭧ 1995, American Society for Microbiology

Intraerythrocytic Presence of henselae

DORSEY L. KORDICK AND EDWARD B. BREITSCHWERDT* Department of Companion Animal and Special Species Medicine, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606

Received 21 November 1994/Returned for modification 3 January 1995/Accepted 1 March 1995

Recent reports in the medical literature emphasize the risk of zoonotic disease and the high degree of prevalence of asymptomatic feline infection with Bartonella (Rochalimaea) henselae. While investigating Bar- tonella bacteremia in cats, we used transmission electron microscopy to demonstrate B. henselae in the erythrocytes of persistently bacteremic cats.

Bartonella species (emended to include those organisms pre- 1,220 erythrocytes from cat A identified 35 (2.9%) infected viously classified as Rochalimaea species) have been reported cells, while 40 of 645 (6.2%) erythrocytes from cat B contained to cause cat scratch disease, , Carrion’s intracellular B. henselae. No epicellular or extracellular organ- disease, endocarditis, , and fever of unknown or- isms were observed in samples derived from either cat. Al- igin (3). Bartonella henselae appears to be the predominant though they were potentially altered during processing, failure cause of cat scratch disease in people. to identify any epicellular organisms from the electron micro- (the agent of Carrion’s disease) has been reported to reside graphs of either cat supports an intraerythrocytic presence for within erythrocytes (1). However, other species of Bartonella B. henselae. The sizes and morphologies of these intraerythro- (B. quintana, B. vinsonii) and the more distantly related feline cytic were consistent with our earlier transmission blood parasite, Hemobartonella felis, are described as maintain- electron microscopic observations of Bartonella organisms cul- ing an epicellular location adjacent to the erythrocyte (13). tivated on blood agar and were not dissimilar to photomicro- Bartonella organisms are difficult to isolate by conventional graphs of B. bacilliformis published elsewhere (2, 4). blood culture. However, by using the lysis centrifugation The intraerythrocytic presence of B. henselae is not surpris- method, the bacteria can be isolated from whole blood on ing. The mechanism of entry is unknown. In Fig. 2, it appears artificial medium supplemented with blood or hemin to satisfy that the erythrocyte membrane is engulfing the bacteria. Be- the hemin requirement of Bartonella species (11). The culture cause erythrocytes are not normally capable of endocytosis, we technique involves drawing blood into a collection tube con- suspect a bacterium-induced or forced endocytosis. Salmonella taining a lysing solution of sodium dodecyl sulfate–saponin– typhimurium enters cells assisted by the rotation of its flagella polypropylene glycol (Isolator; Wampole Laboratories, Cran- (7). By using specialized staining techniques, flagella are bury, N.J.). Following centrifugation and removal of the clearly present on B. bacilliformis. The flagella probably per- supernatant, the lysed erythrocyte concentrate is applied to form an important role in cellular entry. Flagella have not yet blood agar plates and incubated in a CO2-enriched environ- been observed on B. henselae, however. Invasion studies with ment at 37ЊC. Cultivation of B. henselae is enhanced by cellular B. bacilliformis have demonstrated decreased erythrocyte pen- lysis or the addition of hemin for growth. These observations etration following bacterial exposure to antiflagellin antibody suggest an intimate relationship with or residence within the (12). Figure 3 illustrates the existence of a pore between the erythrocyte. bacterium and the extracellular fluid space. This communica- Blood was aseptically obtained by jugular venipuncture from tion with the surrounding plasma, identified in electron micro- two cats with suspected B. henselae bacteremia. Persistent bar- graphs from both cats, may represent a nutrient channel or tonella bacteremia was subsequently documented by obtaining perhaps a route of entry or egress for the bacteria into or from positive blood cultures at 1- to 3-month intervals for approxi- the cell. mately 1 year. At the time of the initial isolation, the species of B. bacilliformis also reportedly secretes a protein termed the organisms were determined by 16S rRNA gene sequencing deformation factor which deforms the erythrocyte membrane and restriction endonuclease digestion of chromosomal DNA. and presumably aids in the subsequent binding and entry of the Identical morphologic and growth characteristics provided bacteria (10). To date, deformation factor has not been re- phenotypic identifications of subsequent isolates from each ported in association with B. henselae or any Bartonella species cat. When the original lysis centrifugation culture from cat A other than B. bacilliformis. and a second culture from cat B were performed, one drop of Traditionally, cat scratch disease has been considered a self- whole blood from each cat was also immediately placed into limiting illness, and patients are infrequently treated with an- McDowell and Trump’s 4F:1G fixative and was processed for tibiotics. B. henselae is susceptible to several antimicrobial transmission electron microscopy (5). agents in vitro; however, in vivo infection in humans has been Electron micrographs revealed organisms within erythro- quite refractory to antimicrobial treatment (8, 9). The inacces- cytes (Fig. 1). Occasional erythrocytes contained multiple or- sibility of many routinely prescribed antibiotics to the in- ganisms that were transected as well as cut longitudinally traerythrocytic bacteria, as observed in the electron micro- within the same cell. Electron microscopic examination of graphs presented here, provides a potential explanation for poor antimicrobial efficacy. Although the clinical course of cat scratch disease is typically * Corresponding author. Phone: (919) 829-4234. Fax: (919) 829- mild, disease manifestations in immunocompromised and oc- 4336. casional immunocompetent individuals can be severe (6).

