Prolonged Bartonella Bacteremia in Cats Associated with Cat-Scratch ...

JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1995, p. 3245–3251 0095-1137/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 33, No. 12

Prolonged Bartonella Bacteremia in Cats Associated with Cat-Scratch Disease Patients DORSEY L. KORDICK,1 KENNETH H. WILSON,2,3 DANIEL J. SEXTON,2 TED L. HADFIELD,4 HERMAN A. BERKHOFF,5 AND EDWARD B. BREITSCHWERDT1* Departments of Companion Animal and Special Species Medicine1 and Microbiology, Pathology, and Parasitology,5 College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606; Division of Infectious Diseases, Duke University Medical Center,2 and the Veterans Affairs Medical Center,3 Durham, North Carolina 27710; and the Department of Infectious and Parasitic Diseases Pathology, Armed Forces Institute of Pathology, Washington, DC 203064 Received 5 June 1995/Returned for modification 16 August 1995/Accepted 12 September 1995

Recent evidence supports a causal relationship between Bartonella (Rochalimaea) henselae, cat-scratch disease (CSD), and bacillary angiomatosis. Cats appear to be the primary reservoir. Blood from 19 cats owned by 14 patients diagnosed with CSD was cultured. Blood samples from cats owned by veterinary students (n 5 25) having no association with CSD or bacillary angiomatosis were cultured as controls. Eighty-nine percent (17 of 19) of cats associated with CSD patients and 28% (7 of 25) of controls were bacteremic with Bartonella species (chi-square 5 16.47; P < 0.001). Twenty-three isolates were characterized as B. henselae, while one isolate from the cat of a CSD patient appeared to be a new Bartonella species. Thirteen cats remained culture positive during the ensuing 12-month period. Our results support the conclusion that B. henselae is the predominant species involved in CSD and is transmitted by cats. The incidence of Bartonella bacteremia in control cats suggests that B. henselae bacteremia is prevalent among the domestic cat population in the United States. Cat-scratch disease (CSD), cat-scratch fever, or benign nonbacterial lymphadenitis was recognized by Robert Debre´ in 1931, but it was first reported in 1950 (5). A presumptive diagnosis of CSD is made when a patient has an inoculation wound at the site of a cat bite or scratch, regional lymphadenopathy, a positive CSD skin test, and negative test results for brucellosis, infectious mononucleosis, mycobacteria, syphilis, and tularemia. The search for the etiologic agent was intensified after Wear et al. (40) described the presence of small, silver-staining bacilli in lymph nodes from 28 CSD patients. In 1990, Relman et al. (29) used PCR to amplify Bartonella DNA from bacillary angiomatosis (BA) lesions of four immunocompromised individuals, including one woman who had recently experienced a severe cat scratch. The causative agent eluded isolation attempts until concurrent reports by Welch et al. (42) and Regnery et al. (26) described the recovery of small gram-negative rods from both immunocompetent and immunocompromised patients by the lysis-centrifugation blood culture technique. The organisms were determined to be closely related to Bartonella quintana and were subsequently named Bartonella henselae. The molecular characterization and cultivation of B. henselae facilitated development of an indirect fluorescentantibody (IFA) test which was used by Regnery et al. (27, 28) to identify a seropositive B. henselae bacteremic cat. Soon thereafter, Koehler et al. (16) reported the isolation of Bartonella species from the BA lesions of four immunocompromised patients. B. quintana was found in three patients denying cat or arthropod contact, while B. henselae was cultured from the blood of the fourth patient who had obtained several scratches from his flea-infested cat and kitten. In 1993, Dolan et al. (7) described two immunocompetent patients with B.

