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Bartonella henselae, a bacterial pathogen first identified in 1990, is the cause of an expanding spectrum of clinical syndromes in human patients, including cat scratch disease, bacillary angiomatosis, bacillary peliosis hepatis, endocarditis, and relapsing bacteremia. Central nervous system (CNS) involvement can occur in conjunction with many of these disorders. Neurologic sequelae of cat scratch disease include encephalopathy, myelopathy, meningitis, cerebral arteritis, optic neuritis, and radiculopathy (2, 4, 21, 27, 32). Cerebral bacillary angiomatosis has been reported in human immunodeficiency virus (HIV)-infected patients (34), and aseptic meningitis has been described in association with relapsing B. henselae bacteremia (22). In addition, two recent studies suggest a role for B. henselae in the development of AIDS-related neurological disease: B. henselae-specific antibodies have been identified in the cerebrospinal fluid (CSF) of HIV patients presenting with neurological disease (30), and HIV-associated dementia has been associated with serum antibodies to B. henselae (31). Despite the growing number of reports implicating B. henselae in the development of neurological disease, the pathogenesis of CNS dysfunction remains unclear.

Epidemiological studies have established that cat exposure is a risk factor for B. henselae-associated disease in humans (35, 39) and that Bartonella infection in cats is widespread. Seroprevalence of B. henselae antibodies in cats has been reported to be as high as 54% (13). In regions of the world where fleas are endemic, the prevalence of bacteremia in cats is high and cats can remain infected for months to years (19). However, the clinical significance of B. henselae infection in cats remains controversial. Several studies have not identified disease manifestations in experimentally infected cats (1, 28). Others have documented clinical abnormalities, including fever, anorexia, lethargy, lymphadenopathy, muscle pain, and neurologic dysfunction, in cats following experimental infection (12, 18, 26). It has been suggested that these disparate results are due to differences in virulence among strains of B. henselae (26). The pathogenesis of B. henselae infection in cats and the determinants of organism virulence for this species require further elucidation.

In the present study, we utilized in vitro culture systems to examine B. henselae's ability to invade and persist in feline fetal brain cells. We investigated the relative susceptibilities of astrocytes and microglial cells to infection with B. henselae and assessed the ultrastructural effects of infection.

(This study was presented in part at the 18th Annual Forum of the American College of Veterinary Internal Medicine, 25 to 28 May 2000, Seattle, Wash.)

Fetuses with a gestational age of 40 to 60 days were obtained from random-source pregnant cats following routine ovariohysterectomy. Brains were aseptically removed in toto and placed in a petri dish containing Hanks' balanced salt solution (Mediatech, Herndon, Va.). The meninges were carefully removed, and the brains were washed three times in Hanks' balanced salt solution. Brain tissue was minced with scissors in Dulbecco's modified enriched medium (Mediatech) supplemented with 10% fetal bovine serum (Mediatech) and mechanically dissociated by passage through a 40-mesh tissue sieve (Bellco Glass, Vineland, N.J.). The dissociated tissue was centrifuged at 3,000 × g for 5 min and the supernatant was discarded. The pellet was resuspended in medium and seeded into 75-cm² polystyrene tissue culture flasks (Fisher Scientific, Pittsburgh, Pa.). Cells were incubated at 37°C in 5% CO₂. The cultures were left undisturbed for 5 days, at which time the medium was changed to remove cell debris. The medium was changed every 3 to 5 days thereafter.

The mixed cell culture reached confluence after a minimum of 14 to 21 days, with the astrocytes forming a monolayer that tightly adhered to the flask. The microglia were located more superficially and remained loosely adhered to the underlying astrocyte monolayer. Microglia were removed by mechanical shaking. Flasks were placed on an orbital shaker at 150 rpm for 1 h, after which the culture supernatant containing microglial cells was aspirated and centrifuged at 300 × g for 5 min. The pellet was resuspended in medium and seeded onto single-chamber slides (Nunc, Naperville, Ill.) and into 25-cm² flasks (Fisher Scientific) to yield cultures enriched (>95%) in microglia. Cells were confirmed to be microglia based on characteristic morphology, uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (Biomedical Technologies, Stoughton, Mass.), and positive staining with nonspecific esterase (Sigma Diagnostic, St. Louis, Mo.) (8).

The astrocyte monolayer remaining in the flasks was briefly incubated in 0.25% trypsin (Mediatech) and reseeded onto single-chamber slides and into 25-cm² flasks to yield cultures enriched (90 to 95%) in astrocytes. Cells were confirmed to be astrocytes by characteristic morphology and positive immunohistochemical staining with glial fibrillary acidic protein (Dako, Santa Barbara, Calif.) utilizing an ABC immunoperoxidase technique (Vector Laboratories, Burlingame, Calif.).

