Sequence variation in the ftsZ gene of Bartonella henselae isolates and clinical samples

In a search for methods for subtyping of Bartonella henselae in clinical samples, we amplified and sequenced a 701-bp region in the 3' end of the ftsZ gene in 15 B. henselae isolates derived from cats and humans in the United States and Europe. The ftsZ sequence variants that were discovered were designated variants Bh ftsZ 1, 2, and 3 and were compared with 16S rRNA genotypes I and II of the same isolates. There was no ftsZ gene variation in the strains of 16S rRNA type I, all of which were Bh ftsZ 1. The type II strains constituted two groups, with nucleotide sequence variation in the ftsZ gene resulting in amino acid substitutions at three positions, one of which was shared by the two groups. One 16S rRNA type II isolate had an ftsZ gene sequence identical to those of the type I strains. Variants Bh ftsZ 1 and 2 were detected in tissue specimens from seven Swedish patients with diagnoses such as chronic multifocal osteomyelitis, cardiomyopathy, and lymphadenopathy. Patients with similar clinical entities displayed either Bh ftsZ variant. The etiological role of B. henselae in these patients was supported by positive Bartonella antibody titers and/or amplification and sequencing of a part of the B. henselae gltA gene. B. henselae ftsZ gene sequence variation may be useful in providing knowledge about the epidemiology of various B. henselae strains in clinical samples, especially when isolation attempts have failed. This report also describes manifestations of atypical Bartonella infections in Sweden.     

Coinfection with Bartonella clarridgeiae and Bartonella henselae and with different Bartonella henselae strains in domestic cats.

Bartonella clarridgeiae and several strains of Bartonella henselae, the agent of cat scratch disease, with variations in the 16S rRNA gene have been found to infect the blood of cats. An epidemiologic study of Bartonella infection in domestic French cats revealed that of 436 cats sampled, 5 cats (1.1%) were coinfected with B. henselae and B. clarridgeiae and 2 cats (0.5%) were coinfected with two strains of B. henselae with variations in the 16S rRNA gene, B. henselae type I and type II. In an indirect immunofluorescence assay, coinfected cats tested positive for both Bartonella species at titers of > or = 128. Identification of the colonies was achieved by preformed enzyme analysis, PCR-restriction fragment length polymorphism analysis of the citrate synthase gene, and 16S rRNA gene sequencing. Colony size differences in mixed culture allowed differentiation of the Bartonella species. The coinfection of cats with two Bartonella species or variants of the same species raises concern about the possibility of dual infection in humans. The development of a polyvalent vaccine targeted against the most pathogenic or invasive strains may be a means of protecting cats and man from infection.     

Prevalence of Bartonella henselae and Bartonella clarridgeiae in stray cats.

The aim of the present work was to determine by blood culture the prevalence of blood infection with Bartonella species in a well-defined, European, urban stray cat population. Therefore, 94 stray cats were trapped from 10 cat colonies. Blood samples of these cats were cultured on both blood agar and liquid medium in order to raise the likelihood of bacterial detection. Fifty blood samples (53%) gave a positive culture result for Bartonella species. Isolate identification was performed by sequencing the first 430 bases of the 16S ribosomal DNA. Three types of sequences were thus obtained. The first type (17 isolates; 34%) was identical to that of B. henselae Houston-1 and the corresponding strains were referred as B. henselae type I. The second sequence type (18 isolates; 36%) was identical to that initially described as "BA-TF," and the corresponding strains were referred to as B. henselae type II. The third sequence type (15 isolates; 30%) was identical to that of the Bartonella clarridgeiae type strain (ATCC 51734). Our study points out the major role of stray cats as a reservoir of Bartonella spp. which can be transmitted to pet cats and, consequently, to humans. The study also highlights the high prevalence of B. clarridgeiae (16%) in the blood of stray cats.     

Genetic variability and prevalence of Bartonella henselae in cats in Berlin, Germany, and analysis of its genetic relatedness to a strain from Berlin that is pathogenic for humans

Nineteen Bartonella henselae strains and one Bartonella clarridgeiae strain were isolated from blood samples of 97 pet cats and 96 stray cats from Berlin, Germany, indicating prevalence rates of 1 and 18.7%, respectively, for B. henselae and 0 and 1%, respectively, for B. clarridgeiae. Eighteen of 19 B. henselae isolates corresponded to 16S rRNA type II. Pulsed-field gel electrophoresis (PFGE) analysis revealed seven different PFGE types among the feline B. henselae strains. Interestingly, all feline isolates displayed low genetic relatedness to B. henselae strain Berlin-1, which is pathogenic for humans.     

