Bartonella clarridgeiae, a Newly Recognized Zoonotic Pathogen Causing Inoculation Papules, Fever, and Lymphadenopathy (Cat Scratch Disease)

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, July 1997, p Vol. 35, No /97/$ Copyright 1997, American Society for Microbiology Bartonella clarridgeiae, a Newly Recognized Zoonotic Pathogen Causing Inoculation Papules, Fever, and Lymphadenopathy (Cat Scratch Disease) DORSEY L. KORDICK, 1 EDWARD J. HILYARD, 2 TED L. HADFIELD, 2 KENNETH H. WILSON, 3,4 ARNOLD G. STEIGERWALT, 5 DON J. BRENNER, 5 AND EDWARD B. BREITSCHWERDT 1 * Department of Companion Animal and Special Species Medicine, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina ; Department of Infectious and Parasitic Diseases Pathology, Armed Forces Institute of Pathology, Washington, DC ; The Veterans Affairs Medical Center 3 and Division of Infectious Diseases, 4 Duke University Medical Center, Durham, North Carolina 27710; and Emerging Bacterial and Mycotic Diseases Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia Received 21 January 1997/Returned for modification 3 March 1997/Accepted 11 April 1997 Shortly after adopting a 6-week-old cat, a veterinarian was bitten on the left index finger. Within 3 weeks, he developed headache, fever, and left axillary lymphadenopathy. Initial blood cultures from the cat and veterinarian were sterile. Repeat cultures from the cat grew Bartonella-like organisms with lophotrichous flagella. Sera from the veterinarian were not reactive against Bartonella henselae, B. quintana, or B. elizabethae antigens but were seroreactive (reciprocal titer, 1,024) against the feline isolate. Sequential serum samples from the cat were reactive against antigens of B. henselae (titer, 1,024), B. quintana (titer, 128), and the feline isolate (titer, 2,048). Phenotypic and genotypic characterization of this and six additional feline isolates, including microscopic evaluation, biochemical analysis, 16S rrna gene sequencing, DNA-DNA hybridization, and PCR-restriction fragment length polymorphism of the 16S gene, 16S-23S intergenic spacer region, and citrate synthase gene identified the isolates as B. clarridgeiae. This is the first report of cat scratch disease associated with B. clarridgeiae. Since the early 1900s, various diverse microbiologic agents have been implicated in the etiology of cat scratch disease (CSD). During the 1980s, it appeared that the long-sought causative agent had been found. A gram-negative bacterium was observed by Wear and colleagues in lymph node sections obtained from CSD patients and stained by the Warthin-Starry silver stain or Brown-Hopps tissue Gram stain (43). In 1988, English et al. (19) isolated a bacterial agent from clinical specimens of CSD patients which was subsequently named Afipia felis by Brenner et al. (9). During the early 1990s, two groups independently described a newly recognized gram-negative rod associated with fever, lymphadenopathy, and cat contact (i.e., CSD) which was named Rochalimaea henselae (37, 44) and later reclassified as Bartonella henselae (10). Regnery and coworkers developed an indirect fluorescent-antibody (IFA) assay to assess seroreactivity against B. henselae antigens in patients diagnosed with CSD (36). The relative role of A. felis and/or B. henselae in the clinical syndrome known as CSD has been controversial for several years. Recently, investigators performed serologic analysis on samples obtained from patients satisfying the clinical criteria for a presumptive diagnosis of CSD. Comparison of seroreactivity data indicates that an overwhelming number of patients have antibodies to B. henselae but are seronegative for A. felis (17, 33 36, 40). In addition, Anderson et al. and Bergmans et al. detected B. henselae DNA but not A. felis DNA in samples of CSD skin test antigen and lymph node aspiration biopsy * Corresponding author. Mailing address: Department of Companion Animal and Special Species Medicine, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, NC specimens from CSD patients (1, 2, 4). Although the role of A. felis in the clinical syndrome known as CSD has not been clarified, B. henselae appears to be the predominant cause of CSD. Seroreactivity to B. quintana antigen has been observed in several CSD patients, but B. quintana has not been recovered from any patient with this presentation or from any associated cats. B. clarridgeiae was recently isolated from a cat owned by a human immunodeficiency virus-positive person bacteremic with B. henselae (14, 29). Other currently classified species of Bartonella have not been associated with CSD. A previous study in our laboratory investigating the duration of Bartonella bacteremia