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Bartonella henselae causes cat scratch disease (CSD), bacillary angiomatosis, visceral peliosis, and endocarditis in humans (1, 5, 14, 22, 23). The domestic cat is a confirmed reservoir for B. henselae (15); >90% of CSD cases are associated with cat contact, typically with superficial trauma in the form of a bite or scratch. While B. henselae is not readily detectable by PCR in the saliva or nail clippings of infected cats (8), cat fleas (Ctenocephalides felis) can harbor B. henselae in the gut, shed the organism in their feces (12), and transmit infections to specific-pathogen-free kittens (7). These findings raise the possibility that B. henselae infections are acquired by scratching contaminated flea feces into the skin at sites of flea bites, in much the same way as Bartonella quintana, the agent of trench fever, is transmitted in the feces of the human body louse.

The clinical course of experimental B. henselae infection in cats includes persistent bacteremia accompanied by a specific antibody response (9, 17). Bacteremic cats are generally asymptomatic (25) but can experience histopathological lesions in multiple organs (10). The prevalence of B. henselae bacteremia or Bartonella-specific antibody in cat populations is striking (4, 6, 11, 13, 18, 24) and may represent a significant public health threat, particularly for individuals with frequent cat contact. For example, in a study of 205 feral, clinic, pet, and impounded cats in northern California, 5% were associated with human cases of CSD or bacillary angiomatosis. However, 40% were positive for blood culture of B. henselae, with prevalences ranging from 4 to 47% in the pet group, 53% in impounded cats, and 70% in the feral cat group, and antibodies to B. henselae were detected in 81% of the cats tested (6). A similar pattern was observed in a study of Australian cat populations, in which 22 of 77 cats tested (35%) were culture positive for B. henselae (4); 40% of these were feral and 16% were domestic cats.

Bartonella spp. are believed to require hemin for growth (20), and they exhibit an affinity for erythrocytes (RBC) that is best characterized with Bartonella bacilliformis, the agent of Carrión's disease and verruga peruana (19). B. bacilliformis is transmitted to humans by nocturnal sand flies in parts of South America, and upon reaching the blood it invades and ultimately lyses most circulating RBC, leading to severe anemia. B. henselae likewise exhibits an affinity for RBC, although the extent of RBC invasion varies with the host. In contrast to the bacteremia that is common in cats, blood cultures are rarely positive for humans, in which the organism appears to localize primarily in regional lymph nodes (9, 10, 17, 18, 25). Kordick and Breitschwerdt (16) described the intraerythrocytic location of B. henselae during persistent bacteremia in cats. Electron micrographs revealed bacteria within 2.9 to 6.2% of the RBC examined, but no bacteria were found to be free of RBC or associated with the RBC surface. The significance of RBC invasion to long-term infections in cats, acquisition by cat fleas, and transmission to humans is unknown. Detailed investigation of the RBC invasion process, including the potential isolation of noninvading mutants, will require a suitable model for RBC invasion in vitro; this was the focus of the present study.

B. henselae invasion of feline RBC was analyzed in vitro as described for B. bacilliformis invasion of human RBC (3), with modifications. B. henselae Houston-1 was supplied by R. Regnery (Centers for Disease Control and Prevention, Atlanta, Ga.) and tested by PCR (21) and indirect fluorescent antibody binding (9). Bartonella cultures were grown on plates of heart infusion agar supplemented with 5% defibrinated rabbit blood (HIBA) in 4% CO₂ for 6 to 7 days at 35°C. Sterile feline blood, preserved and anticoagulated with EDTA, was obtained from Harlan Bioproducts (Indianapolis, Ind.). One-milliliter aliquots were each supplemented with 0.14 ml of acid-citrate-dextrose anticoagulant solution (Sanofi Animal Health, Inc., Overland Park, Kans.) to help maintain RBC integrity. Sterility was confirmed by inoculating HIBA plates and incubating them for up to 7 days at 37°C.

Feline RBC in EDTA and acid-citrate-dextrose were washed three times in Hanks balanced salt solution without bicarbonate and resuspended at approximately 1.7 × 10⁸ to 2 × 10⁸/ml, based on direct hemocytometer counts. For each invasion assay, one HIBA plate containing a lawn of B. henselae cells (6 to 7 days of growth) was overlaid with 1 ml of Bartonella liquid growth medium (RPMI 1640 without bicarbonate, supplemented with 1% L-glutamine, HEPES buffer, sodium pyruvate, nonessential-amino-acid solution, and 2 mg of hemin/ml [prepared in 0.01 N NaOH]; Sigma Chemical Co., St. Louis, Mo.) (30). Bacterial cells were suspended in the liquid growth medium, 2- or 20-µl aliquots were added to sterile 1.5-ml microcentrifuge tubes, and the total volume in each tube was adjusted to 100 µl with Bartonella liquid growth medium. A 100-µl aliquot from each HIBA plate was also serially diluted and plated to determine the viable-cell count (typically approximately 6.0 × 10⁹ B. henselae CFU/ml). Feline RBC in Hanks balanced salt solution were added to tubes containing the B. henselae suspension and incubated at 35°C for various lengths of time, ranging from 1 to 24 h. In some experiments, the samples were centrifuged at 900 × g for 4 min to increase bacterial association with RBC (3), but this step did not appear to affect the results. Feline RBC were separated from unassociated bacteria by washing with phosphate-buffered saline (PBS), since Percoll gradient centrifugation (3) resulted in substantial lysis of these RBC.