Study of genotypes and virB4 secretion gene of Bartonella henselae strains from patients with clinically defined cat scratch disease

Bartonella henselae is the causative agent of cat scratch disease (CSD), which usually presents as a self-limiting lymphadenopathy. Occasionally, the bacteria will spread and be responsible for tissue and visceral involvement. Two B. henselae genotypes (genotypes I and II) have been described to be responsible for uncomplicated CSD on the basis of 16S rRNA sequence analysis. A type IV secretion system (T4SS) similar to the virulence-associated VirB system of Agrobacterium tumefaciens was recently identified in the B. henselae Houston-1 genotype I strain. We studied the correlations of the B. henselae genotypes with the clinical presentations and with the presence of T4SS. Isolates originated from CSD patients whose lymph nodes were prospectively analyzed. B. henselae genotype I was identified in 13 of 42 patients (30%). Among these, two teenage twins presented with hepatosplenic CSD and one immunocompetent adult presented with osteomyelitis. Genotype II was detected in 28 of 42 patients (67%), all of whom presented with uncomplicated CSD. The last patient was infected with both genotypes. T4SS was studied by PCR amplification of the virB4 gene. Amplification of virB4 codons 146 to 256, 273 to 357, and 480 to 537 enabled us to detect 66, 90, and 100% of the B. henselae isolates, respectively. Sequence analysis revealed sequence variations that correlated with genotype distribution. Our studies suggest that B. henselae genotype I strains harbor virB4 genes that are different from those harbored by genotype II strains and that genotype I strains might be more pathogenic.     

Limited diversity among human isolates of Bartonella henselae

A study of 59 isolates of Bartonella henselae reveals relatively limited diversity among those of human origin (n = 28). Either of two distinct alleles of both gltA and 16S ribosomal DNA (rDNA) was found in all isolates, with a high level of congruity between 16S and gltA inheritance among proven human pathogens. Human isolates from all over Eastern Australia were most commonly 16S rDNA (Bergmans) type I, with the same gltA allele as the type strain (Houston-1). Comparable feline isolates were more commonly 16S type II, with less congruity of inheritance between 16S and gltA alleles. Previously described arbitrarily primed PCR and EagI-HhaI infrequent restriction site PCR fingerprinting techniques separated Bartonella species effectively but lacked discriminating power within B. henselae. Examination of the 16-23S intergenic spacer region revealed for several strains several point mutations as well as a repeat sequence of unknown significance which is readily detected by HaeIII restriction fragment length polymorphism analysis. The bacteriophage-associated papA gene was present in all isolates. Enterobacterial repetitive intergenic consensus PCR proved to be a useful and robust typing tool and clearly separated human isolates (including imported strains) from the majority of feline isolates. Our data are consistent with published evidence and with previous suggestions of intragenomic rearrangements in the type strain and suggest that human isolates come from a limited subset of B. henselae strains. They strengthen arguments for careful exploration of genotype-phenotype relationships and for the development of a multilocus enzyme electrophoresis and multilocus sequence typing-based approach to the phylogeny of B. henselae.     

16S/23S rRNA intergenic spacer regions for phylogenetic analysis, identification, and subtyping of Bartonella species

Species of the genus Bartonella are currently recognized in growing numbers and are involved in an increasing variety of human diseases, mainly trench fever, Carrion's disease, bacillary angiomatosis, endocarditis, cat scratch disease, neuroretinitis, and asymptomatic bacteremia. Such a wide spectrum of infections makes it necessary to develop species and strain identification tools in order to perform phylogenetic and epidemiological studies. The 16S/23S rRNA intergenic spacer region (ITS) was sequenced for four previously untested species, B. vinsonii subsp. arupensis, B. tribocorum, B. alsatica, and B. koehlerae, as well as for 28 human isolates of B. quintana (most of them from French homeless people), six human or cat isolates of B. henselae, five cat isolates of B. clarridgeiae, and four human isolates of B. bacilliformis. Phylogenetic trees inferred from full ITS sequences of the 14 recognized Bartonella species using parsimony and distance methods revealed high statistical support, as bootstrap values were higher than those observed with other tested genes. Five well-supported lineages were identified within the genus and the proposed phylogenetic organization was consistent with that resulting from protein-encoding gene sequence comparisons. The ITS-derived phylogeny appears, therefore, to be a useful tool for investigating the evolutionary relationships of Bartonella species and to identify Bartonella species. Further, partial ITS amplification and sequencing offers a sensitive means of intraspecies differentiation of B. henselae, B. clarridgeiae, and B. bacilliformis isolates, as each strain had a specific sequence. The usefulness of this approach in epidemiological investigations should be highlighted. Among B. quintana strains, however, the genetic heterogeneity was low, as only three ITS genotypes were identified. It was nevertheless sufficient to show that the B. quintana population infecting homeless people in France was not clonal.     