in cats associated with CSD patients identified a patient (patient 10) fulfilling the diagnostic criteria for classical CSD, yet acute- and convalescent-phase serum samples were minimally reactive against antigens derived from three characterized Bartonella species (25). As a result of the subsequent characterization of isolate 94-F40, we describe the isolation of B. clarridgeiae from a cat associated with human CSD. Case report. A 6-week-old female domestic shorthair cat was adopted from a North Carolina pet placement group by a 27-year-old male veterinarian. Shortly after acquiring the cat, he sustained a bite wound on his left index finger. A papular lesion approximately 0.5 cm in diameter developed at the site of the bite wound and persisted for approximately 1 month (Fig. 1). The cat was healthy but infested with fleas. Within 3 weeks, the patient developed headache, fever (maximum temperature, 38.5 C) lasting 1 week, and left axillary lymphadenopathy (Fig. 2). Lysis centrifugation blood cultures performed 5 days after the onset of febrile illness and before a 2-week course of doxycycline (500 mg every 12 h) failed to grow bac- 1813

2 1814 KORDICK ET AL. J. CLIN. MICROBIOL. TABLE 1. Blood culture results and seroreactivity of the veterinarian and his cat at various time points Time of sample collection Blood culture result IFA serology (reciprocal titer) 94-F40 B. henselae B. quintana B. elizabethae FIG. 1. Inoculation papule from the cat bite. teria. The headache and fever abated during doxycycline therapy, and after 1 month, the axillary lymphadenopathy resolved. Initial lysis centrifugation cultures from the cat and the patient failed to yield bacteria. Two subsequent blood cultures from the cat (1.5 and 4 months later) grew Bartonella-like organisms. Acute-phase and 1- and 5-month convalescentphase serum samples from the patient were analyzed by IFA for Bartonella species-specific immunoglobulin G (IgG) (Table 1). He did not seroconvert to B. henselae, B. quintana, or B. elizabethae antigens but was seroreactive at a reciprocal titer of 1,024 against the Bartonella-like isolate obtained from his cat. Sera from the cat were reactive against B. henselae (reciprocal titer, 1,024), B. quintana (titer, 128), and the homologous isolate (titer, 2,048). MATERIALS AND METHODS Bacterial strains. The type strains of B. henselae (Houston 1; ATCC 49882), B. elizabethae (F9251; ATCC 49927), B. vinsonii subsp. vinsonii (Baker; ATCC VR-152), and B. clarridgeiae (ATCC 51734) were obtained from the American Type Culture Collection (Rockville, Md.). The type strains of B. grahamii (NCTC 12860) and B. doshiae (NCTC 12862) were obtained from the National Collection of Type Cultures (London, England). The B. quintana type strain (Fuller; ATCC VR-358) was provided by one of us (T.L.H.), the B. bacilliformis type strain (KC583; ATCC 35685; NCTC 12138) was obtained from Robert E. Weaver, Centers for Disease Control and Prevention; and the B. vinsonii subsp. berkhoffii type strain (ATCC 51672) was isolated in our laboratory (7, 26). NCSU 94-F40 ( ATCC ) was isolated from the cat described in this report at the College of Veterinary Medicine, North Carolina State University, Raleigh, N.C. FIG. 2. Axillary lymphadenopathy in the patient. Veterinarian 11/18/93 Negative 1, /20/93 ND a 1, /12/94 ND 1, Cat 11/18/93 Negative ND b 512 ND ND 1/6/94 ND 2,048 1, ND 1/31/94 Positive 2,048 1, ND 4/7/94 Positive ND ND ND ND a ND, not done. b Several samples from the cat were of insufficient volume to test. Six feline Bartonella isolates obtained during previous studies at the College of Veterinary Medicine were included in the genotypic analysis (25). Isolation of bacteria. Aseptically collected blood was cultured by using an Isolator tube (10 ml) for the veterinarian s sample and the Pediatric Isolator (1.5 ml) for all samples from the cat (Wampole Laboratories, Cranbury, N.J.). The samples were processed in accordance with the manufacturer s recommendations and plated on Trypticase soy agar (TSA) containing 5% rabbit blood (BBL, Becton Dickinson, Cockeysville, Md.). Inoculated plates were incubated at 35 C in 5% CO 2 with humidity. Microscopic and biochemical analysis. Bartonella organisms were visualized by light microscopy following Gram or Gimenez staining. To determine whether flagella were present, 6-day-old subcultures were gently scraped from agar plates in phosphate-buffered saline. Samples were applied to grids, negatively stained with 2% phosphotungstic acid, and examined by using a transmission electron microscope (18). Motility was assessed by incubation of the isolate in M agar and hanging drop slides. A drop of a saline suspension of bacteria was