Conservation of the 17-Kilodalton Antigen Gene within the Genus Bartonella

The 17-kDa antigen of Bartonella henselae has previously been shown to elicit a strong humoral immune response in patients with cat scratch disease (CSD) and to be useful in screening human serum samples for CSD. In this study, PCR amplification of genes homologous to the 17-kDa antigen gene of B. henselae was performed using genomic DNAs from several species of Bartonella, including the currently recognized human pathogens. Amplicons of similar size were demonstrated using the following chromosomal DNA templates: B. henselae (two strains), B. quintana (two strains), B. elizabethae, B. clarridgeiae, B. vinsonii subsp. vinsonii, and B. vinsonii subsp. berkhoffii. No evidence of a B. bacilliformis homolog of the 17-kDa antigen gene was obtained using multiple primer pairs. DNA sequencing revealed open reading frames capable of coding for proteins with sizes similar to that of the 17-kDa antigen of B. henselae in all of the amplicons; however, extensive sequence divergence across the genus was noted. Cloning of the amplified products into pUC19 resulted in recombinants that directed synthesis of homologs of the 17-kDa protein. Immunoblot analysis using human sera from CSD cases demonstrated very little cross-reactivity among different species for this protein. In contrast, immunoblots using rabbit serum raised to the recombinant B. henselae antigen showed extensive cross-reactivity with the proteins of other Bartonella species. The data suggest that the use of the 17-kDa antigen as a serologic reagent may allow the development of more specific diagnostic assays. Furthermore, the nucleotide sequences from the various versions of the 17-kDa antigen gene should be useful for rapid identification of Bartonella at the species level.     

Bartonella sp. bacteremia in patients with neurological and neurocognitive dysfunction

We detected infection with a Bartonella species (B. henselae or B. vinsonii subsp. berkhoffii) in blood samples from six immunocompetent patients who presented with a chronic neurological or neurocognitive syndrome including seizures, ataxia, memory loss, and/or tremors. Each of these patients had substantial animal contact or recent arthropod exposure as a potential risk factor for Bartonella infection. Additional studies should be performed to clarify the potential role of Bartonella spp. as a cause of chronic neurological and neurocognitive dysfunction.     

Molecular Evidence of Perinatal Transmission of Bartonella vinsonii subsp. berkhoffii and Bartonella henselae to a Child

Bartonella vinsonii subsp. berkhoffii, Bartonella henselae, or DNA of both organisms was amplified and sequenced from blood, enrichment blood cultures, or autopsy tissues from four family members. Historical and microbiological results support perinatal transmission of Bartonella species in this family. It is of clinical relevance that Bartonella spp. may adversely influence human reproductive performance.     

Comparison of In-House and Commercial Slides for Detection by Immunofluorescence of Immunoglobulins G and M against Bartonella henselae and Bartonella quintana

We compared the sensitivities and specificities of indirect fluorescent antibody tests developed in our laboratory and commercially available from Focus Technologies (FT; formerly MRL Diagnostic) for detection of serum antibodies to Bartonella spp. Serum samples tested were from patients with culture- or PCR-confirmed Bartonella quintana or B. henselae infections causing cat scratch disease (CSD), chronic bacteremia, or endocarditis. At a cutoff titer of 64, the FT test had higher sensitivity than our in-house test in detecting anti-B. henselae immunoglobulin G (IgG) antibodies in CSD patients (91.2 versus 52.9%; P < 0.001). The specificity in serum samples from 85 control patients was, however, lower with the FT test (87%) than with the in-house test (98.8%) (P = 0.002). A cutoff titer of 128 improves the specificity for the FT test but lowers the sensitivity to 85%. For patients infected with B. henselae, our in-house test, but not the FT test, enabled endocarditis to be detected more reliably. With the in-house test, titers of IgG against B. henselae of ≥1,024 were found only in endocarditis patients and not in CSD patients. With the FT test, 19.1% of CSD patients had titers of IgG against B. henselae of ≥1,024 (P < 0.001). Our in-house technique also improved detection of anti-B. quintana antibodies in homeless patients with endocarditis. IgG titers of ≥1,024 were present in 75% of serum samples, but only in 16.7% of serum samples with the FT test (P = 0.004). Since each test has advantages over the other, the serological diagnosis of Bartonella infections would benefit if both tests were used concurrently.     

