Molecular characterization of Bartonella vinsonii subsp. berkhoffii genotype III

The molecular characterization of a Bartonella vinsonii subsp. berkhoffii genotype III strain (NCSU strain 06-CO1) isolated from the blood of a military working dog diagnosed with endocarditis is reported in this study. Several genes were amplified and sequenced for comparative sequence similarity with other strains.     

Bartonella vinsonii subsp. berkhoffii and Bartonella henselae as potential causes of proliferative vascular diseases in animals

Bartonella species are highly fastidious, vector borne, zoonotic bacteria that cause persistent intraerythrocytic bacteremia and endotheliotropic infection in reservoir and incidental hosts. Based upon prior in vitro research, three Bartonella sp., B. bacilliformis, B. henselae, and B. quintana can induce proliferation of endothelial cells, and each species has been associated with in vivo formation of vasoproliferative tumors in human patients. In this study, we report the molecular detection of B. vinsonii subsp. berkhoffii, B. henselae, B. koehlerae, or DNA of two of these Bartonella species simultaneously in vasoproliferative hemangiopericytomas from a dog, a horse, and a red wolf and in systemic reactive angioendotheliomatosis lesions from cats and a steer. In addition, we provide documentation that B. vinsonii subsp. berkhoffii infections induce activation of hypoxia inducible factor-1 and production of vascular endothelial growth factor, thereby providing mechanistic evidence as to how these bacteria could contribute to the development of vasoproliferative lesions. Based upon these results, we suggest that a fourth species, B. vinsonii subsp. berkhoffii, should be added to the list of bartonellae that can induce vasoproliferative lesions and that infection with one or more Bartonella sp. may contribute to the pathogenesis of systemic reactive angioendotheliomatosis and hemangiopericytomas in animals.     

Molecular characterization of the sucB gene encoding the immunogenic dihydrolipoamide succinyltransferase protein of Bartonella vinsonii subsp. berkhoffii and Bartonella quintana

Members of the genus Bartonella have historically been connected with human disease, such as cat scratch disease, trench fever, and Carrion's disease, and recently have been recognized as emerging pathogens causing other clinical manifestations in humans. However, because little is known about the antigens that elicit antibody production in response to Bartonella infections, this project was undertaken to identify and molecularly characterize these immunogens. Immunologic screening of a Bartonella vinsonii subsp. berkhoffii genomic expression library with anti-Bartonella antibodies led to the identification of the sucB gene, which encodes the enzyme dihydrolipoamide succinyltransferase. Antiserum from a mouse experimentally infected with live Bartonella was reactive against recombinant SucB, indicating the mounting of an anti-SucB response following infection. Antigenic cross-reactivity was observed with antiserum against other Bartonella spp. Antibodies against Coxiella burnetti, Francisella tularensis, and Rickettsia typhi also reacted with our recombinant Bartonella SucB. Potential SucB antigenic cross-reactivity presents a challenge to the development of serodiagnostic tests for other intracellular pathogens that cause diseases such as Q fever, rickettsioses, brucelloses, tularemia, and other bartonelloses.     

Bartonella vinsonii subsp. berkhoffii as an agent of afebrile blood culture-negative endocarditis in a human

We report a case of endocarditis in a human infected with Bartonella vinsonii subsp. berkhoffii, which causes bacteremia and endocarditis in dogs. Bacterial identification was established by PCR amplification and sequencing of an intergenic spacer region (ITS1), 16S ribosomal DNA, and a gene encoding citrate synthase (gltA). Bartonella antibodies were detected by immunofluorescence.     

Isolation of Bartonella vinsonii subsp. berkhoffii Genotype II from a Boy with Epithelioid Hemangioendothelioma and a Dog with Hemangiopericytoma

In this report, we describe isolation of Bartonella vinsonii subsp. berkhoffii genotype II from a boy with epithelioid hemangioendothelioma and a dog with hemangiopericytoma. These results suggest that B. vinsonii subsp. berkhoffii may cause vasoproliferative lesions in both humans and dogs.     