placed on single-well Teflon-coated microscope slides. A coverslip was placed over the well, and the preparation was examined. Commercial bacterial identification systems (UniScept 20E, An-IDENT, and API-ZYM [Analytab, Sherwood Medical, Plainview, N.Y.]) were used in conjunction with standard microbiological methods to characterize the isolate (30). Biochemical tests were performed in triplicate on 12-day-old cultures. Microimmunofluorescent serology. Vero cell cultures were inoculated with Bartonella organisms that were initially cultivated on solid media. Infected cells were harvested between 3 and 8 days postinoculation when 80% infected and were used as a whole-cell antigen in the IFA. Sera were diluted in 0.5% bovine serum albumin in phosphate-buffered saline before being applied to Tefloncoated microscope slides in 5- l aliquots. The protocol has been described in detail previously (7). Cellular fatty acid composition. Organisms were cultivated on TSA-rabbit blood at 35 C in 5% CO 2 for 4 to 9 days. Cells were harvested from an average of three plates and saponified 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 by using a Microbial Identification System (Microbial ID, Inc., Newark, Del.). DNA hybridization. Bartonella strains were cultivated on TSA supplemented with 5% rabbit blood at 37 C with 5% CO 2 for 12 to 15 days. Colonies were removed from media with a cell scraper and suspended in 1% NaCl. DNA for use in the hydroxyapatite hybridization experiments was extracted, purified, and labeled with [ 32 P]dCTP as previously described (8). Radioactively labeled DNA was heat denatured, combined with sheared, unlabeled DNA, and allowed to reassociate at 55 C (optimal incubation temperature) and/or 70 C (stringent incubation temperature). Double-stranded DNA was detected in a scintillation counter, and relative binding ratios were calculated. Divergence in related sequences was approximately 1% for each degree of decreased thermal stability in a heterologous reassociated DNA duplex compared with the homologous reassociated DNA duplex. Divergence was calculated to the nearest 0.5%. Extraction of DNA used for PCR amplification. DNA used for PCR amplification of the 16S gene and the 16S-23S rrna intergenic spacer (ITS) region was extracted in the following manner. Subcultures of the blood isolates and type strains were grown on TSA-rabbit blood plates, recovered in glucose-tris- EDTA, and kept on ice until subjected to proteinase K digestion. The bacterial lysate was purified with hexadecyltrimethylammonium bromide-nacl at 65 C, and genomic DNA was precipitated with isopropanol. The resulting pellet was washed in ethanol, air dried, and reconstituted in Tris-EDTA solution (10 mm Tris, 1 mm EDTA, ph 8). DNA used in the evaluation of the citrate synthase gene was extracted by an alternative method. Eight to 10 isolated colonies from

3 VOL. 35, 1997 CAT SCRATCH DISEASE DUE TO B. CLARRIDGEIAE 1815 FIG. 3. B. henselae (A) and isolate 94-F40 (B) stained with 2% phosphotungstic acid, ph 7.2. Magnification, 31,000. subcultures were harvested with a 10- l loop and placed into 1 N NaOH for 30 min. An equal volume of 1 M Tris-HCl, ph 8.4, was added to adjust the ph. The lysate was diluted 1:10 with sterile PCR grade H 2 O, and 1, 5, or 10 l of the DNA preparation was used per PCR. PCR amplification. PCR amplification of the 16S rrna gene was accomplished by using eubacterial primers P0-C and PC-5A as reported elsewhere (7, 45). The 16S-23S ITS region was amplified by using the primers and cycling parameters described by Roux and Raoult (38). Citrate synthase amplicons were produced by using primers BHCS.781 (GGG GAC CAG CTC ATG GTG G) and BHCS.1137 (AAT GCA AAA AGA ACA GTA AAC A). Amplification of the citrate synthase gene was carried out in 50- l reaction volumes. A standard PCR mixture consisted of the following: 1 l of template DNA, 1UofTaq polymerase, 5 l of10 Taq buffer, 3 l of MgCl 2 (25 mm), 0.3 l ofdeoxynucleoside triphosphates (100 mm each), 50 pmol of each primer, and sterile distilled water to a final volume of 50 l. Cycling parameters were 30 cycles of denaturation at 95 C for 20 s, annealing of primers at 60 C for 30 s, and primer extension at 72 C for 1 min. All PCRs were performed in a Gene-Amp 9600 thermal cycler (Perkin-Elmer Cetus, Norwalk, Conn.). Restriction endonuclease digestion of PCR-amplified DNA. 16S amplicons from the novel isolate and type strains were digested by restriction endonucleases DdeI and MnlI in accordance with the manufacturer s (New England BioLabs, Beverly, Mass.) recommendations. PCR-amplified DNA from the ITS region was digested with HaeIII, AluI, and TaqI. Four microliters of template DNA and 10 U