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.     

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.     

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.     

Bartonella henselae Invasion of Feline Erythrocytes In Vitro

Bartonella henselae, the causative agent of cat scratch disease, establishes long-term bacteremia in cats, in which it attaches to and invades feline erythrocytes (RBC). Feline RBC invasion was assessed in vitro, based on gentamicin selection for intracellular bacteria or by laser confocal microscopy and digital sectioning. Invasion rates ranged from 2 to 20% of the inoculum, corresponding to infection of less than 1% of the RBC. Invasion was a slow process, requiring >8 h before significant numbers of intracellular bacteria were detected. Pretreatment of the bacteria with trypsin, or of the RBC with trypsin or neuraminidase, had no effect, but pronase pretreatment of RBC resulted in a slight increase in invasion frequency. The ability to model B. henselae invasion of feline RBC in vitro should permit identification of bacterial surface components involved in this process and elucidate the significance of RBC invasion to transmission and infection in cats.     

Characterization of Human Immunoglobulin (Ig) Isotype and IgG Subclass Response to Bartonella henselae Infection

Serologic parameters of cat scratch disease (CSD) were evaluated by Western blot analysis. Sera from patients with serologically confirmed CSD antigen were screened for immunoglobulin (Ig) isotype-specific as well as IgG subclass-specific reactivity against Bartonella henselae whole-cell antigen. Bartonella-negative control sera were used to determine baseline antibody activity. Heterogeneous B. henselae-specific IgG reactivity with numerous protein bands, ranging from >150 to <17 kDa, was observed. Though individual banding patterns were variable, one approximately 83-kDa B. henselae protein (Bh83) was immunoreactive with all CSD sera tested, suggesting it is a conserved antigen during infection. Bh83 was not recognized by reference human antisera against Rickettsia rickettsii, Chlamydia group positive, Treponema pallidum, Orientia tsutsugamushi, Fransciscella tularensis, Ehrlichia chaffeensis, Mycoplasma pneumoniae, and Escherichia coli, although other cross-reactive proteins were evident. Significantly, CSD sera failed to recognize the 83-kDa protein when tested against Bartonella quintana antigen, though sera from B. quintana-infected patients did react to Bh83. This cross-reactivity suggests epitope conservation during infection with B. henselae or B. quintana. Western blot analysis further revealed similar banding patterns when B. henselae was reacted against the Ig isotypes IgG and IgG₁ and both secretory and alpha chains of IgA. Neither IgM nor IgE reacted significantly to Bartonella antigen by our Western blot analysis. Dissection of the antibody response at the IgG subclass level indicated that prominent antigen recognition was limited to IgG₁. These observations provide insight into induced immunity during CSD and provide evidence for conserved epitope expression during infection with B. henselae or B. quintana.

The spectrum of human disease and pathologic syndromes observed to be associated with Bartonella henselae, an alpha-2 proteobacterium, has been progressively expanding since its identification in 1992 (18, 27). Granulomatous and vasculoproliferative diseases stemming from this emerging pathogen have since been described in both immunosuppressed and immunocompetent patients. Implicated in the etiology of cutaneous bacillary angiomatosis, bacillary hepatic peliosis and its parenchymal variant, endocarditis, and fever with persistent bacteremia (2, 9, 10, 12, 23, 24, 28), B. henselae is most notably recognized for its role as the primary etiologic agent of cat scratch disease (CSD) (5, 14). Afflicting an estimated 24,000 persons in the United States annually (8), CSD is characterized by a broad range of clinical symptoms manifested in varying degrees of severity depending largely on the immune status of the host. Infected patients present with subacute regional lymphadenopathy after inoculation, low-grade fever, anorexia, and malaise. Such manifestations are typically self-limiting and resolve untreated within several weeks in the immunocompetent host. It has become clear, though, that individuals with a depressed cellular immune response succumb to more-severe, atypical manifestations of CSD, including systemic complications of multiorgan involvement, particularly of the spleen and liver, and involvement of the central nervous system (2, 21, 28). Although B. henselae is a cause of human disease with a wide spectrum of severity, little is known regarding pathogenicity and immunity induced during infection.B. henselae is a fastidious, gram-negative bacillus that may require an incubation period as long as 5 weeks to culture axenically. Consequently, serologic methods, such as indirect fluorescent-antibody assay (IFA) and enzyme immunoassay (EIA), have been the most-practical and least-invasive means of clinical diagnosis (3, 19). Widely accepted as a diagnostic assay, IFA is routinely used to confirm B. henselae infection (4). However, when the whole bacterial cell antigen is used, IFA is unable to differentiate species-specific serologic reactivity from cross-reactivity with other antigens of phylogenetic proximity, namely, Bartonella quintana (4, 11). Modifications to improve the efficacy of serologic detection methods are pending a more-comprehensive understanding of the factors influencing both the pathogenesis of infection due to B. henselae and the evoked human immune response.The purpose of this study was to dissect the humoral immune response to B. henselae antigen in patients with clinically and laboratory-diagnosed CSD (positive by IFA) by Western blot analysis. In evaluation of the B. henselae proteins recognized following infection, an 83-kDa immunodominant protein was identified that was recognized by all seropositive patient samples tested. Furthermore, we have provided an in-depth characterization of the immunoglobulin (Ig) isotype and IgG subclass response in CSD patients. The findings, which elucidate serologic responses to B. henselae infection, provide insight into the immunity induced by this pathogen.(This work was presented in part at the 13th Sesqui-Annual Meeting of the American Society for Rickettsiology [abstract 14], September 1997, Champion, Pa.)     