Molecular genetic techniques for typing of Bartonella isolates

Primer sets for PCR detection of four housekeeping genes of Bartonella spp. isolated from clinical material were developed and tested. We tested a strategy of the specific restriction fragment length polymorphism typing of bartonella species using as an example two strains isolated for the first time in Russia from patients with endocarditis and fever of uncertain origin. The results of typing were confirmed by sequencing of the obtained amplicons. The results of sequencing confirmed the affiliation of these isolates to the subspecies B. vinsonii subsp. arupensis. The necessity of molecular epidemiological analysis of bartonellosis in Russia was substantiated.     

Severe Anemia,Panserositis, and Cryptogenic Hepatitis in an HIV Patient Infected with Bartonella henselae

Bartonella spp. constitute emerging pathogens of worldwide distribution. Bacillary angiomatosis is the most frequent skin manifestation of bartonelloses; nevertheless, B. henselae infection should always be considered systemic, especially in immunodeficient individuals. The authors report the case of an AIDS patient with bacillary angiomatosis, who had concurrent severe anemia, hepatitis, peritonitis, pleuritis, and pericarditis. Clinical manifestation, electronic microscopic examination of erythrocytes, and histopathology of a papule biopsy suggested a Bartonella sp. infection. Multiple genes were target by PCR and B. henselae DNA was amplified and sequenced (GenBank accession number EF196804) from the angiomatous papule. Treatment with clarithromycin resulted in resolution of the bacillary angiomatosis, fever, anemia, panserosites, and hepatitis.     

Serodiagnosis of Cat Scratch Disease: Response to Bartonella henselae in Children and a Review of Diagnostic Methods

 In this study, sera from 116 children without clinical evidence of cat scratch disease (CSD) and sera from 19 children with CSD were investigated by two different immunofluorescent assays (test A and test B) to evaluate the seroprevalence of Bartonella henselae antibodies in this population. Antibodies against Bartonella henselae were found in noninfected children only in low titers: 13% and 3.5% of serum samples were positive by test A and test B, respectively. IgG titers as determined by test A ranged from 1 : 64 to 1 : 256, whereas test B did not yield titers  1 1 : 64. Of the 19 children with CSD, 7 (37%) and 5 (26%) had low antibody titers (1 : 64–1 : 256) on admission by test A and test B, respectively. In such cases, IgM antibodies against Bartonella henselae and/or a titer increase in the following weeks are required to prove suspected CSD. The results indicate that the seroprevalence of Bartonella henselae antibodies in children without evidence of CSD is low, and that the results may differ depending on the tests being used. Low antibody titers, however, were found not only in healthy children but also among patients with CSD, which could be indicative of the beginning or of the end of an illness. Currently, only few data are available concerning the seroprevalence of antibodies to Bartonella spp. among children. Although children are often affected by CSD, the specificity of most tests has been evaluated by investigating blood donors, who are usually adults. Several different serological tests are used for the diagnosis of CSD, including the immunofluorescence assay, the enzyme-linked immunosorbent assay, and Western blot (WB) analysis. The sensitivities of different IFAs range from 14 to 100%, depending on the antigen used, the cut-off chosen, and the test procedures. The current diagnostic value of different serological tests for diagnosis of CSD is reviewed.     

Evaluation of an in-house cat scratch disease IgM ELISA to detect <Emphasis Type="Italic">Bartonella henselae</Emphasis> in a routine laboratory setting

Cat scratch disease (CSD) is caused by Bartonella henselae infection and is a common cause of regional lymphadenopathy. The diagnosis of CSD largely depends on serology, but detection of B. henselae in an affected lymph node by PCR is also an important diagnostic tool. We evaluated an IgM in-house ELISA protocol and analyzed its performance in routine CSD serology. Serum samples from PCR-positive patients (n = 126), PCR-negative patients (n = 123), and age-matched controls (n = 126) were used for evaluation. The sensitivity of the IgM ELISA was only 56%, showing that the performance of B. henselae serology under routine laboratory settings is low, probably caused by the wide variability in disease duration in patients suspected of CSD whose samples were submitted to our laboratory. Most patients (46%) with a positive IgM response were between 0 and 20 years of age. We conclude that the serodiagnosis of B. henselae is hampered by the low sensitivity and specificity of the assays when used in a routine laboratory setting. For this reason, a negative IgM or PCR result can never exclude CSD, especially with late sample collection.     