of enzyme were used in each reaction. Digestions were performed at 37 C (DdeI, MnlI, HaeIII, and AluI) or 65 C ( TaqI) for 2 h, and products were separated by electrophoresis through a 3% gel (2% NuSieve [FMC BioProducts, Rockland, Maine], 1% agarose [Sigma Chemical Co., St. Louis, Mo.]) at 200 V. Restriction endonuclease digestion of the PCR-amplified 600-base portion of the citrate synthase gene was performed with TaqI (Life Technologies, Bethesda, Md.) at 60 C for 2 h. Products derived from the digestion of the citrate synthase gene were electrophoresed through 2% agarose gels at 100 V for 2.5 h. All gels were stained with ethidium bromide and viewed by using UV light to assess differences (21). DNA sequencing of the 16S rrna gene. Dideoxy terminator sequence reactions of purified PCR products were performed in a PE 9600 in preparation for use in an ABI 373A automated DNA sequencer (Applied Biosystems Inc., Foster City, Calif.) (7). The 16S rrna nucleotide sequence of the Bartonella-like isolate was compared with sequences from the Ribosomal Database Project (31) for B. henselae, B. quintana, B. elizabethae, B. vinsonii subsp. vinsonii, B. vinsonii subsp. berkhoffii, B. clarridgeiae, B. bacilliformis, B. grahamii, B. doshiae, and B. taylorii. Phylogenetic analysis was performed by using the Genetic Data Environment (39) on a Sun Sparcstation II (Sun Microsystems, Mountain View, Calif.). A dendrogram prepared from 16S rdna sequences was based on the maximum-likelihood method (20). Nucleotide sequence accession number. The 16S rrna gene sequence for 94-F40 was deposited in the GenBank, EMBL, and DDBJ databases under accession number U RESULTS Blood culture isolates. The blood culture obtained from the patient remained negative for 63 days. Blood was obtained from the associated cat on three occasions at approximately 2.5-month intervals. The initial culture was sterile; however, two subsequent blood cultures grew a Bartonella-like organism (designated isolate 94-F40) within 10 to 23 days of incubation. The bacterial colonies were smooth with tan to pale pink pigmentation and slightly raised centers. Colonies were loosely adherent to, but not embedded in, the medium. There was no hemolysis of the blood agar. Biochemical testing. Isolate 94-F40 was negative in oxidase, catalase, urease, and Voges-Proskauer tests. It utilized arginine and hydrolyzed indoxyl-acetate. Positive reactions were obtained for the production of arginine aminopeptidase, alanine aminopeptidase, glycine aminopeptidase, esterase, esterase lipase, leucine arylamidase, and naphthol-as-bi-phosphohydrolase. Microscopic analysis. Isolate 94-F40 stained faintly as a gram-negative rod but was easier to visualize when cultivated in Vero cells and stained by the Gimenez technique. The isolate was tinctorially similar to other Bartonella species by both the Gram and Gimenez methods. After 1 week of incubation, motility was not detected in M agar; however, twitching motion was seen in a hanging drop preparation. Negative staining with 2% phosphotungstic acid revealed the presence of lophotrichous flagella when examined with a transmission electron microscope (Fig. 3). Seroreactivity of patient and cat. An acute-phase and two convalescent-phase serum samples from the patient analyzed by IFA were not reactive against B. henselae, B. quintana, orb. elizabethae but did react with isolate 94-F40. Sequential serum samples from the cat reacted with antigens of B. henselae, B. quintana, and isolate 94-F40 (Table 1). Cellular fatty acid analysis. Isolate 94-F40 clustered among other feline Bartonella isolates and Bartonella type strains as described previously (25). DNA hybridization. Radiolabeled DNA from Bartonella-like isolate 94-F40 was tested with other isolates obtained from cats and the type strains of Bartonella species (Table 2). Strain 94-F40 DNA was 89% related to B. clarridgeiae DNA and 34 to 47% related to DNAs from the other Bartonella type strains TABLE 2. DNA relatedness of Bartonella-like strain 94-F40 and other feline isolates to Bartonella type strains a Source of unlabeled DNA Result obtained with labeled 94-F40 DNA RBR b, 55 C %D c RBR, 70 C Bartonella-like strain 94-F (ATCC ) Bartonella-like strain Lea Bartonella-like strain Bishop Bartonella-like strain Snowy Bartonella-like strain Jazz Bartonella clarridgeiae ATCC Bartonella henselae ATCC Bartonella quintana ATCC VR Bartonella vinsonii subsp. berkhoffii ATCC Bartonella vinsonii subsp. vinsonii ATCC VR-152 Bartonella elizabethae ATCC Bartonella bacilliformis ATCC KC (NCTC 12138) Bartonella grahamii NCTC Bartonella doshiae NCTC a All reactions were run in duplicate. b RBR, relative binding index. c D, divergence.

