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.     

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.     

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.     

Production of Bartonella Genus-Specific Monoclonal Antibodies

Monoclonal antibodies (MAbs) which react with heat-resistant proteins with molecular masses of 32 to 33 kDa of 14 different Bartonella species were produced. These antibodies did not react with antigens of 26 diverse bacterial strains by microimmunofluorescence assay except MAb B3D4, which reacted with Chlamydia psittaci and Chlamydia trachomatis at low titers. The identification of a common Bartonella antigenic protein will make it possible to later produce a diagnostic antigen by cloning and expressing it in Escherichia coli. Moreover, these MAbs allow all Bartonella species to be identified to the genus level.     

Molecular method for Bartonella species identification in clinical and environmental samples

A new, efficient molecular method for detection of Bartonella, based on the 16S-23S rRNA intergenic spacer and 16S rRNA amplification by multiplex PCR combined with reverse line blotting, was designed. This assay could simultaneously detect 20 different known species and other Bartonella species not described previously.     

Identification of Brucella by Ribosomal-Spacer-Region PCR and Differentiation of Brucella canis from Other Brucella spp. Pathogenic for Humans by Carbohydrate Profiles

Molecular and chemical characteristics often provide complementary information in the differentiation of closely related organisms. The genus Brucella consists of a highly conserved group of organisms. Identification of the four species pathogenic in humans (Brucella melitensis, Brucella abortus, Brucella suis, and Brucella canis) is problematic for many clinical laboratories that depend primarily on serology and phenotypic characteristics to differentiate species. PCR amplification of the 16S-23S ribosomal DNA interspace region was evaluated for species-specific polymorphism. B. abortus, B. melitensis, B. suis, and B. canis produced identical PCR interspace profiles. However, these PCR products were unique to brucellae, allowing them to be readily distinguished from other gram-negative bacteria (including Bartonella spp. and Agrobacterium spp.). Carbohydrate profiles differentiated B. canis from the other three Brucella species due to the absence of the rare amino sugar quinovosamine in the three other species. PCR of the rRNA interspace region is useful in identification of the genus Brucella, while carbohydrate profiling is capable of differentiating B. canis from the other Brucella species.     

Identification of Bartonella-Specific Immunodominant Antigens Recognized by the Feline Humoral Immune System

The seroreactivities of both naturally and experimentally infected cats to Bartonella henselae was examined. Serum samples collected weekly from nine cats experimentally infected with B. henselae LSU16 were tested by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. The magnitude and isotype of the antibody response were investigated by ELISA. Western blot analysis allowed the identification of at least 24 Bartonella-specific antigens recognized by the cats during infection. Antibody titers to specific antigens, as determined by Western blot analysis, ranged from 10 to 640 and varied among the different antibody-antigen interactions. Absorption of sera from an experimentally infected cat, using whole cells and cell lysates of various Bartonella species and other bacteria that commonly colonize cats, supported the identification of those Bartonella-specific antigens recognized by the experimentally infected cats. Furthermore, a number of possible species- and type-specific antigens were identified. Finally, sera obtained from cats at local animal shelters were screened for the presence of antibodies directed against the Bartonella-specific bands identified in the experimentally infected cats. A number of Bartonella-specific antigens have been identified to which strong antibody responses are generated in both experimentally and naturally infected cats, some of which may be useful in diagnosing species- and/or type-specific infections. In addition, the results from these experiments will lead to the development of monoclonal antibodies targeted against those genus-, species-, and type-specific antigens.     

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.     

Use of the Cell Division Protein FtsZ as a Means of Differentiating among Bartonella Species

Genes coding for homologs of the highly conserved cell division protein FtsZ were isolated from Bartonella henselae and Bartonella quintana, the causative agents of cat scratch disease and trench fever, respectively. DNA fragments coding for the ftsZ open reading frames (ORFs) were cloned into Escherichia coli following PCR amplification with primers based on the ftsZ sequence of the closely related species Bartonella bacilliformis. The amino acid sequences predicted from the cloned B. henselae and B. quintana ftsZ ORFs are 81 to 83% identical to the corresponding protein in B. bacilliformis. Like the FtsZ protein of B. bacilliformis, the B. henselae and B. quintana homologs are about twice as large as the FtsZ proteins reported in most other organisms. Localized sequence differences within the C-terminal coding regions of the Bartonella ftsZ genes were used as the basis for species-specific identification of these organisms at both the DNA and protein levels. Oligonucleotide primers which permit the amplification of an ftsZ fragment from each of the Bartonella species without amplifying DNA from the other two species were designed. Anti-FtsZ antisera raised in rabbits against synthetic peptides corresponding to the relatively divergent C-terminal regions were shown via Western blot analysis to react only with the FtsZ protein from the cognate Bartonella species. These observations raise the possibility that the differences in ftsZ sequences can be used as the basis for diagnostic tests to differentiate among these closely related pathogens.     