Detection by Immunofluorescence Assay of Bartonella henselae in Lymph Nodes from Patients with Cat Scratch Disease

Laboratory diagnosis of Bartonella henselae infections can be accomplished by serology or PCR assay on biopsy samples. The purpose of our work was to assess immunofluorescence detection (IFD) in lymph node smears using a specific monoclonal antibody directed against B. henselae and a commercial serology assay (IFA) compared with PCR detection. Among 200 lymph nodes examined from immunocompetent patients, 54 were positive for B. henselae by PCR, of which 43 were also positive by IFD. Among the 146 PCR-negative lymph nodes, 11 were positive by IFD. Based on PCR results, the specificity of this new technique was 92.5%, the sensitivity was 79.6%, and the positive predictive value was 79.6%. At a cutoff titer of 64, the sensitivity of the IFA was 86.8% and the specificity was 74.1%. Diagnosis of cat scratch disease (CSD) may be improved, with a specificity of 100%, when the two tests (IFD and IFA) were negative; the sensitivity was 97.4% if one of the two tests was positive. Since PCR-based detection with biopsy samples is available only in reference laboratories, we suggest using IFD coupled with the commercial serology test for the diagnosis of CSD.     

Bartonella henselae Infects Human Erythrocytes

Bartonella henselae, a facultative intracellular bacterium, has been known as the agent of cat scratch disease, bacillary angiomatosis, peliosis hepatis, endocarditis, and bacteremic syndrome in humans. Bartonella species can cause intraerythrocytic infections and have been isolated from the bloodstream of patients by several methods. It was demonstrated that B. bacilliformis and B. quintana infect human endothelial cells and human erythrocytes and B. henselae infects erythrocytes of cats. The aim of this study was to investigate through transmission electron microscopy whether B. henselae infects mature human erythrocytes. One red blood cell (RBC) unit received an experimentally standard strain of B. henselae. Blood aliquots were collected from the infected unit immediately after inoculation, at 30 min and 1, 5, 10, and 72 h for ultrastructural evaluation. B. henselae was seen adhering to human erythrocytes 10 h after inoculation and inside the erythrocyte after 72 h. This study demonstrates that B. henselae adheres to and invades mature human erythrocytes. The results favor the possibility that erythrocytes can serve as a primary target in Bartonella spp. infections. From this observation, further studies are warranted to prevent Bartonella spp. transfusional transmission.     

Occurrence of Bartonella henselae and Borrelia burgdorferi sensu lato co-infections in ticks collected from humans in Germany

Bartonella (B.) henselae is the zoonotic agent of cat scratch disease. B. henselae has been associated with therapy-resistant Lyme disease in humans suggesting that B. henselae and Borrelia burgdorferi sensu lato might be transmitted concurrently by ticks. In the present study we found that 16 (6.9%) of 230 Ixodes ricinus collected from humans harboured DNA of Bartonella spp. Fifteen positive ticks were infected with B. henselae and one tick with B. clarridgeiae. Twenty-five percent of the 16 Bartonella positive ticks were co-infected with Borrelia burgdorferi sensu lato. Our data show that B. henselae is present in Ixodes ricinus and that ticks may serve as source of infection for humans.     

Value of Microimmunofluorescence for Diagnosis and Follow-up of Bartonella Endocarditis

Bartonella endocarditis is a disease of emerging importance that causes serious complications and high rates of mortality. Due to the fastidious nature of Bartonella species and their high degrees of antibiotic susceptibility, cultures of clinical samples most often remain sterile and valvular biopsy specimens, the best specimens for PCR amplification, are seldom available. Therefore, serology appears to be the easiest diagnostic tool. In order to determine the best cutoff value for serology and its predictive values for the detection of Bartonella endocarditis, we studied 48 patients with culture- and/or PCR-confirmed Bartonella endocarditis. We also applied these serological criteria to 156 patients with blood culture-negative endocarditis. Furthermore, we compared the kinetics of the antibody responses to Bartonella spp. in order to estimate the value of serology for prediction of the occurrence of relapses. A titer of ≥1:800 for immunoglobulin G antibodies to either Bartonella henselae or B. quintana has a positive predictive value of 0.810 for the detection of chronic Bartonella infections in the general population and a value of 0.955 for the detection of Bartonella infections among patients with endocarditis. When this cutoff was applied to 156 patients with blood culture-negative endocarditis, we were able to diagnose Bartonella infections in an additional 45 patients with definite endocarditis for whom a positive Bartonella serology was the only evidence of infection. On follow-up, the kinetics of the decrease in antibody titers were significantly delayed in two patients with relapses. In conclusion, we recommend the determination of antibodies to both B. quintana and B. henselae and the use of a cutoff value of 1:800 for the diagnosis of Bartonella endocarditis. We propose that this criterion, which may also help with the detection of late relapses, be included as a major criterion in the Duke criteria for the diagnosis of infective endocarditis.     