Bartonella quintana and Bartonella vinsonii subsp. vinsonii bloodstream co-infection in a girl from North Carolina,USA

Medical Microbiology and Immunology - The genus Bartonella consists of globally distributed and highly diverse alpha-proteobacteria that infect a wide-range of mammals. Medically, Bartonella spp....     

Coyotes (Canis latrans) as the reservoir for a human pathogenic Bartonella sp.: molecular epidemiology of Bartonella vinsonii subsp. berkhoffii infection in coyotes from central coastal California

Bartonella vinsonii subsp. berkhoffii was originally isolated from a dog suffering infectious endocarditis and was recently identified as a zoonotic agent causing human endocarditis. Following the coyote bite of a child who developed clinical signs compatible with Bartonella infection in Santa Clara County, Calif., this epidemiological study was conducted. Among 109 coyotes (Canis latrans) from central coastal California, 31 animals (28%) were found to be bacteremic with B. vinsonii subsp. berkhoffii and 83 animals (76%) had B. vinsonii subsp. berkhoffii antibodies. These findings suggest these animals could be the wildlife reservoir of B. vinsonii subsp. berkhoffii. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the gltA and 16S rRNA genes for these 31 isolates yielded similar profiles that were identical to those of B. vinsonii subsp. berkhoffii. Partial sequencing of the gltA and 16S rRNA genes, respectively, indicated 99.5 and 100% homology between the coyote isolate and B. vinsonii subsp. berkhoffii (ATCC 51672). PCR-RFLP analysis of the 16S-23S intergenic spacer region showed the existence of two different strain profiles, as has been reported in dogs. Six (19%) of 31 Bartonella bacteremic coyotes exhibited the strain profile that was identified in the type strain of a canine endocarditis case (B. vinsonii subsp. berkhoffii ATCC 51672). The other 25 bacteremic coyotes were infected with a strain that was similar to the strains isolated from healthy dogs. Based on whole bacterial genome analysis by pulsed-field gel electrophoresis (PFGE) with SmaI restriction endonuclease, there was more diversity in fingerprints for the coyote isolates, which had at least 10 major variants compared to the two variants described for domestic dog isolates from the eastern United States. By PFGE analysis, three Bartonella bacteremic coyotes were infected by a strain identical to the one isolated from three healthy dog carriers. Further studies are necessary to elucidate the mode of transmission of B. vinsonii subsp. berkhoffii, especially to identify potential vectors, and to determine how humans become infected.     

Bartonella vinsonii subsp. berkhoffii and Related Members of the Alpha Subdivision of the Proteobacteria in Dogs with Cardiac Arrhythmias, Endocarditis, or Myocarditis

Cardiac arrhythmias, endocarditis, or myocarditis was identified in 12 dogs, of which 11 were seroreactive to Bartonella vinsonii subspecies berkhoffii antigens. Historical abnormalities were highly variable but frequently included substantial weight loss, syncope, collapse, or sudden death. Fever was an infrequently detected abnormality. Cardiac disease was diagnosed following an illness of short duration in most dogs, but a protracted illness of at least 6 months' duration was reported for four dogs. Valvular endocarditis was diagnosed echocardiographically or histologically in eight dogs, two of which also had moderate to severe multifocal myocarditis. Four dogs lacking definitive evidence of endocarditis were included because of seroreactivity to B. vinsonii antigens and uncharacterized heart murmurs and/or arrhythmias. Alpha proteobacteria were not isolated from the blood by either conventional or lysis centrifugation blood culture techniques. Using PCR amplification and DNA sequencing of a portion of the 16S rRNA gene, B. vinsonii was identified in the blood or heart valves of three dogs. DNA sequence alignment of PCR amplicons derived from blood or tissue samples from seven dogs clustered among members of the alpha subdivision of the Proteobacteria and suggested the possibility of involvement of one or more alpha proteobacteria; however, because of the limited quantity of sequence, the genus could not be identified. Serologic or molecular evidence of coinfection with tick-transmitted pathogens, including Ehrlichia canis, Babesia canis, Babesia gibsonii, or spotted fever group rickettsiae, was obtained for seven dogs. We conclude that B. vinsonii subsp. berkhoffii and closely related species of alpha proteobacteria are an important, previously unrecognized cause of arrhythmias, endocarditis, myocarditis, syncope, and sudden death in dogs.     