Does a Feline Leukemia Virus Infection Pave the Way for Bartonella henselae Infection in Cats?

Domestic cats serve as the reservoir hosts of Bartonella henselae and may develop mild clinical symptoms or none after experimental infection. In humans, B. henselae infection can result in self-limiting cat scratch disease. However, immunocompromised patients may suffer from more-severe courses of infection or may even develop the potentially lethal disease bacillary angiomatosis. It was reasoned that cats with immunocompromising viral infections may react similarly to B. henselae infection. The aim of our study was to investigate the influence of the most important viruses known to cause immunosuppression in cats—Feline leukemia virus (FeLV), Feline immunodeficiency virus (FIV), and Feline panleukopenia virus (FPV)—on natural B. henselae infection in cats. Accordingly, 142 cats from animal shelters were necropsied and tested for B. henselae and concurrent infections with FeLV, FIV, or FPV by PCR and immunohistochemistry. A significant association was found between B. henselae and FeLV infections (P = 0.00028), but not between B. henselae and FIV (P = 1.0) or FPV (P = 0.756) infection, age (P = 0.392), or gender (P = 0.126). The results suggest that susceptibility to B. henselae infection is higher in cats with concurrent FeLV infections, regardless of whether the infection is latent or progressive. Histopathology and immunohistochemistry for B. henselae failed to identify lesions that could be attributed specifically to B. henselae infection. We conclude that the course of natural B. henselae infection in cats does not seem to be influenced by immunosuppressive viral infections in general but that latent FeLV infection may predispose cats to B. henselae infection or persistence.The outcome of Bartonella henselae infection for human patients depends mostly on their immune status. In immunocompetent patients, B. henselae causes the self-limiting cat scratch disease, characterized by local granulomatous to abscedating lymphadenitis (10). Immunocompromised hosts, in contrast, are prone to suffer from disseminated cat scratch disease with bacteremia and may develop angioproliferative lesions, including peliosis hepatis or the potentially lethal disease bacillary angiomatosis (38, 39). Domestic cats are one well-known reservoir host of B. henselae and transmit the agent to humans through scratch marks or bite wounds. The prevalence of feline infection is high in animal shelters and populations of stray cats, since fleas transmit the agent among cats (6). A recent epidemiologic study revealed a B. henselae bacteremia prevalence of 18.7% among cats from animal shelters, whereas only 1% of pet cats in the same area were found to be bacteremic (4). The clinical course and pathology of feline bartonellosis differ from those for human infections. In domestic cats, natural infection has been associated with gingivitis, stomatitis, lymphadenopathy, uveitis, and urinary tract diseases (13, 21, 42). Experimental infection, on the other hand, leads to prolonged and relapsing intraerythrocytic bacteremia with either no clinical symptoms or mild unspecific symptoms (1, 6, 19, 35). Histologically, specific-pathogen-free cats with acute B. henselae infections had marked generalized lymphatic hyperplasia (15, 20). In chronic experimental infections, a variety of additional unspecific histological lesions, including lymphocytic interstitial nephritis, cholangitis, hepatitis, and lymphoplasmacytic myocarditis, were observed (20).In contrast to the availability of data on bartonellosis in otherwise healthy experimentally infected cats, no systematic investigations of the significance and lesions of natural B. henselae infection in cats with immunosuppressive diseases have been conducted.The aim of the present study was to investigate systematically the influence of immunosuppressive infectious diseases on natural B. henselae infection in cats. We hypothesized that, as with humans, immunosuppressive diseases in cats may alter both susceptibility to B. henselae and the course of infection. Accordingly, we focused on the three most important agents known to cause immunosuppression in cats: feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), and feline panleukopenia virus (FPV).     