henselae-induced lymphadenopathy; both of the patients owned cats. The most compelling evidence that cats are a primary reservoir of B. henselae was presented by Koehler et al. (15) in a study which identified seven of seven Bartonella bacteremic cats belonging to or associated with four patients with BA. Recently, multiphasic studies resulted in the reclassification of Rochalimaea and Grahamella species into the genus Bartonella and the removal of the family Bartonella from the order Rickettsiales (1, 3). The spectrum of clinical manifestations associated with Bartonella infection in people includes classical CSD (7, 15, 46), Carrio ´n’s disease (30), trench fever (45), cutaneous BA (12, 16, 22, 25, 29, 38, 42), vasoproliferative conditions of the liver and spleen (21), lymphadenopathy (7, 8, 10, 29), and endocarditis (6, 9, 13, 36). Reports of Bartonellainduced neurologic disease (32, 33, 37), relapsing fever (18, 26, 35, 41), granulomatous hepatosplenic syndrome (8, 10, 31, 34, 38, 39), osteolytic lesions (16, 39), pulmonary granulomas (4), and neuroretinitis (11, 23) are also in the literature. Exposure to cats is an important risk factor for B. henselae infection (15, 46). With a population of approximately 60 million domestic cats in the United States (44), there is a large potential zoonotic reservoir for introducing B. henselae infection into the human population. In view of this, we investigated the incidence of Bartonella bacteremia in cats associated with CSD patients. Determination of the prevalence and degree of persistence of Bartonella bacteremia in cats is necessary to assess the correlation between feline bartonellosis and disease in humans. (The study was presented in part at the 34th Interscience Conference on Antimicrobial Agents and Chemotherapy, Orlando, Fla., 4 to 7 October 1994 [17a].)

* Corresponding author. Mailing address: College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough St., Raleigh, NC 27606. Phone: (919) 829-4234. Fax: (919) 829-4336.

Our study compared cats historically associated with patients with diagnosed cases of CSD and healthy cats owned by veterinary students with no related history of disease. All patients were exposed to one or more cats and had a

MATERIALS AND METHODS

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presumptive clinical diagnosis of CSD or serological evidence of Bartonella exposure. The diagnosis of B. henselae infection was confirmed in one patient by PCR amplification of Bartonella DNA from pulmonary granulomas (4). Cats (n 5 19) owned by patients (n 5 14) with known or suspected CSD were examined and tested at the College of Veterinary Medicine, North Carolina State University. Twenty-five cats owned by veterinary students were used as controls. Control cats appeared healthy, tested negative for feline immunodeficiency virus and feline leukemia virus, and had no association with human CSD or BA. Questionnaires were distributed to all participants in the study. Attending physicians provided specific details relative to each patient’s presentation and course of illness, including criteria for diagnosis, antimicrobial treatment, and patient outcome. Owners of control animals completed questionnaires which included historical details about the cat including whether the cat regularly scratched, bit, licked, or slept with anyone in the household. Strain sources. The type strains of B. henselae (Houston-1; ATCC 49882) and Bartonella elizabethae (F9251; ATCC 49927) were obtained from the American Type Culture Collection (Beltsville, Md.). The B. quintana type strain (Fuller; ATCC VR-358) was provided by one of us (T.L.H.). Twenty-four feline isolates described in this report, in addition to NCSU-F01, a B. henselae isolate used as an antigen for serological testing, were obtained at the College of Veterinary Medicine, North Carolina State University. An avian isolate of Chlamydia psittaci (strain NCSU-165) was provided by Kevin Flammer. Blood culture isolation of microorganisms. Blood was collected aseptically from the external jugular vein, either 8 ml was injected into Wampole Isolator 10 microbial tubes (Wampole Laboratories, Cranbury, N.J.) or 1.5 ml was injected into Pediatric Isolator 1.5 tubes (Wampole Laboratories), and the tubes were inverted several times. Wampole Isolator 10 tubes were centrifuged at 2,300 3 g for 30 min at room temperature, the supernatant was removed, and the concentrate was applied to Trypticase soy agar (TSA) plates supplemented with 5% defibrinated rabbit blood (BBL, Becton Dickinson, Cockeysville, Md.). The Pediatric Isolator 1.5 tubes were vortexed for 15 s, and the entire sample was streaked onto TSA-rabbit blood plates. Cultures were incubated at 358C in 5% CO2 and were examined daily for bacterial growth. Cultures were monitored for at least 2 months before they were deemed negative and discarded. Microscopic and biochemical analyses. Bartonella organisms were visualized by light microscopy following Gram or Gimenez staining. Commercial bacterial identification systems which assay for preformed enzymes and carbohydrate utilization (UniScept 20E, An-IDENT, and API-ZYM; Analytab, Sherwood Medical, Plainview, N.Y.), the presence of oxidase (Dryslide; Difco Laboratories, Detroit, Mich.), and urease production (Urea agar; BBL, Becton Dickinson) were used in conjunction with standard microbiological methods to characterize the isolates (19). Cellular fatty acid composition. The organisms were grown on rabbit blood agar at 358C in 5% CO2 for 4 to 9 days. Cells were harvested from an average of three plates and were processed as follows. Briefly, the cells were saponified in methanol-NaOH with heat. The liberated fatty acids were methylated, and the methyl esters were extracted into an ether-hexane mixture. One microliter of the fatty acid methyl ester mixture was injected onto the column. Fatty acid profiles were determined with the Microbial Identification System (Microbial ID, Inc., Newark, Del.). The system consists of an HP5290 gas chromatograph equipped with a flame ionization detector, 5% methylphenyl silicone fused-silica capillary column, auto sampler, integrator, computer, and printer. The run begins at a temperature of 1708C and increases by 58C/min to 2708C. Fatty acids are identified and relative proportions are determined by computer analysis. Serological analysis. Sera were analyzed by microimmunofluorescence assay for immunoglobulin G (IgG) reactive for B. henselae Houston-1 and NCSU-F01, B. quintana Fuller, and C. psittaci NCSU-165. Bartonella organisms were cultivated in Vero cells and were harvested when the cells were .80% infected (3 to 5 days postinoculation). Antigen for IFA testing was prepared by pelleting and resuspending the microorganisms in 0.5% bovine serum albumin in phosphatebuffered saline (PBS). Five-microliter aliquots of crude antigen were applied to 24-well Teflon-coated slides (Cel-line Associates, Newfield, N.J.), air dried, acetone fixed, and frozen until they were used. The C. psittaci organisms were similarly processed following culture in McCoy’s cells, a mouse kidney fibroblast cell line. Slides used for the IFA test were blocked in 5% skim milk prior to the application of diluted test sera. Twofold dilutions of serum in PBS ranging from 1:16 to 1:1,024 were applied to slides in 10-ml aliquots; this was followed by 30-min incubation (378C) and wash (PBS) steps. Fluorescein isothiocyanate (FITC)-conjugated goat anti-cat IgG (heavy and light chains; Cappel, Organon Teknika Corp., Durham, N.C.) was diluted 1:200 in 0.5% BSA-PBS and was applied to each well containing feline serum in order to detect IgG-reactive antibodies in the cats. In some cases, the reactivity of human serum to B. henselae Houston-1 and B. quintana Fuller was determined in our laboratory by using FITC conjugated goat anti-human IgG (heavy and light chains; Cappel) at a working dilution of 1:20 in 0.5% BSA–PBS. The slides were incubated for an additional 30 min, washed in PBS, and examined at 340 magnification with a fluorescence microscope. Human sera were assayed by the IFA test in our laboratory or at the Centers for Disease Control and Prevention, Atlanta, Ga., by previously reported methods (28).