4 1816 KORDICK ET AL. J. CLIN. MICROBIOL. taylorii, B. grahamii, and B. doshiae (6), were 97.7, 99.4, and 99.7% similar to isolate 94-F40, respectively. A phylogenetic tree depicting the relationship among these Bartonella species is shown in Fig. 5. DISCUSSION FIG. 4. PCR-RFLP profiles of Bartonella type strains and feline isolates. The 16S rrna gene was digested with DdeI. tested. Four of the six isolates from other cats were assayed, and they were 86 to 88% related to 94-F40. The type strain of B. taylorii was unavailable (21a), and the type strains of B. peromysci and B. talpae are no longer extant. PCR-RFLP analysis. Restriction fragment length polymorphism (RFLP) analysis performed on the PCR-amplified 16S gene and the 16S-23S ITS region demonstrated unique restriction patterns. Endonuclease digestion of the 16S gene with DdeI resulted in fragments of approximately 410 and 210 bp for each type strain. In all type strains except 94-F40, a third fragment migrated at approximately 380 bp. Other speciesspecific products ranged from 100 to 280 bp. Digestion of the ITS region with HaeIII did not yield any fragment common to all type strains. Examples of restriction profiles are shown in Fig. 4. Comparison of the products generated by endonuclease digestion of the citrate synthase gene revealed a band of 300 bp in all of the species tested. B. quintana had a band at 120 bp, B. henselae had a species-specific band at approximately 150 bp, and isolate 94-F40 had a band at 400 bp. DNA sequence of the 16S rrna gene. DNA sequence analysis of the 16S rrna gene disclosed several areas of variability among the Bartonella species currently listed in GenBank. Strain 94-F40 had 99.8% sequence similarity with B. clarridgeiae, 97.3% with B. henselae, 97.7% with B. quintana, 97.9% with B. elizabethae, 97.2% with B. bacilliformis, 98.1% with B. vinsonii subsp. vinsonii, and 97.2% with B. vinsonii subsp. berkhoffii. The 16S rrna gene sequences from recently described Bartonella species isolated from rodents in England, B. B. clarridgeiae isolated from a cat induced fever, lymphadenopathy, and an inoculation papule (classic CSD) in a human. Our patient failed to develop IFA seroreactivity to B. henselae, B. quintana, and B. elizabethae, despite overwhelming clinical evidence supporting a diagnosis of CSD. We were unable to confirm bacteremia in the patient; however, blood cultures from the cat responsible for inflicting the puncture wound on the patient s finger yielded a Bartonella organism on two of three culture attempts. Although the lysis centrifugation blood culture technique enhances recovery of Bartonella isolates, anecdotal reports indicate that Bartonella is more difficult to culture from human blood than from feline blood. Our inability to obtain a positive culture from the cat on the initial attempt may be due to the fact that we were able to collect only 0.75 ml, as opposed to the recommended 1.5 ml, in the Pediatric Isolator system. At this dilution, the lysis solution contained in the tube may have lysed not only the blood cells but also the bacteria. Alternatively, as we observed during a recent transmission study, bacteremia in experimentally infected cats appears to be relapsing in nature (27). When strain 94-F40 was cultivated in Vero cells and used as antigen in an IFA, the patient was seropositive for the isolate obtained from his cat. Serum obtained from the cat was reactive with antigens of B. henselae, B. quintana, and strain 94- F40. Given the high seroprevalence of B. henselae in the domestic cat population throughout the world (3, 11 13, 16, 22, 23, 32, 41), it is probable that the cat had been previously exposed to B. henselae, as well as B. clarridgeiae. However, extensive cross-reactivity, which is highly variable in degree, occurs among cats, dogs, and humans exposed to Bartonella species (3, 11, 12, 24). Therefore, in this instance, the feline IgG antibody response to B. clarridgeiae may be less specific than the human IgG response to the organism. Genotypic analysis, including DNA sequencing and PCR- RFLP, of portions of the rrna and citrate synthase genes identified isolate 94-F40 as B. clarridgeiae. DNA-DNA hybridization is considered by most to be the ultimate determinant of taxonomic relationships among organisms. Under optimal conditions, the DNA relatedness to isolate 94-F40 was 86 to 89% FIG. 5. Phylogenetic tree depicting the relationship of strain 94-F40 to the other known Bartonella species.