Bartonella koehlerae sp. nov., isolated from cats

Two of the 25 Bartonella isolates recovered during a prevalence study of Bartonella henselae bacteremia in domestic cats from the greater San Francisco Bay region were found to differ phenotypically and genotypically from all prior B. henselae isolates. These isolates, C-29 and C-30, which were recovered from the blood of two pet cats belonging to the same household, grew on chocolate agar as pinpoint colonies following 14 days of incubation at 35 degrees C in a candle jar but failed to grow on heart infusion agar supplemented with 5% rabbit blood. Additional phenotypic characteristics distinguished the isolates C-29 and C-30 from other feline B. henselae isolates. The restriction patterns obtained for C-29 and C-30 by citrate synthase PCR-restriction fragment length polymorphism (RFLP) analysis as well as by genomic RFLP could not be distinguished from each other but were distinctly different from that of the B. henselae type strain. In reciprocal reactions, DNAs from strains C-29 and C-30 were 97 to 100% related under optimal and stringent DNA reassociation conditions, with 0 to 0.5% divergence within related sequences. Labeled DNA from the type strain of B. henselae was 61 to 65% related to unlabeled DNAs from strains C-29 and C-30 in 55 degrees C reactions, with 5.0 to 5.5% divergence within the related sequences, and 31 to 41% related in stringent, 70 degrees C reactions. In reciprocal reactions, labeled DNAs from strains C-29 and C-30 were 68 to 92% related to those of the B. henselae type strain and other B. henselae strains, with 5 to 7% divergence. The 16S rRNA gene sequence of strain C-29 was 99.54% homologous to that of the type strain of B. henselae. On the basis of these findings, the two isolates C-29 and C-30 are designated a new species of Bartonella, for which we propose the name Bartonella koehlerae. The type strain of Bartonella koehlerae is strain C-29 (ATCC 700693).     

Detection and identification of two Bartonella henselae variants in domestic cats in Germany.

To determine the prevalence of bacteremia caused by Bartonella henselae in domestic cats in the region of Freiburg, Germany, we investigated culture of blood from 100 cats from 89 different households over a 12-month period. B. henselae could be isolated from 13% (13 of 100) of these cats. In eight households with two cats each and in one household with three cats, B. henselae bacteremia was found either in all of the animals or in none of the animals. Positive cultures were more likely to be found for female, young (24 months of age or younger) cats than for male or older cats. Identification of the Bartonella isolates was made by colony morphology, by Gram staining, biochemically by RapID ANA II or Rapid ID 32 A systems, and by whole-cell fatty acid analysis. Differentiation between B. henselae and Bartonella quintana was only possible by 16S rRNA sequencing, enterobacterial repetitive intergenic consensus (ERIC)-PCR and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Genomic fingerprinting of the B. henselae isolates by ERIC-PCR yielded two different patterns based on three distinct bands.     

Prevalence of Bartonella species in domestic cats in The Netherlands.

Cats have been shown to provide the only known reservoir of Bartonella henselae, the causative agent of cat scratch disease. To determine the prevalence of Bartonella bacteremia and antibodies in Dutch cats, blood samples from 113 cats from shelters (sheltered cats), 50 pet cats, and 25 specific-pathogen-free (SPF) cats were analyzed. Culture and subsequent PCR-restriction fragment length polymorphism (RFLP) analysis of the 16S-23S rRNA intergenic region and 16S rRNA gene PCR-hybridization assays revealed a prevalence of Bartonella bacteremia in 22% of the sheltered cats and showed no bacteremia in the SPF cats. Three spacer RFLP types were found: types A, B, and G, with type B being predominant over types A and G. An important finding was the existence of mixtures of different Bartonella species. Bartonella DNA was detected in 7 of 27 DNA extracts from fleas combed from the sheltered cats (26%). Seropositivity was 50% for sheltered cats and 56% for pet cats, as determined by a B. henselae enzyme-linked immunoassay.     