J. CLIN. MICROBIOL. All feline sera were tested for the presence of feline leukemia virus antigen and feline immunodeficiency virus antibodies by using the CITE Combo enzymelinked immunosorbent assay test kit (IDEXX, Portland, Maine). DNA extraction. Subcultures of the blood isolates were grown on TSA-rabbit blood plates and were recovered in filter-sterilized PBS with a cell scraper. The harvested bacteria were frozen at 2708C until extraction of the DNA. DNA extraction for PCR amplification and subsequent sequencing was performed by a standard phenol-chloroform protocol following agitation with glass beads in a mini-beadbeater (Biospec, Bartlesville, Okla.). Dedicated equipment and reagents were used to assemble the PCR mixtures in a biological containment hood, and extreme care was taken to prevent cross-contamination of samples. High-molecular-weight chromosomal DNA for restriction endonuclease digestion was obtained by an alternative method. Bartonella colonies were recovered from agar plates in glucose-Tris-EDTA and were kept on ice until they were subjected to proteinase K digestion. The bacterial lysate was purified with hexadecyltrimethylammonium bromide-NaCl at 658C, and genomic DNA precipitated with isopropanol. PCR amplification. PCR amplification of the 16S rRNA gene was accomplished with P0-C and PC-5A primers, which are modifications of previously described eubacterial primers (43). Reactions were performed in a Gene-Amp 9600 thermal cycler (Perkin-Elmer Cetus, Norwalk, Conn.) by a protocol reported elsewhere (2). DNA sequencing of 16S rRNA gene. Sequencing reactions of the purified PCR products were performed in a PE 9600 apparatus in preparation for use in an automated DNA sequencer (model 373A; Applied Biosystems Inc., Foster City, Calif.) as described previously (2). Published data confirm the existence of a ‘‘signature region’’ in the 16S rRNA gene between nucleotide positions 1101 and 1109 (Escherichia coli numbering) that is species specific for B. henselae, B. elizabethae, and B. quintana (16). The inclusion of sequence data from nucleotide positions 976 through 990 can further distinguish between Bartonella vinsonii and Bartonella bacilliformis. Primer PC4 (59-TTGACGTCATCCCCACCTTCC TC-39) initiates the reaction at position 1175 and generates the sequence in the desired region of residues 976 to 1109. The nucleotide sequence of each isolate in this signature area was compared with GenBank data for the Bartonella organisms B. henselae, B. quintana, B. elizabethae, B. vinsonii, B. bacilliformis, B. talpae, B. peromysci, B. grahamii, B. taylorii, and B. doshiae. Restriction digest of chromosomal DNA. B. henselae chromosomal DNAs obtained from 10 isolates from felines were subjected to digestion with restriction endonucleases MspI and XhoI according to the manufacturer’s recommendations (New England Biolabs, Beverly, Mass.). DNA was digested for 2 h at 378C, and the products were separated by electrophoresis through a 0.65% agarose gel to assess strain differences.