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.     

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.     

Diagnostic value of the indirect immunofluorescence assay in cat scratch disease with Bartonella henselae and Afipia felis antigens.

Serum samples from 35 cat scratch disease (CSD) patients, 180 control patients (123 without lymph node enlargement and 57 with lymph node enlargement not evoking CSD), and 102 nonpatient subjects (35 with cat contact and 67 without cat contact) were tested by semiquantitative indirect immunofluorescence assay for the presence of antibodies directed to Afipia felis (ATCC 53690T) or Bartonella henselae (ATCC 49882T). The CSD group had statistically higher antibody titers against B. henselae than the control groups (P < 10(-5)), whereas no difference in A. felis antibody titers was evidenced among all groups tested. Among the 317 serum samples studied, the three with high A. felis antibody titers ( > or = 64) also had high antibody titers against other alpha-2 proteobacteria. The value of the indirect immunofluorescence assay with B. henselae antigen for the diagnosis of CSD was as follows: for a cutoff of 32, sensitivity was 0.80, specificity was 0.85, and the likelihood ratio was 5.1; for a cutoff of 64, the likelihood ratio was 12.1. In summary, in France, CSD is associated with high antibody titers against B. henselae, as previously described in the United States. However, the causes for B. henselae seronegativity in CSD patients and those for high antibody titers outside the typical nosological frame of CSD still have to be identified.     

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.     

Development of an Immunoglobulin M Capture-Based Enzyme-Linked Immunosorbent Assay for Diagnosis of Acute Infections with Bartonella henselae