Comparison of Different DNA Fingerprinting Techniques for Molecular Typing of Bartonella henselae Isolates

Seventeen isolates of Bartonella henselae from the region of Freiburg, Germany, obtained from blood cultures of domestic cats, were examined for their genetic heterogeneity. On the basis of different DNA fingerprinting methods, including pulsed-field gel electrophoresis (PFGE), enterobacterial repetitive intergenic consensus (ERIC)-PCR, repetitive extragenic palindromic (REP) PCR, and arbitrarily primed (AP)-PCR, three different variants were identified among the isolates (variants I to III). Variant I included 6 strains, variant II included 10 strains, and variant III included only one strain. By all methods used, the isolates could be clearly distinguished from the type strain, Houston-1, which was designated variant IV. A previously published type-specific amplification of 16S rDNA differentiated two types of the B. henselae isolates (16S rRNA types 1 and 2). The majority of the isolates (16 of 17), including all variants I and II, were 16S rRNA type 2. Only one isolate (variant III) and the Houston-1 strain (variant IV) comprised the 16S rRNA type 1. Comparison of the 16S rDNA sequences from one representative strain from each of the three variants (I to III) confirmed the results obtained by 16S rRNA type-specific PCR. The sequences from variant I and variant II were identical, whereas the sequence of variant III differed in three positions. All methods applied in this study allowed subtyping of the isolates. PFGE and ERIC-PCR provided the highest discriminatory potential for subtyping B. henselae strains, whereas AP-PCR with the M13 primer showed a very clear differentiation between the four variants. Our results suggest that the genetic heterogeneity of B. henselae strains is high. The methods applied were found useful for typing B. henselae isolates, providing tools for epidemiological and clinical follow-up studies.     

Differentiation of Bartonella species by a microimmunofluorescence assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western immunoblotting

Bartonella species can be differentiated by microimmunofluorescence assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting with murine polyclonal antisera to Bartonella henselae, B. quintana, B. elizabethae, and B. bacilliformis. A pairwise comparison on the basis of SDS-PAGE protein profiles demonstrated similarity values for proteins of different Bartonella species ranging from 28.6 to 86.4%. Antigenic relationships revealed by immunoblotting with murine antisera were equivalent to those of proteins observed by SDS-PAGE. A dendrogram obtained on the basis of protein bands of SDS-polyacrylamide gels showed that Bartonella species could be divided into three groups. B. bacilliformis was distinct from all other Bartonella species; B. grahamii, B. taylorii, B. doshiae, and B. vinsonii formed a cluster, as did B. henselae, B. quintana, B. elizabethae, and B. clarridgeiae. These relationships were consistent with those revealed by parsimony trees derived from 16S rRNA and gltA gene sequencing. SDS-PAGE analysis showed that 120-, 104-, 85-, 71-, 54-, 47-, 40-, 33-, 30-, and 19-kDa proteins were present in all species, with the 54-kDa protein being the most dominant. Proteins with a molecular mass of less than 54 kDa allow the differentiation of species and are a possible target for future species-specific antibodies and antigens.     

Detection of Rickettsia rickettsii and Bartonella henselae in Rhipicephalus sanguineus ticks from California

Sixty-two questing adult Rhipicephalus sanguineus (Latreille) ticks were collected by direct removal from blades of turfgrass and adjacent concrete walkways at a suburban home in Riverside County, CA, and tested for the presence of Rickettsia, Bartonella, and Ehrlichia DNA. Polymerase chain reaction (PCR) was used to amplify fragments of the 17-kDa antigen gene and the rOmpA gene of the spotted fever group rickettsiae. One male tick contained R. rickettsii DNA; its genotype differed from R. rickettsii isolates found in Montana and Arizona that cause Rocky Mountain spotted fever and from Hlp#2 and 364D serotypes. One male tick and one female tick contained B. henselae DNA. No Ehrlichia platys or Ehrlichia canis DNAs were detected using nested PCR for their 16S rRNA genes. These findings extend the area where Rickettsia rickettsii may be vectored by Rh. sanguineus. Rh. sanguineus also may be infected with Bartonella henselae, a human pathogen that is typically associated with fleas and causes cat scratch disease.     