RESULTS CSD patients. The ages, sexes, immune status, clinical signs, criteria for diagnosis, duration of illness, and treatment regimens for CSD patients are summarized in Table 1. Recovery of Bartonella species from cat blood. The signalment of the cats (age, breed, sex), date of initial culture, and relationship to the onset of CSD are summarized in Table 2. Lysis-centrifugation blood cultures were examined daily for growth. Bartonella colonies appeared within 7 to 31 days of incubation. Most isolates (19 of 24) appeared within the range of 7 to 13 days, 3 of 24 isolates required 17 to 20 days of incubation, and 2 of 24 isolates did not show evidence of growth until 31 days had passed. Two colonial forms were recovered and occasionally, for some strains, coexisted within the same culture. One form consisted of rough, very adherent, cream-colored colonies and the other was mucoid, nonadherent, and opaque to cream in coloration. Both colonial forms ranged from ,1 to 2.5 mm in diameter. Subcultures of all isolates on TSA-rabbit blood plates yielded colonies of one phenotype within 3 to 6 days. The rough, adherent form appeared to be dominant. Hemolysis of the rabbit blood was not observed with any isolate. Persistent bacteremia was documented in 13 cats from which blood was cultured for various periods of time (Fig. 1). Blood samples from six cats belonging to a patient with pulmonary bartonellosis were initially cultured, and the cats were found to have Bartonella bacteremia 6 months after the patient became noticeably ill. The cats remained bacteremic for an additional year. Another patient in our study experienced classical CSD shortly after acquiring two kittens during January 1993. Cul-

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TABLE 1. Clinical characteristics of CSD patients Patient

Summarya

Characteristic 1

Sexb Age (yr) Immune compromised Cat contact Clinical signs Fever Myalgia Hyporexia Weight loss Inoculation wound Lymphadenopathy Parenchymal Encephalopathy Biopsy resultc Serology titer B. henselae Houston-1 B. quintana Fuller Antimicrobial treatmente Duration of illnessf

2

3

4

5

6

7

8

9

10

11

12

13

14

F 9 2 1

M 13 2 1

M 51 2 1

M 23 2 1

F 19 1 1

F 8 2 1

M 36 2 1

F 9 2 1

M 36 2 1

M 27 2 1

F 39 2 1

M 28 2 1

M 20 2 1

F 23 2 1

6F, 8M Median, 23 11, 132 141, 02

1 1 1 1 1 1 1 1 2

1 2 2 2 1 1 2 2 2

1 1 1 1 1 1 2 2 2

1 1 1 1 2 1 1 2 1

1 2 1 1 1 2 1 2 1

2 2 2 2 1 1 1 2 1

2 2 2 2 1 1 2 2 2

1 2 1 2 1 1 2 2 1

1 2 2 2 1 1 2 2 2

1 2 2 2 1 1 2 2 2

1 2 2 2 1 1 2 2 2

1 2 2 2 2 1 2 2 1

2 2 2 2 1 1 2 2 2

1 2 2 2 1 1 2 2 2

111, 32 31, 112 51, 92 41, 102 121, 22 131, 12 41, 102 11, 132 51, 92

64 128 cefd, cefp 4w

128 64 nt 6w

64 128 naf 8w

1,025 1,025 pen 4w

1,025 1,025 dox 5m

NDd ND ceph 4w

ND ND ery ND

ND ND amcl, tris 7w

256 ND dox 4w

32 ,16 dox 2w

128 128 nt 12m

,64 ND cip 2w

128 2,048 nt 4w

ND ND nt 4w

a

Numbers indicate numbers of patients. F, female; M, male. c Patient 5, lung tissue; other patients, lymph node. d ND, not determined. e cefd, cefadroxil; cefp, cefproxadine; naf, nafcillin; pen, penicillin; dox, doxycycline; ceph, cephalexin; ery, erythromycin; amcl, amoxicillin-clavulanate potassium; tris trimethoprim-sulfamethoxazole; cip, ciprofloxacin; nt, not treated. f w, weeks; m, months. b