We describe the development of an immunoglobulin M-specific enzyme-linked immunosorbent assay for the detection of an early antibody response to Bartonella henselae, the causative agent of cat scratch disease, bacillary angiomatosis, and endocarditis. This assay discriminates between B. henselae-positive and -negative patient samples with sensitivity and specificity values of 100% and 97.1%, respectively.Bartonella henselae is an emerging pathogen of epidemiological and pathological concern. The wide spectrum of diseases caused by this gram-negative bacterium appears to be linked to the state of the host immune system (11). For example, infection with B. henselae causes cat scratch disease (CSD) in immunocompetent individuals (14); angioproliferative illnesses, including bacillary angiomatosis, in immunocompromised patients (13); and endocarditis in patients with preexisting heart valve lesions (11).Most common in children and young adults, CSD is widely accepted as the result of a bite or scratch of a cat infested with cat fleas (Ctenocephalides felis) (10, 18) harboring this pathogen. Bartonella henselae has also been detected in Ixodes scapularis ticks collected in New Jersey (1), supporting the notion that ticks may play a role in the transmission of CSD. Typical disease sequelae include regional lymphadenopathy, low-grade fever, and malaise, with cutaneous lesions occasionally being detected at the site of the bite or scratch (15). Approximately 24,000 new cases of CSD are reported each year, with up to 10% of cases requiring hospitalization resulting from atypical CSD manifesting as neuroretinitis, granulomatous hepatitis, and osteomyelitis (17). Less-well-defined disease conditions possibly associated with B. henselae bacteremia are a diverse number of neurological illnesses, such as encephalitis and fatal meningitis (9). In a recent study, Bartonella henselae and B. vinsonii subsp. berkhoffii were detected in six immunocompetent individuals who presented with seizures, ataxia, and memory loss (6). The high rates of carriage by domestic cats, combined with the close proximity in which humans and cats live, increase the likelihood of human exposure to B. henselae. In a recent study, an immunoglobulin G (IgG) seroprevalence rate of 53.3% was reported among cat owners in Poland (8).Current diagnostic tests for infection with B. henselae include PCR and serology-based assays, such as immunofluorescence assays (IFAs). IFAs employ whole bacterial cells as the antigen and are currently the most widely used diagnostic tool. However, IFAs are highly reader dependent, generally lack overall specificity, and are not quantitative.The predicted high seroprevalence to Bartonella henselae within the general population as a result of the high rates of cat ownership makes the availability of an assay capable of distinguishing between current or acute infections and prior seroconversion necessary. The availability of a test for the detection of an early response to B. henselae antigens would therefore greatly facilitate the better diagnosis and treatment of infected patients.We recently reported on the use of a recombinant B. henselae 17-kDa antigen (the r17-kDa antigen), first described by Anderson et al. (3), for the development of an IgG enzyme-linked immunosorbent assay (ELISA) with a sensitivity and a specificity of 71.1% and 93.0%, respectively (13). In that report, recombinant antigen was coated onto ELISA plates and reacted with patient sera. We have now further characterized this antigen and optimized the conditions for its use in an IgM capture-based ELISA for the detection of an IgM antibody response during the acute stages of infection.As previously reported, we expressed the r17-kDa protein in a prokaryotic expression system as a histidine-tagged fusion protein which was purified by use of a nickel-agarose column (13). For the IgM capture assay, purified r17-kDa protein (1 mg/ml) was biotinylated with a Sulfo-NHS biotin kit (Pierce, Rockford, IL). MaxiSorp 96-well plates (Nunc, Rochester, NY) were coated with anti-human IgM (Kirkegaard & Perry Laboratories, Gaithersburg, MD) at a concentration of 1 μg/ml in coating buffer (0.015 M Na₂CO₃, 0.035 M NaHCO₃ [pH 9.6]) and incubated overnight at 4°C. The plates were washed with phosphate-buffered saline-Tween 20 and blocked with 1% bovine serum albumin (BSA) at room temperature. IgM-positive and -negative patient sera, as initially determined by testing done by Focus Diagnostics and Specialty Laboratory, were confirmed as positive or negative for B. henselae in-house by using an IgM IFA kit, according to the manufacturer''s (Focus Diagnostics, Cypress, CA) recommended protocol. The sera were diluted 1:100 in 1% BSA with 0.05% Tween 20, prior to being run in duplicate, and reacted with the plates for 1 h at room temperature. The biotinylated r17-kDa protein was added to the plates at 0.1 μg/ml in dilution buffer, and the plate was incubated at room temperature for 1 h with shaking. Streptavidin-horseradish peroxidase (Southern Biotech, Birmingham, AL) was used to detect the plates, followed by development with 3,3′,5,5′-tetramethylbenzidine (Moss, Pasadena, MD) for 30 min. The reaction was stopped with 2 N HCl, and the absorbance at 450 nm was recorded after a period of 5 to 30 min.This assay clearly discriminates between B. henselae IgM IFA-positive (n = 13) and IFA-negative (n = 34) patient samples, as shown in Fig. ​Fig.1,1, with sensitivity and specificity values of 100% and 97.1%, respectively. Receiver operating characteristic (ROC) curve analysis was used to evaluate the performance of the IgM capture method. ROC curve analysis (data not shown) of the calibrated slopes revealed an area under the curve of 0.998 (95% confidence interval, 0.919 to 1.000).Open in a separate windowFIG. 1.Results obtained from analysis of B. henselae IFA-positive (n = 13) (▪) and IFA-negative (n = 34) (⧫) patient samples by the IgM capture-based ELISA described in the text. Index values represent the ratio between the optical density at 450 nm of the sample and the optical density at 450 nm of the calibrator. Index values above 1.1 are reported as positive, index values less than 0.9 are reported as negative, and values of 0.9 to 1.1 reported as equivocal.Lyme borreliosis has been reported to be the most prevalent human vector-borne disease in the United States (7). Such data, along with the growing awareness that Borrelia burgdorferi-infected Ixodes ticks can be coinfected with pathogens such as Bartonella, Anaplasma, and, more commonly, Babesia (5, 12), raise a question concerning the ability of our assay to discriminate between infection with Bartonella henselae and infection with Borrelia burgdorferi. This question is of clinical significance, particularly in light of the finding of other studies that showed that patients infected with multiple tick-borne pathogens present with more severe illnesses of longer durations (5). When our IgM ELISA was tested with 54 Lyme spirochete-positive (confirmed by the Viramed Western blot assay) and Bartonella henselae-negative (confirmed by IFA [Focus Diagnostics]) patient serum samples, no cross-reactivity was detected in 53/54 samples (data not shown), suggesting that this ELISA would be a valid serological test for the diagnosis of infections with B. henselae, even with patient samples coinfected with Borrelia burgdorferi. The single cross-reactive sample was confirmed to be Bartonella henselae negative by several rounds of testing by IFA. Additional analysis of this sample indicated that it was IgM positive by IFA (Fuller Laboratories, Fullerton, CA) for Babesia microti (data not shown). Further studies will need to be performed to confirm the coinfection of this particular sample with B. burgdorferi and B. microti and to determine what effects, if any, this might have on the specificity capabilities of our IgM ELISA. Finally, we acknowledge that the remarkably high sensitivity and specificity values of this assay are unusual for an IgM response. We believe that these values, particularly the specificity value, can be explained at least in part by the antigen used in this study. As a component of the type IV secretion system, the 17-kDa antigen likely plays an important role in the early stages of infection. This protein is a homolog of the VirB5 family, and there is increasing evidence suggesting that these proteins function as specialized adhesins (4). Several key structural and functional studies strongly suggest that VirB5 proteins are likely involved in host recognition by the type IV secretion system pilus, most likely through protein-protein interactions involving amino acids within the C terminus (2, 4, 19). Consistent with a role in the early stages of infection of host cells, this antigen has been shown to be expressed at minimal levels by B. henselae organisms cultivated on cell-free laboratory medium but is induced to high levels in the presence of human endothelial cells (16). We believe that our data are consistent with those from studies implicating VirB5 proteins in protein-protein interactions involved in host cell recognition (2, 4, 19). In light of the findings of those studies as well as our own observations (unpublished data) showing the importance of the C terminus in host recognition, the very high specificity obtained with the 17-kDa antigen is perhaps not surprising after all. It is reasonable to speculate that the important role played by the 17-kDa antigen in the type IV secretion system contributes in large part to why it is one of only a few antigens, with BadA being another, shown to be beneficial as a serological marker for B. henselae infection.In conclusion, this is the first demonstration of an ELISA for the laboratory diagnosis of early infection with Bartonella henselae. The high sensitivity and the high specificity achievable with this assay make it a diagnostic tool capable of distinguishing between patients with active or very recent infection from those with past exposure to this organism.     