Evaluation of RBC invasion by confocal microscopy. Infected RBC were analyzed by laser scanning confocal microscopy to discriminate between intracellular and epicellular B. henselae cells. RBC invasion samples prepared as described above were diluted 1:10 or 1:100 in PBS, and 10-µl volumes were added to individual wells of an eight-well glass slide. The slides were air dried, heat fixed, stained for 20 min with a 1:100 dilution of acridine orange (0.5 mg/ml), washed in distilled water, and again air dried. A 1:100 dilution of fluorescein isothiocyanate (1 mg/ml) was used as a secondary stain for 20 min, after which the slides were rinsed and air dried. Coverslips were sealed with Citifluor mounting fluid, and slides were viewed at the University of Georgia Center for Ultrastructural Research, using a Bio-Rad (Hercules, Calif.) MRC 600 laser scanning confocal microscope with an excitation wavelength of 488 nm and an emission wavelength of 617 nm. Z scans were performed to section RBC images digitally in depth in increments of 0.5 µm. Feline RBC are rounded (as opposed to biconcave) and, with a diameter of 3.9 to 5.5 µm, can be completely traversed in 8 to 11 digital sections taken in 0.5-µm increments.

View larger version (99K): [in this window] [in a new window]   FIG. 1.   Digital sections of feline RBC after infection with B. henselae, as viewed by laser scanning confocal microscopy. Shown are sections 1 (a), 5 (b), 8 (c), and 11 (d); sections were taken in 0.5-µm increments from top to bottom. Note that the fluorescence intensity of the B. henselae cell (arrowhead) was low in panels a and b and increased in panel c. The B. henselae cell can be seen clearly within the RBC membrane and was probably intracellular. In panel d, the fluorescence intensity of the Bartonella cell once again diminished. Bar, 5 µm; magnification, ×1,000.

View larger version (93K): [in this window] [in a new window]   FIG. 2.   Digital sections of feline RBC after infection with B. henselae, as viewed by laser scanning confocal microscopy. Sections were taken as described for Fig. 1; shown are sections 2 (a), 6 (b), 8 (c), and 10 (d). The arrowhead indicates a likely intracellular B. henselae cell, while the arrows indicate probable epicellular B. henselae cells. Note that the fluorescence intensity of the organism indicated by the arrowhead in panel a increased in panel b and then faded in panels c and d. Bar, 5 µm; magnification, ×1,000.

Assessment of RBC invasion by gentamicin selection. Intracellular B. henselae cells were quantitated on the basis of protection from gentamicin. Invasion mixtures prepared and incubated from 1 to 24 h as described above were treated with gentamicin sulfate (250 µg/ml) for 3 h at 35°C to kill extracellular B. henselae cells. RBC were centrifuged at 900 × g for 4 min, washed three times in PBS to remove residual antibiotic, and serially diluted in PBS. Aliquots (0.1 ml) were each combined with 0.3 ml of sterile water to lyse the RBC, and 100-µl volumes of each dilution were individually inoculated onto HIBA plates that were subsequently incubated at 35°C in 4% CO₂ for 6 to 7 days. Bartonella viable-cell counts consistently reflected substantial RBC invasion (Table 1). No CFU were recovered when RBC infection was carried out at 4°C, reflecting a temperature-dependent process. Invasion was also dose dependent, but variability in CFU recovery between experiments was high. CFU counts after gentamicin selection (reported as percentages of the initial inoculum) ranged from 2 to 17%, averaging 7% for the 24-h incubation. In some experiments, total CFU before and after the 24-h incubation, prior to the gentamicin treatment, were compared. Little change in viable numbers was observed during this incubation, consistent with the slow growth of these bacteria in culture (data not shown). No CFU were recovered when RBC were omitted, and no spontaneous gentamicin resistance was observed at the gentamicin concentration used.

Characterization of B. henselae-feline RBC interaction. To analyze biochemically the interaction between B. henselae and feline RBC, we assessed the effects of protease or neuraminidase pretreatment of the RBC, or protease pretreatment of the bacteria, on RBC invasion. Treatments were carried out as described previously for B. bacilliformis (3), with minor changes. Briefly, trypsin (15, 25, or 50 µg) was added to 1.5-ml microcentrifuge tubes containing feline RBC or B. henselae cells in Bartonella liquid growth medium, and the tubes were incubated at 37°C for 10 min. Antitrypsin was added in a twofold molar excess, and the tubes were placed on ice. Trypsinized RBC suspensions were evaluated visually for hemolysis, while trypsinized B. henselae cells were diluted and inoculated on HIBA plates to confirm bacterial viability. In some experiments, both the bacteria and the RBC were treated with trypsin (50 µg). Alternatively, RBC were pretreated with type XIV bacterial protease from Streptomyces griseus (pronase, 1 mg/ml in PBS; Sigma Chemical Co.) or neuraminidase (C. perfringens neuraminidase type VIII, 1 U/ml; Sigma Chemical Co.) for 30 min at 37°C. RBC were washed twice with PBS after each treatment and then used in invasion assays as described above. Trypsin pretreatment of feline RBC, B. henselae cells, or both had no detectable effect on invasion, but a 23 to 50% increase in invasion was observed with pronase-pretreated RBC (data not shown). Pretreatment of feline RBC with neuraminidase had no effect on invasion, indicating that sialic acid moieties on the RBC surface are probably not required. Significantly, the same pattern of resistance to enzymatic pretreatment of bacteria or RBC was reported for B. bacilliformis (3, 19, 29), suggesting that similar surface structures may be functional in the interplay between bacteria and RBC in each case.