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.     

Comparative microbiological features of Bartonella henselae infection in a dog with fever of unknown origin and granulomatous lymphadenitis

We report the first documented case of Bartonella henselae infection in a dog from France and the first isolation of B. henselae from a dog with fever of unknown origin. This observation contributes to the “One Health” concept focusing on zoonotic pathogens emerging from companion animals. A 1-year-old female German shepherd dog was referred for evaluation of fever of unknown origin of 1 month duration. Diagnostic investigations confirmed diffuse pyogranulomatous lymphadenitis. The dog became afebrile, and lymph node size normalized in response to a 6-week course of doxycycline. Retrospectively, Bartonella DNA was amplified from an EDTA-anticoagulated blood sample obtained before antimicrobial therapy, with the gtlA fragment sharing 99 % identity with the 350-bp gtlA fragment of the B. henselae Houston-1 strain. The same strain was isolated in the blood of three healthy cats from the household. Two months after discontinuation of doxycycline, the dog experienced a febrile relapse. Bartonella DNA was again amplified from blood prior to and immediately after administration of a 6-week course azithromycin therapy. However, without administration of additional medications, PCR was negative 9 months after azithromycin therapy and the dog remains clinically healthy 12 months following the second course of antibiotics. The medical management of this case raises several clinically relevant comparative infectious disease issues, including the extent to which Bartonella spp. contribute to fever of unknown origin and pyogranulomatous inflammatory diseases in dogs and humans, and the potential of doxycycline and azithromycin treatment failures. The possibility that dogs could constitute an underestimated reservoir for B. henselae transmission to people is also discussed.     

IgM to Bartonella henselae in cat-scratch disease and during acute Epstein-Barr virus infection

The diagnostic value of IgM to Bartonella henselae was evaluated in 20 children with cat-scratch disease (CSD) and controls consisting of 20 blood donors and 20 children with enlarged lymph nodes without CSD by two indirect immunofluorescence assays (IFA). One was based on B. henselae cocultivated with Vero cells (host cell-associated IFA), and the other on B. henselae grown on agar (host cell-free IFA). With the host cell-associated IFA, 18 of 20 children with CSD revealed IgM, whereas only 14 did so with the host cell-free IFA. Sera of two blood donors as well as sera from three children with enlarged lymph nodes without CSD showed also positive IgM to cell-associated B. henselae. This study reveals that the IFA applied had sensitivities of 70 – 90% and specificities of 87.5 – 100% for detecting IgM to B. henselae. Additionally, 20 patients with IgM to Epstein-Barr virus (EBV) capsid antigen were tested for IgM to B. henselae. Sera of 16 and 9 of these patients revealed IgM to B. henselae with the host cell-associated and the host cell-free IFA, respectively. Using Western blot these sera were demonstrated to react against linearized proteins of Vero cells and of B. henselae. Thus, since acute EBV infection may substantially reduce the specificity of B. henselae-specific IgM tests, we conclude that diagnosis of CSD should be confirmed by a significant IgG titer to B. henselae or by detection of this pathogen. Received: 7 May 1997     

Cat Scratch Disease Due to Bartonella henselae Infection Mimicking Parotid Malignancy

An unusual Bartonella henselae infection presenting clinically as a putative parotid cancer was diagnosed based on serological tests, histomorphology and amplification of a 16S-rDNA sequence of Bartonella henselae. The patient improved greatly upon antibiotic treatment and did not require surgery. Although uncommon, infection with Bartonella spp., particularly Bartonella henselae, should be included in the differential diagnosis of parotid tumors. Electronic Publication     

Seroprevalence of Antibodies to Bartonella henselae in Patients with Cat Scratch Disease and in Healthy Controls: Evaluation and Comparison of Two Commercial Serological Tests

