Molecular detection of Bartonella henselae for the diagnosis of cat scratch disease and bacillary angiomatosis of the conjunctiva.

PURPOSE: The purpose of this study was to evaluate clinical cases of cat scratch disease (CSD) and bacillary angiomatosis involving the conjunctiva by special stains and transmission electron microscopy (TEM) and to compare these findings with the results from species-specific polymerase chain reaction (PCR) analysis of the same specimens. METHODS: Six potential cases of CSD and 2 possible cases of bacillary angiomatosis of the conjunctiva were analyzed by light microscopy, the Warthin-Starry technique, TEM, and PCR. DNA isolated from cultured Bartonella henselae, B. bacilliformis, B. quintana, and B. elizabethae were used as control templates for establishment of the PCR sensitivity and specificity. Cultured DNA was also used as appropriate positive controls during analysis of the clinical specimens. RESULTS: The histological studies, electron microscopy, and the PCR analysis confirmed the identification of the bacilli within the involved tissues. Furthermore, molecular diagnosis by PCR allowed for speciation of the infecting Bartonella organisms in 6 of the 8 cases and correlated with the histological findings. CONCLUSIONS: The PCR-based identification of Bartonella correlated well with the results of light microscopy and TEM and provided a simple and rapid method of diagnosis to the species level. The molecular analysis may prove to be beneficial in enhancing the current diagnostic techniques for CSD and bacillary angiomatosis.

[Application of Warthin-Starry stain, immunohistochemistry and transmission electron microscopy in diagnosis of cat scratch disease].

OBJECTIVE: To evaluate the diagnostic utility of Warthin-Starry silver stain, immunohistochemistry and transmission electron microscopy in the detection of human Bartonella henselae infection and pathologic diagnosis of cat scratch disease (CSD). METHODS: The paraffin-embedded lymph node tissues of 77 histologically-defined cases of cat scratch disease collected during the period from January, 1998 to December, 2008 were retrieved and studied using Warthin-Starry silver stain (WS stain) and mouse monoclonal antibody against Bartonella henselae (BhmAB stain). Five cases rich in bacteria were selected for transmission electron microscopy. RESULTS: Under electron microscope, the organisms Bartonella henselae appeared polymorphic, round, elliptical, short rod or bacilliform shapes, ranged from 0.489 to 1.110 microm by 0.333 to 0.534 microm and often clustered together. Black short rod-shaped bacilli arranged in chains or clumps were demonstrated in 61.0% (47/77) of CSD by WS stain. The organisms were located outside the cells and lie mainly in the necrotic debris, especially near the nodal capsule. In 72.7% (56/77) of the cases, dot-like, granular as well as few linear positive signals were observed using BhmAB immunostain and showed similar localization. Positive results for both stains were identified in 59.7% (46/77) of the cases. When applying both stains together, Bartonella henselae was observed in 74.0% (57/77) of the case. The difference between the results obtained by WS stain and BhmAB immunostain was of statistical significance (P < 0.05). CONCLUSIONS: Bartonella henselae is the causative pathogen of cat scratch disease. WS stain, BhmAB immunostain and transmission electron microscopy are helpful in confirming the histologic diagnosis. Immunostaining using BhmAB can be a better alternative than WS stain in demonstrating the organisms.

Blood cell findings resembling Bartonella spp.

Some Bartonella species are able to invade red blood cells (RBC) and may cause persistent infection in the susceptible host. Use of transmission electron microscopy (TEM) demonstrates, inside erythrocytes, the typical triple-walled agents. However, when examining ultrathin sections of blood cells, the authors have, on several occasions, detected intraerythrocytic abnormalities that mimic but are not typical of Bartonella spp. Small endovesicles, pseudoinclusions, cavities, and irregular hemoglobin granules distribution, resulting in regions of increased or decreased electron density, may be observed in the erythrocytes and platelets, which may be confused with bartonellas. So far, detailed ultrastructural findings of Bartonella spp. in blood cells have not yet been described. Aiming to improve TEM interpretation of blood cells changes, in routine examination of blood sections of patients with suspected bartonellosis, the authors studied the morphological findings they have observed, and present their putative nature, according to information in the literature.

Detrimental effects of Bartonella henselae are counteracted by L-arginine and nitric oxide in human endothelial progenitor cells.

The recruitment of circulating endothelial progenitor cells (EPCs) might have a beneficial effect on the clinical course of several diseases. Endothelial damage and detachment of endothelial cells are known to occur in infection, tissue ischemia, and sepsis. These detrimental effects in EPCs are unknown. Here we elucidated whether human EPCs internalize Bartonella henselae constituting a circulating niche of the pathogen. B. henselae invades EPCs as shown by gentamicin protection assays and transmission electron microscopy (TEM). Dil-Ac-LDL/lectin double immunostaining and fluorescence-activated cell sorting (FACS) analysis of EPCs revealed EPC bioactivity after infection with B. henselae. Nitric oxide (NO) and its precursor l-arginine (l-arg) exert a plethora of beneficial effects on vascular function and modulation of immune response. Therefore, we tested also the hypothesis that l-arg (1-30 mM) would affect the infection of B. henselae or tumor necrosis factor (TNF) in EPCs. Our data provide evidence that l-arg counteracts detrimental effects induced by TNF or Bartonella infections via NO (confirmed by DETA-NO and L-NMMA experiments) and by modulation of p38 kinase phosphorylation. Microarray analysis indicated several genes involved in immune response were differentially expressed in Bartonella-infected EPCs, whereas these genes returned in steady state when cells were exposed to sustained doses of l-arg. This mechanism may have broad therapeutic applications in tissue ischemia, angiogenesis, immune response, and sepsis.

Bartonella henselae infects human erythrocytes.