Western Immunoblotting for Bartonella Endocarditis

To differentiate infectious endocarditis (IE) from other Bartonella infections and to identify infecting Bartonella bacteria at the species level on a serological basis, we used Western immunoblotting to test sera from 51 patients with Bartonella IE (of which 27 had previously benefited from species identification by molecular techniques), 11 patients with chronic Bartonella quintana bacteremia, and 10 patients with cat scratch disease. Patients with IE were Western blot positive in 49 of 51 cases, and significant cross-reactivity with three heterologous Bartonella antigens was found in 45 of 49 cases. Sera from bacteremic patients did not react with more than one heterologous antigen, and sera from patients with cat scratch disease gave negative results. Sera reacted only with B. henselae in four cases of IE, including one with a positive PCR result for valve tissue. Western blot and cross-adsorption performed on serum samples from patients with IE (the identity of the causative species having been determined by PCR) were demonstrated to identify efficiently the causative species in all cases. When applied to patients diagnosed on the basis of serological tests only, this technique allowed identification of the causative species in 20 of 22 cases. The results were in accordance with epidemiological features. Six reactive bands of B. quintana (of molecular sizes from 10 to 83 kDa) demonstrated significant association with sera from patients with B. quintana endocarditis. Overall, Western blotting and cross-adsorption made it possible to identify the causative species in 49 of 51 (96%) IE cases.     

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.     

Small Indian mongooses and masked palm civets serve as new reservoirs of Bartonella henselae and potential sources of infection for humans

The prevalence and genetic properties of Bartonella species were investigated in small Indian mongooses and masked palm civets in Japan. Bartonella henselae, the causative agent of cat-scratch disease (CSD) was isolated from 15.9% (10/63) of the mongooses and 2.0% (1/50) of the masked palm civets, respectively. The bacteraemic level ranged from 3.0 × 10¹ to 8.9 × 10³ CFU/mL in mongooses and was 7.0 × 10³ CFU/mL in the masked palm civet. Multispacer typing (MST) analysis based on nine intergenic spacers resulted in the detection of five MST genotypes (MSTs 8, 14, 37, 58 and 59) for the isolates, which grouped in lineage 1 with MST genotypes of isolates from all CSD patients and most of the cats in Japan. It was also found that MST14 from the mongoose strains was the predominant genotype of cat and human strains. This is the first report on the isolation of B. henselae from small Indian mongooses and masked palm civets. The data obtained in the present study suggest that these animals serve as new reservoirs for B. henselae, and may play a role as potential sources of human infection.     

Wild ruminants in the area of the North-Western Poland as potential reservoir hosts of <Emphasis Type="Italic">Bartonella schoenbuchensis</Emphasis> and <Emphasis Type="Italic">B. bovis</Emphasis>

The aim of this work was to examine if the game species from the north-western Poland, roe deer (Capreolus capreolus), red deer (Cervus elaphus) and wild boar (Sus scrofa), may be reservoir hosts of bacteria from the genus Bartonella, and whether the sheep tick (Ixodes ricinus) is their vector. To this end, the prevalence of Bartonella DNA in the tissues of these game species was measured, just as in sheep ticks (I. ricinus) infesting them, and ticks collected from plants in the hunting area. The prevalence of Bartonella DNA was 39% (23/59) in roe deer and 35% (7/20) in red deer. No Bartonella DNA was detected in any of the 21 wild boars. The presence of Bartonella DNAwas detected in 1.9% of ticks infesting roe deer (2/103), while no pathogen DNA was found in the 20 ticks infesting the red deer and the 3 ticks infesting wild boars, or the 200 ticks collected from plants. Amplicons of two different lengths were obtained; 198 bp, characteristic for B. bovis, and 317 bp, characteristic for B. schoenbuchensis, which were confirmed later by sequencing. The examined ruminants are probably the reservoir hosts of B. schoenbuchensis and B. bovis in the biotope of the Puszcza Wkrzańska Forest, and wild boars do not participate in the Bartonella propagation in the environment. I. ricinus is unlikely to be the main vector of Bartonella species detected in the examined roe deer and red deer; probably other bloodsucking arthropods, parasitizing wild ruminants, play this role.     