Predominance of two Bartonella henselae variants among cat-scratch disease patients in the Netherlands.

Restriction endonuclease analysis of the PCR-amplified 16S-23S rRNA gene spacer region was used to investigate the prevalence of Bartonella henselae variants in samples from cat-scratch disease (CSD) patients. Analysis of spacer PCR fragments from 27 Bartonella DNA-positive samples from Dutch patients with CSD with AluI revealed two restriction fragment length polymorphism (RFLP) patterns, patterns A and B. Twenty samples yielded B. henselae pattern A, and 7 samples yielded B. henselae pattern B. Three samples from North American patients with CSD were shown to contain B. henselae with RFLP pattern B. To be able to detect and differentiate Bartonella DNA in clinical material more sensitively and faster, two B. henselae PCRs which amplify part of the 16S rRNA gene and which can discriminate between two B. henselae variants were developed. Thirty-two of 41 Bartonella DNA-positive samples from Dutch patients with CSD contained type I B. henselae, 7 samples contained type II B. henselae, and two samples were negative in both type-specific PCRs. Two samples from North American patients with CSD both contained type II B. henselae. A 100% correlation was found between the AluI spacer RFLP pattern and the 16S rRNA PCR type. We have shown that Dutch patients with CSD contain a limited number of B. henselae variants, suggesting that, in contrast to systemic bartonellosis, CSD in immunocompetent patients is caused by a limited number of B. henselae variants.     

Inter- and intraspecies identification of Bartonella (Rochalimaea) species.

Species of the genus Rochalimaea, recently renamed Bartonella, are of a growing medical interest. Bartonella quintana was reported as the cause of trench fever, endocarditis, and bacillary angiomatosis. B. henselae has been implicated in symptoms and infections of human immunodeficiency virus-infected patients, such as fever, endocarditis, and bacillary angiomatosis, and is involved in the etiology of cat scratch disease. Such a wide spectrum of infections makes it necessary to obtain an intraspecies identification tool in order to perform epidemiological studies. B. vinsonii, B. elizabethae, seven isolates of B. quintana, and four isolates of B. henselae were studied by pulsed-field gel electrophoresis (PFGE) after restriction with the infrequently cutting endonucleases NotI, EagI, and SmaI. Specific profiles were obtained for each of the four Bartonella species. Comparison of genomic fingerprints of isolates of the same species showed polymorphism in DNA restriction patterns, and a specific profile was obtained for each isolate. A phylogenetic analysis of the B. quintana isolates was obtained by using the Dice coefficient, UPGMA (unweighted pair-group method of arithmetic averages), and Package Philip programming. Amplification by PCR and subsequent sequencing using an automated laser fluorescent DNA sequencer (Pharmacia) was performed on the intergenic spacer region (ITS) between the 16 and 23S rRNA genes. It was found that each B. henselae isolate had a specific sequence, while the B. quintana isolates fell into only two groups. When endonuclease restriction analysis of the ITS PCR product was done, three enzymes, TaqI, HindIII, and HaeIII, allowed species identification of Bartonella spp.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genomic variation of Bartonella henselae strains detected in lymph nodes of patients with cat scratch disease

Bartonella henselae is the primary agent of cat scratch disease (CSD). In order to study the genetic variation of B. henselae and the correlation of the various genotypes with epidemiological and clinical findings, two seminested, groEL- and pap31-based PCR assays were carried out with specimens from 273 patients. Amplicons were sequenced to determine the genotype of the causative Bartonella species. Compared to our reference intergenic spacer region-based PCR, the groEL- and pap31-based assays were 1.7 and 1.9 times more sensitive, respectively. All 107 positive patients were infected with B. henselae; neither Bartonella clarridgeiae nor other species were detected. Based on the groEL and pap31 sequences, B. henselae amplicons were classified into two genogroups, Marseille and Houston-1, and into four variants, Marseille, CAL-1, Houston-1, and a new variant, ZF-1. Patients infected with either one or the other genogroup did not exhibit different epidemiological or clinical characteristics. Our study highlights the genotypic heterogeneity of B. henselae in patients with CSD.     