Seroprevalence of Antibodies to Microorganisms Known To Cause Arterial and Myocardial Damage in Patients with or without Coronary Stenosis

Infections are assumed to play a role in coronary artery disease (CAD) and cardiomyopathies. It is unknown whether the seroprevalence of antibodies to these microorganisms is higher in patients with than without CAD. The seroprevalence of antibodies to Bartonella henselae, Borrelia burgdorferi, Chlamydia pneumoniae, Coxiella burnetii, Helicobacter pylori, human granulocytic Ehrlichia, Leptospira, Rickettsia conorii, and Treponema pallidum was assessed prospectively in patients with exertional dyspnea or anginal chest pain who underwent coronary angiography because of suspected CAD. Patients with normal angiograms (NA) were those in whom no more than 50% stenosis of any coronary artery was found. Patients with CAD were patients who underwent percutaneous transluminal coronary angioplasty. There were 50 patients with CAD (9 female) and 62 with NA (25 female), with a mean age of 62 years. All patients had antibodies to at least one microorganism: to B. henselae, 8% of CAD patients and 5% of NA patients; to B. burgdorferi IgG, 14% CAD and 6% NA; to B. burgdorferi IgM, 6% CAD and 3% NA; to C. pneumoniae lipopolysaccharide (LPS) IgA, 76% CAD and 77% NA; to C. pneumoniae LPS IgG, 80% CAD and 90% NA; to C. burnetii, 0% CAD and 5% NA; to H. pylori, 92% CAD and 68% NA; to human granulocytic Ehrlichia, 8% CAD and 3% NA; to Leptospira IgG, 4% CAD and 2% NA; to R. conorii, 10% in both groups; and to T. pallidum, 2% CAD and 0% NA. The seroprevalence of antibodies to micro-organisms known to induce arterial and myocardial damage does not differ between patients with CAD and NA.     