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FIG. 1. Electron photomicrograph of two B. henselae organisms inside a FIG. 3. Electron photomicrograph of intraerythrocytic B. henselae illustrat- feline erythrocyte. Staining was with methanol uranyl acetate and then lead ing the existence of a pore between the bacterium and the extracellular fluid citrate. Magnification, ϫ16,900. space (arrow). Staining was with methanol uranyl acetate and then lead citrate. Magnification, ϫ38,000.

We acknowledge the Electron Microscopy Laboratory at North Therefore, interest in Bartonella vaccine development is wide- Carolina State University College of Veterinary Medicine for the prep- spread. Knowledge of the habitat of the bacteria and its inter- aration of the electron micrographs. action with the host’s immune system is necessary to achieve REFERENCES success in this endeavor and to attempt to answer other ques- 1. Benson, L. A., S. Kar, G. McLaughlin, and G. M. Ihler. 1986. Entry of tions that currently elude us regarding members of the genus Bartonella bacilliformis into erythrocytes. Infect. Immun. 54:347–353. Bartonella. The demonstration of B. henselae in erythrocytes 2. Breitschwerdt, E. B., D. L. Kordick, D. E. Malarkey, B. Keene, T. L. Had- will, it is hoped, further these efforts. field, and K. Wilson. 1995. Endocarditis in a dog due to infection with a novel Bartonella subspecies. J. Clin. Microbiol. 33:154–160. 3. Brenner, D. J., S. P. O’Connor, H. H. Winkler, and A. G. Steigerwalt. 1993. Proposals to unify the genera Bartonella and Rochalimaea, with descriptions of comb. nov., Bartonella vinsonii comb. nov., Bartonella henselae comb. nov., and comb. nov., and to remove the family Bartonellaceae from the order . Int. J. Syst. Bacteriol. 43:777–786. 4. Cuadra, M., and J. Takano. 1969. The relationship of Bartonella bacilliformis to the red blood cell as revealed by electron microscopy. Blood 33:708–716. 5. Dykstra, M. J. 1985. A manual of applied techniques for biological electron microscopy. Plenum Publishing Corp., New York. 6. Groves, M. G., and K. S. Harrington. 1994. Rochalimaea henselae infections: newly recognized zoonoses transmitted by domestic cats. J. Am. Vet. Med. Assoc. 204:267–271. 7. Jones, B. D., C. A. Lee, and S. Falkow. 1992. Invasion by Salmonella typhi- murium is affected by the direction of flagellar rotation. Infect. Immun. 60:2475–2480. 8. Margileth, A. M. 1992. Antibiotic therapy for cat-scratch disease: clinical study of therapeutic outcome in 268 patients and a review of the literature. Pediatr. Infect. Dis. J. 11:474–478. 9. Maurin, M., and D. Raoult. 1993. Antimicrobial susceptibility of Rochali- maea quintana, Rochalimaea vinsonii, and the newly recognized Rochalimaea henselae. J. Antimicrob. Chemother. 32:587–594. 10. Mernaugh, G., and G. M. Ihler. 1992. Deformation factor: an extracellular protein synthesized by Bartonella bacilliformis that deforms erythrocyte membranes. Infect. Immun. 60:937–943. 11. Myers, W. F., L. D. Cutler, and C. L. Wisseman, Jr. 1969. Role of erythro- cytes and serum in the nutrition of Rickettsia quintana. J. Bacteriol. 97:663– 666. 12. Scherer, D. C., I. DeBuron-Connors, and M. F. Minnick. 1993. Character- ization of Bartonella bacilliformis flagella and effect of antiflagellin antibodies on invasion of human erythrocytes. Infect. Immun. 61:4962–4971. FIG. 2. Intraerythrocytic B. henselae illustrating possible engulfment by 13. Weiss, E., and J. W. Moulder. 1984. Order I. Rickettsiales, p. 687–719. In erythrocyte membrane (arrow). Staining was with methanol uranyl acetate and N. R. Krieg (ed.), Bergey’s manual of systematic bacteriology. The Williams then lead citrate. Magnification, ϫ21,000. & Wilkins Co., Baltimore.