Microglia- and astrocyte-enriched cell cultures were infected 5 days after being established with isolate NCSU 93-F029 of B. henselae. The organism had originally been isolated from the blood of a cat that displayed signs of transient neurological dysfunction subsequent to its owner developing cat scratch disease. The B. henselae isolate was passaged three times in Vero cell culture. Quantification of CFU on trypticase soy agar with 5% rabbit blood (BBL, Becton Dickinson and Co., Cockeysville, Md.) yielded a count of 1.5 × 10¹⁰ CFU/ml. One-milliliter aliquots of the harvest were stored frozen at 70°C. Each aliquot was diluted in 10 ml of medium, and 1 ml of the suspension (1.5 × 10⁹ CFU) was added to coat the bottom of each vessel. The cultures were rocked at room temperature for 4 h, after which the inoculum was removed and replaced with fresh medium. Parallel-inoculated Vero cell cultures served as the positive control and uninfected brain cell cultures derived from the same fetal tissue served as the negative control for each experiment. Following inoculation, all cell cultures were incubated at 37°C with 5% CO₂.

Giménez stains. Chamber slides were harvested at 1, 4, 7, 11, and 14 days after inoculation and were stained by the Giménez method (11). Slides were rinsed in phosphate-buffered saline (PBS), fixed in methanol for 15 min, and air dried. Fixed slides were covered with a solution of 1% basic fuchsin and phenol (Sigma-Aldrich Chemical, St. Louis, Mo.) for 90 s and then rinsed in tap water for 30 s. The slides were counterstained with 1% malachite green oxalate (Sigma-Aldrich Chemical) for 9 s and washed in tap water for 10 s, and then the counterstaining sequence was repeated. The air-dried slides were observed with light microscopy. No organisms were observed in microglial cells at 1 or 4 days postinoculation. However, at 7 days postinoculation, organisms were observed within the cytoplasm of the microglial cell. The bacterial staining imparted a pink color to the cell cytoplasm, and aggregates of organisms could be visualized in a perinuclear location (Fig. 1A). Positive staining persisted in microglial cells at 11 and 14 days postinoculation. There was no evidence of cellular pathology; i.e., there were no changes in cellular morphology or evidence of cell loss in infected cultures. Bacterial organisms were never identified within astrocytes at any time throughout the course of the experiment (Fig. 1B).

View larger version (42K): [in this window] [in a new window]   FIG. 1.   (A) Microglial-cell-enriched cultures, 14 days after inoculation with B. henselae, display evidence of infection based on positive Giménez staining for bacteria. Bacterial aggregates are identified in the perinuclear region of the cells (arrows). (B) Astrocyte-enriched cultures, 14 days after inoculation with B. henselae, reveal no evidence of cellular infection when stained by the Giménez method. Bars, 25 µm.

Isolation of organisms from cell culture. At 14 and 28 days after inoculation, gentamicin sulfate (Mediatech) was added to the flasks at a concentration of 250 µg/ml and the cultures were allowed to incubate for 3 h at 37°C. This was done to selectively remove any extracellular bacteria from the culture system. Gentamicin is not taken up by mammalian cells, and therefore bacteria that have entered cells are presumably protected from its antimicrobial effects. Cultures were then washed with sterile PBS, and cells were scraped from the flasks and spun gently to form a pellet. The pellet was washed twice and resuspended in 1 ml of sterile PBS. The cell suspension was lysed by freezing at 70°C for 3 min followed by a thaw at room temperature. The freeze-thaw sequence was repeated and the lysate was streaked onto trypticase soy plates with 5% rabbit blood (BBL, Becton Dickinson and Co.). The agar plates were incubated at 37°C with 5% CO₂. Bacterial colonies were isolated 7 days after plating lysate from microglial cells harvested at 14 and 28 days postinoculation. Bacterial culture of the cell lysate derived from Bartonella-inoculated astrocyte cultures yielded no growth after 14 days of incubation.

Electron microscopy. Cells were harvested at 7 and 14 days after inoculation, and cultures were fixed with McDowell and Trump's solution (4% formaldehyde and 1% glutaraldehyde in phosphate buffer) and processed according to standard techniques for transmission electron microscopy (9). Examination of microglial cells in culture revealed intracellular bacillary organisms with morphologic characteristics similar to those previously reported for Bartonella species (3, 6, 17) (Fig. 2). The organisms were short, pleomorphic rods measuring 0.1 to 0.4 µm in width and 0.5 to 1.0 µm in length. They were often seen in clusters within the cell cytoplasm. A double membrane was identified surrounding the bacteria in some sections, and vacuoles were present within a small number of the organisms. No morphologic changes were apparent in infected cells compared to cells in uninoculated cultures.

View larger version (76K): [in this window] [in a new window]   FIG. 2.   Electron photomicrographs of bacterial organisms within microglial cells 14 days after inoculation. (A) Intracellular aggregate of bacteria with morphologic characteristics similar to those previously reported for Bartonella species; (B) a group of bacteria adjacent to a mitochondrion. Bars, 0.09 µm.