Serologic testing for the presence of antibodies to Bartonella henselae is a widely accepted diagnostic procedure for laboratory confirmation of the diagnosis of cat scratch disease (CSD). In this study a commercially available indirect immunofluorescence assay (IFA) based on B. henselae-infected human larynx carcinoma cells (test A) was evaluated. Sera from 42 patients with CSD (20 confirmed by PCR) and 270 sera from healthy controls (consisting of 63 cat owners, 65 individuals whose last close contact with cats was >6 months previously, and 142 persons who had never been exposed to cats) were investigated for antibodies to B. henselae. All patients with CSD had titers of immunoglobulin G (IgG) to B. henselae of 128 or higher (test A; sensitivity, 100%). Of the 270 controls 189 (70%) were seronegative (titer, <64), 38 (14.1%) had titers of 64, 30 (11.1%) had titers of 128, 9 (3.3%) had titers of 256, and 4 (1.5%) had high titers, 512 (test A; specificity, 70%). Of the cat owners and individuals who had never had close contact with cats, 71.4 and 71.12%, respectively, were seronegative, and titers of 64, 128, 256, and 512 were found in 14.3 and 16.2%, 1.6 and 10.5%, 9.5 and 0.7%, and 3.2 and 1.4%, respectively. The sera from the patients and from the first 100 healthy adults without a history of close contact with cats were additionally tested with a second commercially available IFA, based on Vero cells infected with B. henselae and Bartonella quintana (test B). The sensitivity and specificity of test B were 93 and 73%, respectively. For patients with CSD the cross-reactivity between B. henselae and B. quintana in this test was 95%. Both systems are highly sensitive but less specific for detection of IgG antibodies to B. henselae in samples from patients with clinically apparent CSD. For detection of IgM antibodies, test A seems to be more sensitive (88%) and more specific (95%) than test B (sensitivity and specificity of 64 and 86%, respectively). The data show that the seroprevalence of antibodies to B. henselae in German individuals is high (30%). Low antibody levels are not sufficient evidence of active or prior infection.     

Prevalence of Bartonella henselae and Bartonella clarridgeiae in an Urban Indonesian Cat Population

We studied evidence of Bartonella henselae and Bartonella clarridgeiae infection in 54 cats living in Jakarta, Indonesia. By using an indirect immunofluorescence assay, we found immunoglobulin G antibody to B. henselae in 40 of 74 cats (54%). The blood of 14 feral cats was cultured on rabbit blood agar plates for 28 days. Bartonella-like colonies were identified as B. henselae or B. clarridgeiae by using restriction fragment length polymorphism analysis and direct sequencing of the PCR amplicons. Of the cats sampled in the study, 6 of 14 (43%; all feral) were culture positive for B. henselae; 3 of 14 (21%; 2 feral and 1 pet) culture positive for B. clarridgeiae. This is the first report that documents B. henselae and B. clarridgeiae infections in Indonesian cats.     

Development of a nested PCR method targeting a unique multicopy element, ISMap02, for detection of Mycobacterium avium subsp. paratuberculosis in fecal samples

This study describes the development of a nested PCR assay that uses a unique element (ISMap02) for Mycobacterium avium subsp. paratuberculosis that is present at six copies within the genome. In addition, the sensitivity of the assay with this element was compared to the sensitivity of detection of the IS900 element in both conventional and real-time PCR assays. The specificity of the ISMap02 element was evaluated by PCR of the DNA extracted from isolates of M. avium subsp. paratuberculosis and M. avium subsp. avium, as well as DNA from M. fortuitum, M. scofulaceum, M. phlei, M. smegmatis, and M. gordonae. Only M. avium subsp. paratuberculosis DNA was detectable after amplification with the ISMap02 primers. The sensitivity of detection for the ISMap02 element in either a conventional or a real-time PCR format was less than 100 fg DNA or 10(2) CFU/ml in serial titration curves with pure bacteria. These results were comparable to those obtained for the IS900 element. Experimental spiking of a negative fecal sample followed by M. avium subsp. paratuberculosis DNA extraction resulted in detection thresholds of 10(2) CFU/g for the IS900 element and 10(3) CFU/g for the ISMap02 element by using a real-time PCR format, but this sensitivity dropped 10-fold for both elements in a conventional PCR format. Analyses of fecal samples obtained from naturally infected animals demonstrated a sensitivity for the detection of M. avium subsp. paratuberculosis DNA by use of the ISMap02 element similar to that achieved by use of the IS900 element when it was used in a conventional PCR format. The real-time PCR format improved the levels of detection of both elements, but not to a significant degree. In conclusion, the ISMap02 element provides a very sensitive and specific alternative as a diagnostic reagent for use in PCR assays for the detection of paratuberculosis.