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

Bartonella adhesin a mediates a proangiogenic host cell response.

Bartonella henselae causes vasculoproliferative disorders in humans. We identified a nonfimbrial adhesin of B. henselae designated as Bartonella adhesin A (BadA). BadA is a 340-kD outer membrane protein encoded by the 9.3-kb badA gene. It has a modular structure and contains domains homologous to the Yersinia enterocolitica nonfimbrial adhesin (Yersinia adhesin A). Expression of BadA was restored in a BadA-deficient transposon mutant by complementation in trans. BadA mediates the binding of B. henselae to extracellular matrix proteins and to endothelial cells, possibly via beta1 integrins, but prevents phagocytosis. Expression of BadA is crucial for activation of hypoxia-inducible factor 1 in host cells by B. henselae and secretion of proangiogenic cytokines (e.g., vascular endothelial growth factor). BadA is immunodominant in B. henselae-infected patients and rodents, indicating that it is expressed during Bartonella infections. Our results suggest that BadA, the largest characterized bacterial protein thus far, is a major pathogenicity factor of B. henselae with a potential role in the induction of vasculoproliferative disorders.

Role of Bartonella henselae endocarditis in the nucleation of aortic valvular calcification.

A 6-year-old boy presented with fatigability, shortness of breath, and bulging neck veins. Echocardiography revealed large vegetations, aortic insufficiency, a dilated left ventricle, and bicuspid aortic valve. There was no history of immunocompromise, fevers, or feline exposures. Blood cultures were negative; antibodies against Bartonella henselae were positive. Gentamicin was administered intravenously. Ross procedure was performed and patient was discharged on antibiotics in 5 days. Native valve was thickened by scar and fibrinous vegetations. Warthin-Starry stain demonstrated coccobacilli. Light and ultrastructural morphology, and monoclonal staining implicated B. henselae. Bacterial membranes contain calcium apatite crystals. Antigenic material was present in bacteria and calcified nodules. This case illustrates calcified protobacteria becoming incorporated into scar tissue during endocarditis.

Ultrastructural changes in a standard strain of Bartonella henselae after passages through BALB/cAn mice.

Human bartonelloses are a group of illnesses of poorly understood pathogenesis. Bartonella henselae is one of the most studied bacterium of its genus. The objective of this study was to observe whether passages of these bacteria, in vivo, would determine ultrastructural changes in them. For this purpose, isogenic mice were inoculated with a standard strain of B. henselae (I). These were initially retrieved from genetically immunodeficient animals (II) and then inoculated in immunocompetent ones. The bacterial colonies obtained (III) were compared, by transmission electron microscopy, with colonies I and II. Loss of fimbriae and an abundant bleb formation were the most common morphological changes found in colony III. Also, on day 6 postinfection, the main histological abnormalities were the endothelial proliferation presented in immunodeficient animals and the incipient granulomata reaction found in one of the immunocompetent inoculated mice, which died spontaneously. These features agree with the Bartonella human disease clinical and histological observations. This study demonstrates that B. henselae in vivo passages induce significant morphological changes in the bacteria and that these abnormalities could explain their seemingly greater virulence. Most of these observations have not been previously described. Thus, further studies on the Bartonella species pathogenesis should consider these data.

Interaction of Bartonella henselae with endothelial cells results in bacterial aggregation on the cell surface and the subsequent engulfment and internalisation of the bacterial aggregate by a unique structure, the invasome.

Vascular colonisation by Bartonella henselae may cause vaso-proliferative tumour growth with clumps of bacteria found in close association with proliferating endothelial cells. By using B. henselae-infected human umbilical vein endothelial cells as an in vitro model for endothelial colonisation, we report here on a novel mechanism of cellular invasion by bacteria. First, the leading lamella of endothelial cells establishes cellular contact to sedimented bacteria and mediates bacterial aggregation by rearward transport on the cell surface. Subsequently, the formed bacterial aggregate is engulfed and internalised by a unique host cellular structure, the invasome. Completion of this sequence of events requires 24 hours. Cortical F-actin, intercellular adhesion molecule-1 and phosphotyrosine are highly enriched in the membrane protrusions entrapping the bacterial aggregate. Actin stress fibres, which are anchored to the numerous focal adhesion plaques associated with the invasome structure, are typically found to be twisted around its basal part. The formation of invasomes was found to be inhibited by cytochalasin D but virtually unaffected by nocodazole, colchicine or taxol, indicating that invasome-mediated invasion is an actin-dependent and microtubuli-independent process. Bacterial internalisation via the invasome was consistently observed with several clinical isolates of B. henselae, while a spontaneous mutant obtained from one of these isolates was impaired in invasome-mediated invasion. Instead, this mutant showed increased uptake of bacteria into perinuclear localising phagosomes, suggesting that invasome-formation may interfere with this alternative mechanism of bacterial internalisation. Internalisation via the invasome represents a novel paradigm for the invasion of bacteria into host cells which may serve as a cellular colonisation mechanism in vivo, e.g. on proliferating and migrating endothelial cells during Bartonella-induced vaso-proliferative tumour growth.

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.

[Bacillary angiomatosis]. / Bazilläre Angiomatose.

Bacillary angiomatosis (BA) is a rare infectious disease usually associated with HIV infection. Recent molecular biologic investigations confirm that both Rochalimaea henselae and Rochalimaea quintana can cause BA. The bacteria can be identified by Warthin-Starry staining and electron microscopy. The typical clinical signs are solitary or multiple dermal or subcutaneous nodules. Bone, liver, spleen and other organs may also be involved. We describe the clinical and histological features of a 39-year-old HIV-infected patient with cutaneous and bony lesions of BA. All manifestations of BA disappeared during therapy with erythromycin.