tures of blood from both cats were positive for B. henselae in October 1994, representing a presumable period of bacteremia spanning 22 months. Interestingly, 7 of 25 (28%) of the control cats were bacteremic. No persistent skin lesions, febrile episodes, lethargy, or lymphadenopathy had been reported in any

household resident in the control group within the previous 18 months. Serology was not performed on the veterinary students. Microscopic and biochemical analyses of the isolates. Pleomorphic gram-negative rods ranged in size from 0.5 to 2.0 mm.

TABLE 2. Blood culture and serology results for cats associated with CSD cases Patient

Cat

Identification no.

Date of onset of illness (mo/yr)

Identification letter

Signalment (age/ sex/breeda)

Initial culture date (mo/yr)

Culture result

1 2 3 4c 5 5 5 5 5 5 6 7 8 9 10 11 12 13 14

11/92 9/93 10/93 3/93 9/92

A B B C D E F G H I J K L M N O P Q R S

1.5 yr/M/DSH 6 mo/M/DLH

5/93 11/93

1 1

1.5 yr/F/DSH 3.5 yr/F/DSH 2 yr/F/DSH 9 mo/M/DSH 2 yr/M/DSH 2 yr/M/DSH 2 yr/F/DSH 5.5 yr/M/DSH 6.5 yr/F/DSH 2.5 yr/F/DSH 1.5 yr/M/DSH 8 wk/F/DSH 2 yr/F/DLH 6 mo/F/DSH 9 mo/M/SiamX 1 yr/M/DSH 6 mo/F/DSH

5/93 3/93 3/93 3/93 3/93 3/93 3/93 4/93 4/93 5/93 6/93 1/94 2/94 2/94 3/94 9/94 9/94

2 1 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1

a b c

1/93 12/91 12/92 4/93 10/93 1/94 10/93 12/93 1/93

Serologyb Bh

F01

Bq

Cp

64 256

64 ,16

128 64

,16 ,16

,16 64 64 128 64 64 128 64 64 128 128 1,024 1,024 128 ,16 256 32

64 64 64 256 64 32 128 ,16 64 128 256 ,16 ,16 ,16 ,16 ,16 ,16

,16 64 32 128 256 256 256 ,16 128 128 128 ,16 64 ,16 512 256 ,16

,16 128 64 128 1,024 512 256 ,16 ,16 ,16 ,16 ,16 ,16 ,16 ,16 ,16 32

Age at onset of illness; M, male; F, female; DSH, domestic shorthair; DLH, domestic longhair; SiamX, Siamese crossbred. Data are reciprocal endpoint titers. Bh, B. henselae Houston-1; F01, B. henselae NCSU-F01; Bq, B. quintana Fuller; Cp, C. psittaci NCSU-165. Only one of three cats in contact with the patient was made available for blood culture.

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FIG. 1. Persistent Bartonella bacteremia in cats. Blood samples for culture were obtained at various time points following the onset of patient illness. *, data derived from the patient with the earliest onset of illness.