Prevalence of Bartonella henselae in Italian stray cats: evaluation of serology to assess the risk of transmission of Bartonella to humans

Bartonella henselae is the major etiological agent of cat scratch disease in humans. Cats act as the natural reservoir of B. henselae and can transmit the infection to humans by a bite or scratch. The prevalence of B. henselae in cat populations was evaluated by serological and bacteriological tests. A total of 769 stray cats from three urban and three rural areas in northern Italy were sampled between January 1999 and December 2000. The positive and the negative predictive values of serological tests with respect to bacteremic status were evaluated. Tests of a total of 140 cats (18%) resulted in detection of bacteremia. A total of 540 cats were tested by serology; 207 (38%) were seropositive. Of the 531 cats tested by both methods, the results for 65 (12.2%) showed both bacteremia detection and seropositivity. The molecular typing of the isolates showed that 20.6% of bacteremic cats were infected with B. henselae type I strain, 61.1% were infected with B. henselae type II, and 18.3% were coinfected with both. A statistically significant difference in antibody and bacteremia prevalences among geographical areas was detected. Statistical analysis showed no association between characteristics such as seroprevalence-bacteremic status, sex, general health status, and the presence of ectoparasites. The negative predictive value of serological test was 84.7%, and the positive predictive value was 31.8%. Receiving operator characteristic analysis of the data showed that serological tests had a low predictive value in relation to the bacteremic status of a cat; in surveys aimed at assessing the real risk of B. henselae infection in a human population, therefore, we suggest the use of blood culture as the reference test. Nevertheless, both blood culture assays and serological tests for Bartonella infection should be performed for a complete evaluation of the health status of cats.     

Cat-scratch disease in Northern Italy: atypical clinical manifestations in humans and prevalence of Bartonella infection in cats

In this paper, we report an investigation on cat-scratch disease (CSD) in Northern Italy. Seventy-four cases of CSD were diagnosed at the San Matteo hospital, Pavia, during the period 2005–2010. Of these 74 patients, 18 (24.3 %) reported atypical clinical manifestations such as ocular papillitis, maculopapular eruptions, vertebral infection, pulmonary infiltrates, and granulomatous hepatitis. Contact with cats was documented for 61 patients (82.4 %), while cat-related trauma was reported for 49 patients (66.2 %). We subsequently investigated the presence of Bartonella infection in cats belonging to the above patients and in other domestic and stray cats from three provinces of Northern Italy. Among the 27 domestic cats tested, nine of the 11 belonging to the CSD patients and two of the remaining 16 were infected by B. henselae (81.8 % vs. 12.5 %). Out of over 1,300 stray cats examined, 23.1 % were seropositive for B. henselae; after culturing and genotyping, 17 % were found to be infected by B. henselae (15.5 %) or B. clarridgeiae (1.5 %).     

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.     

Cytoplasmic, Nuclear, and Platelet Autoantibodies in Human Granulocytic Ehrlichiosis Patients

Serum samples from patients with confirmed human granulocytic ehrlichiosis (HGE) were tested for cytoplasmic, nuclear, and platelet autoantibodies and rheumatoid factor. The indirect fluorescence antinuclear antibody test on Hep-2 cells demonstrated antinuclear titers of ≥40 and ≥160 in 44 and 10%, respectively, of serum samples from HGE patients. Two patients (4%) had anticytoplasmic (mitochondrial and spindle apparatus) antibodies with a titer of 80 and two patients (4%) had anticytoplasmic (mitochondrial) antibodies with a titer of 160 or greater. Flow cytometry was used to demonstrate antiplatelet antibodies in 80% of first serum samples from HGE patients. Rheumatoid factor was not detected. Nuclear and cytoplasmic autoantibodies are a major cause of interference when the indirect fluorescence antibody test is used to detect fluorescence of morulae in Ehrlichia-infected equine neutrophils or HL-60 promyelocytes. Antiplatelet antibodies may contribute to the profound thrombocytopenia which is a characteristic laboratory feature during the acute phase of HGE infection. Whether autoantibodies precede infection or are caused by immune activation of HGE deserves further study.     