Bartonella vinsonii subsp. arupensis as an agent of blood culture-negative endocarditis in a human

We report the case of a patient hospitalized with endocarditis. The etiological diagnosis of Bartonella was suggested by detection of high titers of antibodies by immunofluorescence and Western blotting. Two different nested PCRs performed on sera identified Bartonella vinsonii subsp. arupensis by sequencing.     

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.     

Intraerythrocytic presence of Bartonella henselae.

Recent reports in the medical literature emphasize the risk of zoonotic disease and the high degree of prevalence of asymptomatic feline infection with Bartonella (Rochalimaea) henselae. While investigating Bartonella bacteremia in cats, we used transmission electron microscopy to demonstrate B. henselae in the erythrocytes of persistently bacteremic cats.     

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.     

Bartonella henselae and Bartonella quintana adherence to and entry into cultured human epithelial cells.

Bartonella henselae expresses pili phenotypically similar to type 4 pili. B. henselae pilus expression undergoes phase variation with multiple passages. Low-passage-number, piliated B. henselae adhered to and invaded HEp-2 cells to a greater extent than did multiply passaged B. henselae with reduced pilus expression. Pili may be a pathogenic determinant for Bartonella species.     

Serological cross-reactions between Bartonella quintana, Bartonella henselae, and Coxiella burnetii.

The clinical manifestations of Q fever and bartonelloses can be confused, especially in cases of infectious endocarditis. Differential diagnosis of the diseases is important because the treatments required for Q fever and bartonelloses are different. Laboratory confirmation of a suspected case of either Q fever or bartonelloses is most commonly made by antibody estimation with an indirect immunofluorescence assay. With an indirect immunofluorescence assay, 258 serum samples from patients with Q fever were tested against Bartonella henselae and Bartonella quintana antigens, and 77 serum samples from patients with infection by Bartonella sp. were tested against Coxiella burnetii antigen. Cross-reactivity was observed: more than 50% of the chronic Q fever patients tested had antibodies which reacted against B. henselae antigen to a significant level. This cross-reaction was confirmed by a cross-adsorption study and protein immunoblotting. However, because the levels of specific antibody titers in cases of Bartonella endocarditis are typically extremely high, low-level cross-reaction between C. burnetii antibodies and B. henselae antigen in cases of Q fever endocarditis should not lead to misdiagnosis, provided serology testing for both agents is performed.     

Seroprevalence of Bartonella henselae in cats in Germany.

Bartonella henselae and B. quintana infections in man are associated with various clinical manifestations including cat-scratch disease, bacillary angiomatosis and bacteraemia. While cats are the natural reservoir for B. henselae, the source of B. quintana is unclear. In this study, the sera of 713 cats from Germany were examined for the presence of antibodies against B. henselae, B. quintana or Afipia felis by an indirect immunofluorescence assay (IFA). Bartonella-specific antibody titres of > or =50 were found in 15.0% of the cats. There was substantial cross-reactivity among the various Bartonella antigens, although single sera showed high titres against B. henselae but not against B. quintana and vice versa. Antibodies against A. felis were not detected in any of these cats. Statistical analysis indicated that there is no correlation between Bartonella infections and the sex, age or breed of the cat or its hunting behavior. There was also no correlation between bartonella and toxoplasma infections in cats. However, whereas 16.8% of cats from northern Germany had B. quintana-specific antibodies, only 8.0% of cats from southern Germany were seropositive for B. quintana. No statistically significant difference was found for B. henselae. IFA-positive and IFA-negative sera were used for immunoblot analysis including B. henselae and B. quintana. Marked reactivity was observed with protein bands at 80, 76, 73, 65, 37, 33 and 15 kDa. The results of this study suggest that B. henselae, and possibly a B. quintana-related pathogen, but not A. felis, are common in cats in Germany, and that there are differences in the geographic distribution of bartonella infections in cats.     

Survey of veterinary professionals for antibodies to Bartonella henselae in Japan.