5 VOL. 35, 1997 CAT SCRATCH DISEASE DUE TO B. CLARRIDGEIAE 1817 for B. clarridgeiae and the other four feline B. clarridgeiae isolates obtained in our laboratory. Under stringent conditions, relatedness values ranged from 86 to 93%. The generally accepted definition of a bacterial species is a group of strains with at least 70 and 55% relatedness under optimal and stringent DNA reassociation conditions, respectively, and 5% or less divergence within related sequences (8, 42). Data obtained from isolate 94-F40 and our other feline strains satisfy these guidelines and confirm their identity as B. clarridgeiae. Several recent reports (16, 36, 46) have indicated that 5 to 16% of patients with clinically defined CSD are seronegative for B. henselae. It is possible that some of these seronegative cases of CSD may be due to infection with B. clarridgeiae and the lack of seroreactivity is a result of failure to use the proper antigen for diagnosis. In fact, prevalence of bacteremia with B. clarridgeiae is probably more widespread than currently recognized. During a continuing study investigating the prevalence of feline Bartonella infections, we obtained six additional feline isolates with the same genotype as isolate 94-F40. Of these isolates, two were obtained from cats (cats P and R) with a historical association with CSD (25). Other investigators have also reported the recovery of unique Bartonella isolates (23) or DNA (5) from cat blood. Bergmans and coworkers submitted a partial 16S rrna gene sequence (GenBank accession no. Z69039) derived from bacterial DNA extracted from cat blood (5). Comparison of the 471 nucleotides with corresponding sequences of isolate 94-F40 and B. henselae revealed 100 and 98% similarity, respectively. The cat was associated with a case of CSD in The Netherlands. These data identify B. clarridgeiae as an etiologic agent of CSD and broaden its geographic distribution outside the United States. The type strain of B. clarridgeiae was isolated from the cat of a human immunodeficiency virus-positive patient with B. henselae bacteremia (14, 29). We documented B. henselae and B. clarridgeiae coinfection in a naturally exposed cat used as a blood donor during a transmission study (28). Blood transfusions from this cat transmitted B. henselae to recipients on one occasion and B. clarridgeiae on another. In light of these observations, we propose that cats may be coinfected with more than one species of Bartonella. Although the predominant species involved in CSD is B. henselae, B. clarridgeiae may be responsible for cases of CSD, particularly in patients seronegative for B. henselae. The relevance of B. clarridgeiae in other clinical presentations, such as bacillary angiomatosis (BA), is unknown. In 1991, Cockerell et al. reported the isolation of a Bartonella-like organism from an excised lesion of BA (15). The isolate possessed flagella and appeared to be related to B. bacilliformis by cellular fatty acid analysis. The patient denied having contact with cats, but his pet parakeet frequently perched on the affected arm. Inasmuch as people have become infected with B. henselae without apparent exposure to cats and B. clarridgeiae is the only Bartonella species aside from B. bacilliformis to be identified with flagella, it is possible that the etiologic agent in this case was B. clarridgeiae. Unfortunately, retrospective analysis of the strain is not possible. The isolate was lost during subculture attempts, and genomic DNA became contaminated. Given the prevalence of B. clarridgeiae among cats and the pathogenicity of closely related species to people, additional studies to assess the role of B. clarridgeiae as a cause of CSD, BA, and other disease manifestations in humans appear to be justified. ACKNOWLEDGMENTS We thank Murray Benett for assistance with the editing and alignment of the 16S rrna gene sequence, Ellen D Nicuola for work related to the citrate synthase gene, and Gary Anderson for many helpful discussions. We also acknowledge Brendalyn Bradley-Kerr of the Electron Microscopy Laboratory at the College of Veterinary Medicine, North Carolina State University, for preparation of the electron micrographs. This work was supported by the State of North Carolina, by a grant from SmithKline Beecham Animal Health (currently Pfizer Animal Health), West Chester, Pa. (D.L.K. and E.B.B.), and by the Department of Veterans Affairs (K.H.W.). REFERENCES 1. Anderson, B., C. Kelly, R. Threlkel, and K. Edwards Detection of Rochalimaea henselae in cat-scratch disease skin test antigens. J. Infect. Dis. 168: Anderson, B., K. Sims, R. Regnery, L. Robinson, M. J. Schmidt, S. Goral, C. Hager, and K. Edwards Detection of Rochalimaea henselae DNA in specimens from cat scratch disease patients by PCR. J. Clin. Microbiol. 