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.     

Serologic Evidence of Infection with Ehrlichia spp. in Wild Rodents (Muridae: Sigmodontinae) in the United States

Rodent (Muridae: Sigmodontinae) blood and sera collected from 14 states were tested for seroreactivity to a cultured isolate of the human granulocytic ehrlichiosis (HGE) agent by using an indirect immunofluorescence assay. Of the 1,240 samples tested, 136 (11%) were found to be reactive at titers of ≥32. Rodents with HGE agent-specific antibodies were found in New York (23% of 491 samples; geometric mean endpoint titer [GMT] = 441), Connecticut (11% of 100 samples; GMT = 481), California (9% of 32 samples; GMT = 323), Colorado (2% of 212 samples; GMT = 256), Florida (7% of 27 samples; GMT = 362), Maryland (7% of 15 samples; titer = 64), New Jersey (4% of 76 samples; titer = 256), and Wisconsin (13% of 8 samples; titer = 128). Samples from Georgia (n = 16), Illinois (n = 27), Nevada (n = 27), North Carolina (n = 52), Ohio (n = 57), and Utah (n = 100) were not reactive. The earliest seroreactive sample was from a Peromyscus leucopus mouse collected in June 1986 in Connecticut, and the majority of the seroreactive samples (68%) were from this species. Samples from other Peromyscus species (P. boylii, P. maniculatus, and P. gossypinus) were also found to be reactive, with a GMT for the genus of 410. Several species of Neotoma woodrats (N. fuscipes, N. lepida, N. albigula, and N. mexicana) from California and Colorado had antibodies that reacted with the HGE agent (genus GMT = 194), suggesting that enzootic cycles of Ehrlichia spp. exist outside of the areas of confirmed human disease. Attempts to amplify and detect ehrlichial DNA from the limited tissues available (n = 40 animals) were unsuccessful. Further studies are needed to determine the identity of the organisms inducing antibody production in these rodent species and to elucidate the epidemiology and public health importance of these agents.     

Prevalence of toxic shock syndrome toxin 1 (TSST-1)-producing strains of Staphylococcus aureus and antibody to TSST-1 among healthy Japanese women

Many cases of neonatal toxic shock syndrome (TSS)-like exanthematous disease but few cases of menstrual TSS (mTSS) have been reported in Japan. We determined the prevalence of mucosal colonization with Staphylococcus aureus and of positive antibodies to TSS toxin 1 (TSST-1) among 209 healthy Japanese women in Tokyo. S. aureus isolates from mucosal sites were characterized with respect to TSST-1 production and resistance genotype. Antibody titers were determined for test subjects and for 133 Japanese and 137 Caucasian control women living in the United States. S. aureus was isolated from at least one site in 108 of 209 women (52%) in Tokyo. Of the 159 S. aureus isolates recovered, 14 (9%) were TSST-1 positive (12 unique strains). Twelve of 209 women (6%) were colonized with a TSST-1-producing strain; two (<1%) had vaginal colonization. Only 2 of 12 unique toxigenic strains (14%) were methicillin resistant. Of the 12 TSST-1-positive strains isolated, 6 (50%) were pulsed-field gel electrophoresis type USA200, multilocus sequence type clonal complex 30. Fewer Japanese women in Tokyo (47%) than Caucasian and Japanese women in the United States (89% and 75%, respectively) had TSST-1 antibodies. The prevalences of colonization with TSST-1-producing S. aureus were comparable in Japan and the United States, despite low seropositivity to TSST-1 in Japan. Environmental factors appear to be important in promoting the development of anti-TSST-1 antibodies, as there was a significant difference in titers between Japanese women living in Tokyo and those living in the United States. Most colonizing TSST-1-producing S. aureus strains in Japan were genotypically similar to mTSS strains found in the United States.     

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.