Organisms were also visualized with Gimenez stain. Eight- to 13-day-old subcultures were evaluated in triplicate for various phenotypic characteristics. Twitching motility, assessed by the hanging drop technique and with motility agar (M Medium; bioMerieux Vitek, Inc., Hazelwood, Mo.), was observed in most instances (20 of 24 isolates). All isolates were catalase negative when they were analyzed by the An-IDENT, UniScept 20E, and 3% hydrogen peroxide tests, urease negative when they were streaked onto urea agar, and Voges-Proskauer negative. Tests for indole production, carbohydrate utilization, and nitrate reduction were negative in all cases. Most isolates displayed variability when tested for oxidase by using 1% tetramethyl-p-phenylenediamine and the Dryslide technique. Biochemical analysis of 24 isolates yielded two discrete phenotypic clusters. The biochemical profiles of 21 isolates were nearly identical, while 3 isolates formed a different group. In the larger cluster, which consisted exclusively of B. henselae, the following aminopeptidases were detected: alanine, arginine, glycine, histidine, leucine, phenylalanine, and valine. The three isolates in the group that was different did not contain histidine, leucine, phenylalanine, proline, tyrosine, or valine aminopeptidase and, in addition, possessed indoxyl-acetate, esterase, and esterase lipase and utilized arginine. Cellular fatty acid analysis. Nineteen feline isolates and two American Type Culture Collection Bartonella type cultures were analyzed by fatty acid methyl ester analysis. Six to nine fatty acids were detected in each of the isolates. The predominant fatty acids were 18:1 v7c, 18:0, 16:0, and 17:0, which made up more than 97% of the fatty acid content of the cells. Serological findings. Sera from 17 of 19 CSD-associated cats had reactivity to B. henselae Houston-1 and NCSU-F01 in addition to B. quintana Fuller, with reciprocal titers ranging from 64 to 1,024 (Table 2). In 12 of 25 control cats, seroreactive IgG reciprocal titers ranged from 64 to 512. Three B. henselae bacteremic animals were seronegative for both IgMand IgG-specific antibodies. One cat has been bacteremic but seronegative on repeated occasions. In order to evaluate serological cross-reactivity between Bartonella species and C. psittaci, sera from CSD-associated cats and 3 specific-pathogen-free cats experimentally infected with B. henselae as a component of a separate study were analyzed for C. psittaci-

specific antibodies. Six of 19 CSD-associated cats, all from the same household, were seroreactive to both B. henselae Houston-1 and C. psittaci NCSU-165, while the remainder were seronegative for chlamydia-specific antibodies. Sera from three experimentally infected cats demonstrated Bartonella-reactive IgG titers of 1,024, and the cats were seronegative when their sera were tested against C. psittaci. Sequence of the 16S rRNA gene. Partial sequencing of the 16S rRNA gene was used to identify the species of the Bartonella isolates obtained in blood culture. Of 24 isolates, 23 were identified as B. henselae following analysis of the signature regions between residues 976 and 1109. The identity of one isolate could not be determined because of substantial differences in the nucleotide sequence of this hypervariable region, which may be indicative of a novel Bartonella species or subspecies. Additional genotypic characterization of this isolate is in progress. Restriction digest of DNA. The patterns produced by MspI and XhoI cleavage of chromosomal DNAs from 10 isolates from felines, including 6 isolates from felines in one household, matched the general digestion profile of B. henselae Houston-1 and differed substantially from the profiles of B. quintana Fuller and B. elizabethae F9251. Strain differences among the isolates were suggested by the further definition of several subgroups. This is best illustrated by the restriction patterns derived from isolates obtained from six cats belonging to patient 5 (Fig. 2). DISCUSSION Fourteen patients from North Carolina and Virginia with known or suspected CSD were studied. Six patients were examined by members of the Division of Infectious Diseases at Duke University Medical Center. The remaining patients were examined by family physicians, and the case records were reviewed by the authors. Bartonella bacteremia was documented in 17 of 19 (89%) cats historically associated with 14 cases of CSD, thereby suggesting exposure to Bartonella-infected cats in 12 instances. Aside from a renal transplant patient, all other patients in our study involved previously healthy, immunocompetent people. Two of the cats whose blood was cultured may

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FIG. 2. Analysis of restriction endonuclease digests of Bartonella DNA by MspI. Chromosomal DNAs from six B. henselae isolates from cats and three Bartonella type strains were digested with MspI, and the products were separated by electrophoresis through a 0.65% agarose gel. Isolates D through I were obtained from cats residing in the same household (patient 5). B. henselae strain variations are suggested by differences in the digestion profiles.