32: Baneth, G., D. L. Kordick, B. C. Hegarty, and E. B. Breitschwerdt Comparative seroreactivity to Bartonella henselae and Bartonella quintana among cats from Israel and North Carolina. Vet. Microbiol. 50: Bergmans, A. M. C., J.-W. Groothedde, J. F. P. Schellekens, J. D. A. van Embden, J. M. Ossewaarde, and L. M. Schouls Etiology of cat scratch disease: comparison of polymerase chain reaction detection of Bartonella (formerly Rochalimaea) and Afipia felis DNA with serology and skin tests. J. Infect. Dis. 171: Bergmans, A. M. C., J. F. P. Schellekens, J. D. A. van Embden, and L. M. Schouls Predominance of two Bartonella henselae variants among cat-scratch disease patients in The Netherlands. J. Clin. Microbiol. 34: Birtles, R. J., T. G. Harrison, N. A. Saunders, and D. H. Molyneux 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: Breitschwerdt, E. B., D. L. Kordick, D. E. Malarkey, B. Keene, T. L. Hadfield, and K. Wilson Endocarditis in a dog due to infection with a novel Bartonella subspecies. J. Clin. Microbiol. 33: Brenner, D. J., A. C. McWhorter, J. K. Leete Knutson, and A. G. Steigerwalt Escherichia vulneris: a new species of Enterobacteriaceae associated with human wounds. J. Clin. Microbiol. 15: Brenner, D. J., D. G. Hollis, C. W. Moss, C. K. English, G. S. Hall, J. Vincent, J. Radosevic, K. A. Birkness, W. F. Bibb, F. D. Quinn, B. Swaminathan, R. E. Weaver, M. W. Reeves, S. P. O Connor, P. S. Hayes, F. C. Tenover, A. G. Steigerwalt, B. A. Perkins, M. I. Daneshvar, B. C. Hill, J. A. Washington, T. C. Woods, S. B. Hunter, T. L. Hadfield, G. W. Ajello, A. F. Kaufmann, D. J. Wear, and J. D. Wenger Proposal of Afipia gen. nov., with Afipia felis sp. nov. (formerly the cat scratch disease bacillus), Afipia clevelandensis sp. nov. (formerly the Cleveland Clinic Foundation strain), Afipia broomeae sp. nov., and three unnamed genospecies. J. Clin. Microbiol. 29: Brenner, D. J., S. P. O Connor, H. H. Winkler, and A. G. Steigerwalt 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: Childs, J. E., J. G. Olson, A. Wolf, N. Cohen, Y. Fakile, J. A. Rooney, F. Bacellar, and R. L. Regnery Prevalence of antibodies to Rochalimaea species (cat scratch disease agent) in cats. Vet. Rec. 136: Childs, J. E., J. A. Rooney, J. L. Cooper, J. G. Olson, and R. L. Regnery Epidemiologic observations on infection with Rochalimaea species among cats living in Baltimore, Md. J. Am. Vet. Med. Assoc. 204: Chomel, B. B., R. C. Abborr, R. W. Kasten, K. A. Floyd-Hawkins, P. H. Kass, C. A. Glaser, N. C. Pedersen, and J. E. Koehler Bartonella henselae prevalence in domestic cats in California: risk factors and association between bacteremia and antibody titers. J. Clin. Microbiol. 33: Clarridge, J. E., III, T. J. Raich, D. Pirwani, B. Simon, L. Tsai, M. C. Rodriguez-Barradas, R. Regnery, A. Zollo, D. C. Jones, and C. Rambo Strategy to detect and identify Bartonella species in routine clinical laboratory yields Bartonella henselae from human immunodeficiency virus-positive patient and unique Bartonella strain from his cat. J. Clin. Microbiol. 33: Cockerell, C. J., P. M. Tierno, A. E. Friedman-Kien, and K. S. Kim Clinical, histologic, microbiologic, and biochemical characterization of the causative agent of bacillary epithelioid angiomatosis: a rickettsial illness with features of bartonellosis. J. Invest. Dermatol. 97: Dalton, M. J., L. E. Robinson, J. Cooper, R. L. Regnery, J. G. Olson, and J. E. Childs Use of Bartonella antigens for serologic diagnosis of cat-scratch disease at a national referral center. Arch. Intern. Med. 155: Demers, D. M., J. W. Bass, J. M. Vincent, D. A. Person, D. K. Noyes, C. M.

6 1818 KORDICK ET AL. J. CLIN. MICROBIOL. Staege, C. P. Samlaska, N. H. Lockwood, R. L. Regnery, and B. E. Anderson Cat-scratch disease in Hawaii: etiology and seroepidemiology. J. Pediatr. 127: Dykstra, M. J A manual of applied techniques for biological electron microscopy. Plenum Publishing Corp., New York, N.Y. 19. English, C. K., D. J. Wear, A. M. Margileth, C. R. Lissner, and G. P. Walsh Cat scratch disease: isolation and culture of the bacterial agent. JAMA 259: Felsenstein, J PHYLIP phylogeny inference package (version 3.2). Cladistics 5: Hadfield, T. L., D. Kordick, E. J. Hilyard, M. E. D Nicuola, and E. B. Breitschwerdt Restriction fragment length polymorphism analysis of Bartonella isolates from cats, abstr. C-277, p. 50. Abstracts of the 96th General Meeting of the American Society for Microbiology American Society for Microbiology, Washington, D.C. 21a.Holmes, B. (National Collection of Type Cultures). Personal communication. 