Immune Aspects of Bartonella

Bartonella species have been recognized as important human pathogens only recently. Until the early 1990s, this genus was represented by one species, Bartonella bacilliformis. The recent identification of other Bartonella species as the agents of cat-scratch disease and bacillary angiomatosis has left little doubt of their emerging importance as opportunistic human pathogens. Over the last decade, extensive research has been performed on Bartonella species, resulting in an explosion in our knowledge of the genetic diversity of this genus. Unusual aspects of disease sequelae have fueled worldwide interest in defining the natural history, pathology, and molecular biology of Bartonella species. While much information about these interests has been presented, the advancement of immunological knowledge regarding Bartonella species has been slow. This review discusses immunological data on Bartonella species, focusing on the three primary human pathogens of this genus: B. bacilliformis, B. quintana, and B. henselae.     

Association of Bartonella spp bacteremia with Chagas cardiomyopathy,endocarditis and arrythmias in patients from South America

Infection with Bartonella spp may cause cardiac arrhythmias, myocarditis and endocarditis in humans. The aim of the present study was to evaluate a possible association between Bartonella spp bacteremia and endocarditis, arrhythmia and Chagas cardiomyopathy in patients from Brazil and Argentina. We screened for the presence of bacterial 16S rRNA in human blood by PCR using oligonucleotides to amplify a 185-bp bacterial DNA fragment. Blood samples were taken from four groups of subjects in Brazil and Argentina: i) control patients without clinical disease, ii) patients with negative blood-culture endocarditis, iii) patients with arrhythmias, and iv) patients with chronic Chagas cardiomyopathy. PCR products were analyzed on 1.5% agarose gel to visualize the 185-bp fragment and then sequenced to confirm the identity of DNA. Sixty of 148 patients (40.5%) with cardiac disease and 1 of 56 subjects (1.8%) from the control group presented positive PCR amplification for Bartonella spp, suggesting a positive association of the bacteria with these diseases. Separate analysis of the four groups showed that the risk of a Brazilian patient with endocarditis being infected with Bartonella was 22 times higher than in the controls. In arrhythmic patients, the prevalence of infection was 45 times higher when compared to the same controls and 40 times higher for patients with Chagas cardiomyopathy. To the best of our knowledge this is the first report of the association between Bartonella spp bacteremia and Chagas disease. The present data may be useful for epidemiological and prevention studies in Brazil and Argentina.     

Interaction of bacteria of the genus Bartonella with host cells: Inhibition of apoptosis, induction of proliferation, and oncogenesis

Bacteria of the genus Bartonella are responsible for many previously undetected or recurrent human diseases whose harmfulness increases due to an increasing number of immunocompromised persons. As intracellular parasites, Bartonella species colonize endothelial cells of human and animal vascular tissues and erythrocytes.     

Matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of nonfermenting gram-negative bacilli isolated from cystic fibrosis patients

The identification of nonfermenting gram-negative bacilli isolated from cystic fibrosis (CF) patients is usually achieved by using phenotype-based techniques and eventually molecular tools. These techniques remain time-consuming, expensive, and technically demanding. We used a method based on matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) for the identification of these bacteria. A set of reference strains belonging to 58 species of clinically relevant nonfermenting gram-negative bacilli was used. To identify peaks discriminating between these various species, the profile of 10 isolated colonies obtained from 10 different passages was analyzed for each referenced strain. Conserved peaks with a relative intensity greater than 0.1 were retained. The spectra of 559 clinical isolates were then compared to that of each of the 58 reference strains as follows: 400 Pseudomonas aeruginosa, 54 Achromobacter xylosoxidans, 32 Stenotrophomonas maltophilia, 52 Burkholderia cepacia complex (BCC), 1 Burkholderia gladioli, 14 Ralstonia mannitolilytica, 2 Ralstonia pickettii, 1 Bordetella hinzii, 1 Inquilinus limosus, 1 Cupriavidus respiraculi, and 1 Burkholderia thailandensis. Using this database, 549 strains were correctly identified. Nine BCC strains and one R. mannnitolilytica strain were identified as belonging to the appropriate genus but not the correct species. We subsequently engineered BCC- and Ralstonia-specific databases using additional reference strains. Using these databases, correct identification for these species increased from 83 to 98% and from 94 to 100% of cases, respectively. Altogether, these data demonstrate that, in CF patients, MALDI-TOF-MS is a powerful tool for rapid identification of nonfermenting gram-negative bacilli.     

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.     

Bartonella quintana Aortitis in a Man with AIDS,Diagnosed by Needle Biopsy and 16S rRNA Gene Amplification

A man with newly diagnosed AIDS presented with months of back pain and fever. Computed tomography (CT) results demonstrated aortitis with periaortic tissue thickening. DNA amplification of biopsy tissue revealed Bartonella quintana, and Bartonella serologies were subsequently noted to be positive. The patient improved with prolonged doxycycline and rifabutin treatment. This case illustrates how molecular techniques are increasingly important in diagnosing Bartonella infections.     

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.