not have been the source of infection. In patient 7, illness occurred 17 months prior to culturing of blood of the associated cat. It is possible that these cats were bacteremic at an earlier date but spontaneously eliminated the organism or that the degree of bacteremia was below the level of detection by lysis-centrifugation. Our laboratory has detected relapsing bacteremia of prolonged duration in cats experimentally infected with B. henselae (17b). Cats A and B appear to have eliminated their bacteremia. Cat A initially had a reciprocal titer of 64 to B. henselae Houston-1 that persisted unchanged for 17 months, while cat B maintained reciprocal titers to B. henselae Houston-1 of between 256 to 512 during a 12-month period. In contrast, bacteremia was detected in some cats for periods of up to 18 months following the diagnosis of CSD. Bartonella bacteremia was detected in 7 of 25 (28%) control cats, which were not associated with any known instances of human disease. The incidence of B. henselae bacteremia in CSD-associated cats compared with that in cats not associated with CSD was significant (P , 0.001). Interestingly, within the control cat population, six of seven bacteremic cats occasionally bit, licked, scratched, or slept with their owners, yet clinical signs of CSD were not reported in those households. Since serological evaluation of the owners of control cats was not performed, it may be that they were immune to Bartonella infection or, alternatively, that the strains infecting these particular cats were nonvirulent for humans. Recent studies report that approximately 3% of healthy human controls are B. henselae seropositive and that 12 to 29% of veterinarians are CSD skin test positive (20, 28, 46). Koehler et al. (15) reported Bartonella bacteremia in 25 of 61 (41%) pet and pound cats in the San Francisco area, and Olson et al. (24) cultured B. henselae from 15 of 61 (25%) pet and stray cats in southern California. Given the current domestic cat population in the


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United States of approximately 60 million, 15 million to 25 million cats may potentially be infected with B. henselae. Regardless of the actual numbers of bacteremic cats, recent reports suggest that the potential for zoonotic transmission of B. henselae is considerable. However, it seems important to recognize that the infectivity of B. henselae strains isolated from healthy cats for people has not been established epidemiologically. In the present study, 3 of 15 (20%) B. henselae Houston-1seronegative cats were bacteremic. In addition to the potential for false-negative results, the IFA test is not species specific (10, 17). Because of cross-reactivity among the various Bartonella species, we elected to test sera against B. quintana Fuller as well as two strains of B. henselae strains Houston-1 and NCSU-F01 (Table 2) (39). Serological cross-reactivity is best illustrated by the responses of patient 13 and his cat (cat Q); serum samples from both the patient and his cat contained higher IgG levels against B. quintana Fuller, even though B. henselae, which is directly implicated in the transmission of CSD, was cultured from his cat. These data indicate that IFA serological testing of cats and people is reliable only to the genus level. Knobloch et al. (14) and Drancourt et al. (9) have described human serological cross-reactivity between Bartonella and Chlamydia species. Evaluation of feline serum failed to demonstrate cross-reactivity between these two organisms. In addition to 13 of 19 CSD-associated cats whose sera had variable titers against B. henselae Houston-1, experimentally infected specific-pathogen-free cats with reciprocal IFA test titers of 1,024 were seronegative when their sera were tested against C. psittaci NCSU-165. The six C. psittaci-seropositive cats were from the household of patient 5. It is quite likely that these cats are chronically infected with chlamydia, resulting in persistent conjunctivitis and rhinitis. The diagnosis of Bartonella infection is best accomplished by direct isolation of the organisms from blood or tissue specimens. Feline blood cultures readily grow bartonellae when the cultures are processed and incubated as described above, although in the present study, periods of culture as long as 31 days have passed before colonies were visualized. In our experience, Bartonella species are considerably more difficult to isolate from humans. Whether this observation is due to a more prolonged bacteremia or a higher bacterial titer in the cat is unknown. Determining the etiologic species is also difficult. Following isolation, Bartonella organisms cannot be identified to the species level by conventional methods used in clinical microbiology laboratories. Biochemically, Bartonella species are quite inert. Cellular fatty acid analysis, although useful for many bacterial organisms, may not be a reliable method for determining species of this genus since the fatty acid content of Bartonella species is predominantly composed of only four fatty acids. Presently, 16S rRNA gene sequencing, DNA hybridization, and immunocytochemistry provide the most accurate means of species identification. The increasing use of molecular techniques to detect bacterial DNA will undoubtedly enhance the ability to diagnose these fastidious organisms. There is considerable variation in the clinical presentations and lesions associated with Bartonella infection in people. This variability may be attributable to bacterial strain differences as well as differences in the immunological response of the infected individual. Among our B. henselae isolates, we detected differences in the biochemical constituents, cellular fatty acid contents, and restriction endonuclease digestion patterns of the B. henselae DNAs. These results suggest that several B. henselae strains might contribute to the variations in clinical presentation or cellular inflammatory response. Kittens have been implicated as a risk factor in CSD, yet we