The seroprevalence of Bartonella henselae among veterinary professionals in Japan was investigated by means of an immunoperoxidase (IP) test that used protein A-horseradish peroxidase conjugate. Sera were obtained from 233 veterinary professionals in the Tokyo and Chiba areas. As negative control group, sera from 155 healthy individuals (all medical students) were used. As positive control group, sera from 5 patients highly suspected of cat scratch disease (CSD) by clinical symptoms were tested. Serum antibody titers of > or = 200 to B. henselae were presumed seropositive, because the titer at which about 95.5% of all healthy individuals (148 of the 155) were negative, and 2 of the 5 suspected CSD patients' serum antibody titers were > or = 200. Of the individuals in the veterinary professionals group tested, 35 of the 233 (15.0%) were seropositive for B. henselae. Females were nearly twice as likely as males to have antibodies to B. henselae in the veterinary professionals group. Our data suggest that Japanese veterinary professionals, and in particular younger females who are veterinary assistants and animal beauticians are more often infected by B. henselae.     

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.     

Semiquantitative Species-Specific Detection of Bartonella henselae and Bartonella quintana by PCR-Enzyme Immunoassay

Bartonella henselae is the main causative agent of cat-scratch disease, and both B. henselae and Bartonella quintana cause angioproliferative disorders such as bacillary angiomatosis. To increase the sensitivity of Bartonella detection by PCR and to improve the species differentiation, we developed a semiquantitative, species-specific PCR-based enzyme immunoassay (EIA). The 16S rRNA gene was selected as the target sequence. Internal nucleotide sequences derived from the amplified 16S rRNA region were used to develop species-specific oligonucleotide probes for B. henselae and B. quintana. Biotin-labeled PCR products were immobilized on streptavidin-coated microtiter plates, hybridized to a digoxigenin-labeled probe, and detected with antidigoxigenin peroxidase conjugate. No cross-hybridization with other Bartonella or non-Bartonella species was observed. This EIA was as sensitive as dot blot hybridization and was 10 times more sensitive than visualization of PCR products on agarose gels. Serial dilutions of B. henselae and B. quintana suspensions demonstrated that an optical density (OD) of approximately 0.200 was equivalent to 5 CFU in the reaction mixture. By comparing the OD of the bacterial dilutions with that obtained from clinical specimens we could determine that the number of CFU in clinical samples ranged from 10(3) to 10(6) CFU/ml. The PCR-EIA developed in the present study is a rapid, sensitive, and simple method for the diagnosis of B. henselae and B. quintana infections.     

Experimental transmission of Bartonella henselae by the cat flea.

Bartonella henselae is an emerging bacterial pathogen, causing cat scratch disease and bacillary angiomatosis. Cats bacteremic with B. henselae constitute a large reservoir from which humans become infected. Prevention of human infection depends on elucidation of the natural history and means of feline infection. We studied 47 cattery cats in a private home for 12 months to determine the longitudinal prevalence of B. henselae bacteremia, the prevalence of B. henselae in the fleas infesting these cats, and whether B. henselae is transmitted experimentally to cats via fleas. Vector-mediated transmission of B.henselae isolates was evaluated by removing fleas from the naturally bacteremic, flea-infested cattery cats and transferring these fleas to specific-pathogen-free (SPF) kittens housed in a controlled, arthropod-free University Animal Facility. B. henselae bacteremia was detected in 89% of the 47 naturally infected cattery cats. A total of 132 fleas were removed from cats whose blood was simultaneously cultured during different seasons and were tested individually for the presence of B. henselae DNA by PCR. B. henselae DNA was detected in 34% of 132 fleas, with seasonal variation, but without an association between the presence or the level of bacteremia in the corresponding cat. Cat fleas removed from bacteremic cattery cats transmitted B. henselae to five SPF kittens in two separate experiments; however, control SPF kittens housed with highly bacteremic kittens in the absence of fleas did not become infected. These data demonstrate that the cat flea readily transmits B. henselae to cats. Control of feline infestation with this arthropod vector may provide an important strategy for the prevention of infection of both humans and cats.