22. Jameson, P., C. Green, R. Regnery, M. Dryden, A. Marks, J. Brown, J. Cooper, B. Glaus, and R. Greene Prevalence of Bartonella henselae antibodies in pet cats throughout regions of North America. J. Infect. Dis. 172: Koehler, J. E., C. A. Glaser, and J. W. Tappero Rochalimaea henselae infection: a new zoonosis with the domestic cat as reservoir. JAMA 271: Kordick, D. L., and E. B. Breitschwerdt Comparative reactivity of feline sera to various Bartonella species, p. 16. Abstracts of the 11th Sesqui- Annual Meeting of the American Society for Rickettsiology and Rickettsial Diseases. American Society for Rickettsiology and Rickettsial Diseases, St. Simons Island, Ga. 25. Kordick, D. L., K. H. Wilson, D. J. Sexton, T. L. Hadfield, H. A. Berkhoff, and E. B. Breitschwerdt Prolonged Bartonella bacteremia in cats associated with cat-scratch disease patients. J. Clin. Microbiol. 33: Kordick, D. L., B. Swaminathan, C. E. Greene, K. H. Wilson, A. M. Whitney, S. P. O Connor, D. G. Hollis, G. M. Matar, A. G. Steigerwalt, G. B. Malcolm, P. S. Hayes, T. L. Hadfield, E. B. Breitschwerdt, and D. J. Brenner Description of Bartonella vinsonii subsp. berkhoffii subsp. nov., isolated from dogs, description of Bartonella vinsonii subsp. vinsonii, and emended description of Bartonella vinsonii. Int. J. Syst. Bacteriol. 46: Kordick, D. L., and E. B. Breitschwerdt Relapsing bacteremia following blood transmission of Bartonella henselae to cats. Am. J. Vet. Res. 58: Kordick, D. L., and E. B. Breitschwerdt Unpublished data. 29. Lawson, P. A., and M. D. Collins Description of Bartonella clarridgeiae sp. nov. isolated from the cat of a patient with Bartonella henselae septicemia. Med. Microbiol. Lett. 5: MacFaddin, J. F Biochemical tests for identification of medical bacteria. The Williams & Wilkins Co., Baltimore, Md. 31. Olsen, G. J., R. Overbeek, N. Larsen, T. L. Marsh, M. J. McCaughey, M. A. Maciukenas, W. M. Kuan, T. J. Macke, Y. King, and C. R. Woese The ribosomal database project. Nucleic Acids Res. 20: Olson, P. E., M. R. Wallace, and J. G. Creek Rochalimaea a cosmopolitan zoonosis? West. J. Med. 161:428. (Letter.) 33. Patnaik, M., and J. B. Peter Cat-scratch disease, Bartonella henselae, and the usefulness of routine serological testing for Afipia felis. Clin. Infect. Dis. 21:1064. (Letter.) 34. Pece, S., A. B. Maffione, R. Petruzelli, B. Greco, G. Giuliani, M. R. Partipilo, S. Amarri, F. Schettini, E. Jirillo, and D. Fumarola Rochalimaea henselae organisms possess an elevated capacity of binding to peripheral blood lymphocytes from patients with cat scratch disease. Microbios 77: Raoult, D., H. T. Dupont, and M. Enea-Mutillod Positive predictive value of Rochalimaea henselae antibodies in the diagnosis of cat-scratch disease. Clin. Infect. Dis. 19:355. (Letter.) 36. Regnery, R. L., J. G. Olson, B. A. Perkins, and W. Bibb Serological response to Rochalimaea henselae antigen in suspected cat-scratch disease. Lancet 339: Regnery, R. L., B. E. Anderson, J. E. Clarridge III, M. C. Rodriquez- Barradas, D. C. Jones, and J. H. Carr Characterization of a novel Rochalimaea species, R. henselae sp. nov., isolated from blood of a febrile, human immunodeficiency virus-positive patient. J. Clin. Microbiol. 30: Roux, V., and D. Raoult Inter- and intraspecies identification of Bartonella (Rochalimaea) species. J. Clin. Microbiol. 33: Smith, W. W., C. Wang, P. M. Gillevet, and W. Gilbert Genetic data environment and the Harvard genome database. Genome mapping and sequencing. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 40. Szelc-Kelly, C. M., S. Goral, G. I. Perez-Perez, B. A. Perkins, R. L. Regnery, and K. M. Edwards Serologic responses to Bartonella and Afipia antigens in patients with cat scratch disease. Pediatrics 96: Ueno, H., Y. Muramatsu, B. B. Chomel, T. Hohdatsu, H. Koyama, and C. Morita Seroepidemiological survey of Bartonella (Rochalimaea) henselae in domestic cats in Japan. Microbiol. Immunol. 39: Wayne, L. G., D. J. Brenner, R. R. Colwell, P. A. D. Grimont, O. Kandler, M. I. Krichevsky, L. H. Moore, W. E. C. Moore, R. G. E. Murray, E. Stackebrandt, M. P. Starr, and H. G. Trüper Report of the Ad Hoc Committee on Reconciliation of Approaches to Bacterial Systematics. Int. J. Syst. Bacteriol. 37: Wear, D. J., A. M. Margileth, T. L. Hadfield, G. W. Fischer, C. H. Schlagel, and F. M. King Cat scratch disease: a bacterial infection. Science 221: Welch, D. F., D. A. Pickett, L. N. Slater, A. G. Steigerwalt, and D. J. Brenner Rochalimaea henselae sp. nov., a cause of septicemia, bacillary angiomatosis, and parenchymal bacillary peliosis. J. Clin. Microbiol. 30: Wilson, K. H., R. B. Blitchington, and R. C. Greene Amplification of bacterial 16S ribosomal DNA with polymerase chain reaction. J. Clin. Microbiol. 28: Zangwill, K. M., D. H. Hamilton, B. A. Perkins, R. L. Regnery, B. D. Plikaytis, J. L. Hadler, M. L. Carrter, and J. D. Wenger Cat scratch disease in Connecticut: epidemiology, risk factors, and evaluation of a new diagnostic test. N. Engl. J. Med. 329:8 13.