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did not find a disproportionate number of kittens involved in cases of CSD in our study. Most of the cats associated with our CSD patients were older than 6 months of age (14 of 19 cats) and, therefore, would be classified as adults. Similar to previous reports, most of the bacteremic cats in our study were clinically healthy. The medical histories of CSD-associated cats were unremarkable. Most received routine vaccinations and had only otherwise been seen by a veterinarian for declawing, neutering, and management of bite wounds sustained in cat fights. Six CSD-associated cats originating from one household had conjunctivitis most likely of chlamydial etiology. Another cat presented with prominent generalized lymphadenopathy that gradually resolved without treatment during a 3-month period. This cat also experienced a brief (2-day) episode of mild, nonlocalizing neurologic dysfunction, as might be expected with a metabolic encephalopathy. Since cats can maintain a subclinical bacteremia for extended durations in the presence or absence of circulating antibody, establishing a causal relationship to unusual or rare manifestations of bartonellosis will be difficult. ACKNOWLEDGMENTS This work was supported by a grant from SmithKline Beecham Animal Health, Exton, Pa., the U.S. Department of Veterans Affairs, and the Department of Companion Animal and Special Species Medicine Research Fund. We thank Carol L. Lemons for providing clinical microbiology support and Barbara C. Hegarty for technical assistance in the development of a microimmunofluorescence assay. We are also indebted to Hal Hills, Jenny Simchok, Rhonda Blitchington, and Gary Anderson for generously sharing their expertise in molecular techniques and Vincent T. Andriole and Talmadge T. Brown for critical review of the manuscript. CITE Combo ELISA test kits were a gift of the IDEXX Corp. REFERENCES 1. Birtles, R. J., T. G. Harrison, N. A. Saunders, and D. H. Molyneux. 1995. Proposals to unify the genera Grahamella and Bartonella, with descriptions of Bartonella talpae comb. nov., Bartonella peromysci comb. nov., and three new species, Bartonella grahamii, sp. nov., Bartonella taylorii sp. nov., and Bartonella doshiae sp. nov. Int. J. Syst. Bacteriol. 45:1–8. 2. Breitschwerdt, E. B., D. L. Kordick, D. E. Malarkey, B. Keene, T. L. Hadfield, 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 Bartonella quintana comb. nov., Bartonella vinsonii comb. nov., Bartonella henselae comb. nov., and Bartonella elizabethae comb. nov., and to remove the family Bartonellaceae from the order Rickettsiales. Int. J. Syst. Bacteriol. 43:777–786. 4. Caniza, M. A., D. L. Granger, K. H. Wilson, M. K. Washington, D. L. Kordick, and D. P. Frush. 1995. Bartonella (Rochalimaea) henselae: etiology of pulmonary nodules in a patient with depressed cell-mediated immunity. Clin. Infect. Dis. 20:1505–1511. 5. Carithers, H. A. 1970. Cat scratch disease: notes on its history. Am. J. Dis. Child. 119:200–203. 6. Daly, J. S., M. G. Worthington, D. J. Brenner, C. W. Moss, D. G. Hollis, R. S. Weyant, A. G. Steigerwalt, R. E. Weaver, M. I. Daneshvar, and S. P. O’Connor. 1993. Rochalimaea elizabethae sp. nov. isolated from a patient with endocarditis. J. Clin. Microbiol. 31:872–881. 7. Dolan, M. J., M. T. Wong, R. L. Regnery, J. H. Jorgensen, M. Garcia, J. Peters, and D. Drehner. 1993. Syndrome of Rochalimaea henselae adenitis suggesting cat scratch disease. Ann. Intern. Med. 118:331–336. 8. Doyle, D., S. C. Eppes, and J. D. Klein. 1994. Atypical cat-scratch disease: diagnosis by a serologic test for Rochalimaea species. South. Med. J. 87:485– 487. 9. Drancourt, M., J. L. Mainardi, P. Brouqui, F. Vandenesch, A. Carta, F. Lehnert, J. Etienne, F. Goldstein, J. Acar, and D. Raoult. 1995. Bartonella (Rochalimaea) quintana endocarditis in three homeless men. N. Engl. J. Med. 332:419–423. 10. Golden, S. E. 1993. Hepatosplenic cat-scratch disease associated with elevated anti-Rochalimaea antibody titers. Pediatr. Infect. Dis. J. 12:868–871. 11. Golnik, K. C., M. E. Marotto, M. M. Fanous, D. Heitter, L. P. King, J. I. Halpern, and P. H. Holly. 1994. Ophthalmic manifestations of Rochalimaea

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