Prevalence, risk factors, and genetic diversity of Bartonella henselae infections in pet cats in four regions of the United States

Blood was collected from a convenience sample of 271 pet cats aged 3 months to 2 years (mean age, 8 months, median and mode, 6 months) between May 1997 and September 1998 in four areas of the United States (southern California, Florida, metropolitan Chicago, and metropolitan Washington, D.C.). Sixty-five (24%) cats had Bartonella henselae bacteremia, and 138 (51%) cats were seropositive for B. henselae. Regional prevalences for bacteremia and seropositivity were highest in Florida (33% and 67%, respectively) and California (28% and 62%, respectively) and lowest in the Washington, D.C. (12% and 28%, respectively) and Chicago (6% and 12%, respectively) areas. No cats bacteremic with B. clarridgeiae were found. The 16S rRNA type was determined for 49 B. henselae isolates. Fourteen of 49 cats (28.6%) were infected with 16S rRNA type I, 32 (65.3%) with 16S rRNA type II, and three (6.1%) were coinfected with 16S rRNA types I and II. Flea infestation was a significant risk factor for B. henselae bacteremia (odds ratio = 2.82, 95% confidence interval, 1.1 to 7.3). Cats >or=13 months old were significantly less likely to be bacteremic than cats 相似文献   

Genetic classification and differentiation of Bartonella species based on comparison of partial ftsZ gene sequences

Currently, 19 species are recognized in the genus Bartonella, 7 of which are involved in an increasing variety of human diseases. Development of molecular tools for detection, identification, and subtyping of strains and isolates has promoted research on Bartonella spp. We amplified and sequenced the portion of the ftsZ gene encoding the N-terminal region of the cell division protein for 13 Bartonella species: Bartonella alsatica, B. birtlesii, B. doshiae, B. elizabethae, B. grahami, B. koehlerae, B. schoenbuchensis, B. taylorii, B. tribocorum, Bartonella vinsonii subsp. arupensis, Bartonella vinsonii subsp. berkhoffii, Bartonella vinsonii subsp. vinsonii, and B. bovis Bermond et al.("B. weissii"). Phylogenetically derived trees revealed four statistically supported groups, indicating that sequencing of the ftsZ gene is a useful tool for identifying evolutionary relationships among Bartonella species. Furthermore, we amplified and sequenced the portion of the ftsZ gene encoding the C-terminal region of the protein for 4 B. bacilliformis isolates, 14 B. clarridgeiae isolates, 14 B. quintana isolates, and 30 B. henselae isolates that were obtained from different geographic regions, hosts, and clinical specimens. B. clarridgeiae and B. quintana sequences were highly conserved, while those of the four B. bacilliformis isolates differed from the type strain at 5 positions. Among B. henselae strains isolated from cats and patients, only two genotypes were detected: Houston and Marseille. Among 80 clinical samples we detected Bartonella spp. in 35 (43.75%) and found the assay to be comparable to that of a combined intergenic-spacer-region- and pap31-based PCR assay. Our results show the usefulness of the portion of the ftsZ gene encoding the C-terminal region for diagnosis of Bartonella infections. More samples should be tested to study its usefulness for epidemiological investigations.     

Rapid identification and differentiation of Bartonella species using a single-step PCR assay

Five species of Bartonella have been reported to infect humans and cause a variety of diseases that can be difficult to diagnose. Four species of Bartonella have been reported to infect cats and dogs, and two of these species are considered to be zoonotic pathogens. Diagnosis of Bartonella infections is hampered by the slow, fastidious growth characteristics of Bartonella species. We report on the development of a single-step PCR-based assay for the detection and differentiation of medically relevant Bartonella species. PCR-mediated amplification of the 16S-23S rRNA intergenic region resulted in a product of a unique size for each Bartonella species, thereby allowing differentiation without the necessity of restriction fragment length polymorphism analysis or sequencing of the amplified product. The ability of the single-step PCR assay to differentiate between Bartonella species was determined with characterized isolates and blood samples from animals known to be infected with either Bartonella henselae, B. clarridgeiae, or B. vinsonii subsp. berkhoffii. The sensitivity of the single-step PCR assay relative to that of in vitro culture was determined with blood samples from B. henselae-infected cats. B. henselae target DNA was amplified from 100% of samples with greater than 50 CFU/ml and 80% of samples with 10 to 30 CFU/ml. The single-step assay described in the report expedites PCR-based detection and differentiation of medically relevant Bartonella species.