Burt E. Anderson

Ehrlichia ewingii sp. nov., the etiologic agent of canine granulocytic ehrlichiosis

The 16S rRNA gene was amplified, cloned, and sequenced from the blood of two dogs that were experimentally infected with the etiologic agent of canine granulocytic ehrlichiosis. The 16S rRNA sequence was found to be unique when it was compared with the sequences of other members of the genus Ehrlichia. The most closely related species were Ehrlichia canis (98.0% related) and the human ehrlichiosis agent (Ehrlichia chaffeensis) (98.1% related); all other species in the genus were found to be phylogenetically much more distant. Our results, coupled with previous serologic data, provide conclusive evidence that the canine granulocytic ehrlichiosis agent is a new species of the genus Ehrlichia that is related to, but is distinct from, E. canis and all other members of the genus. We propose the name Ehrlichia ewingii sp. nov.; the Stillwater strain is the type strain.

Cat-scratch disease in Hawaii: Etiology and seroepidemiology

OBJECTIVE To study the etiology and seroepidemiology of cat-scratch disease (CSD) in Hawaii. METHODS Blood and fine-needle aspirate (FNA) from the lymph nodes of 39 consecutive patients with clinical CSD were cultured for Bartonella henselae, and blood samples from index cats, stray cats, and dogs were cultured and their sera were tested by indirect fluorescence antibody test for antibodies to B. henselae and Afipia felis. Sera from age- and sex-matched human subjects without cat exposure served as controls. RESULTS Warthin-Starry staining showed positive results in only 4 of 32 FNAs, and B. henselae was isolated from only one FNA specimen. All of 38 patients who had two or more sera tested had elevated titers of antibody to B. henselae. Only 1 of 48 human control sera had antibody to B. henselae. Of 31 kittens, 21 had positive blood culture results and elevated antibody titers to B. henselae. Of three adult cats, all had negative blood culture results, but they had serologic evidence of past infection. Of 23 adult stray cats, 18 had elevated titers of antibody to B. henselae, but in only one was the blood culture result positive. Results of IFA tests were marginally positive for A. felis in 1 of 29 patients with CSD and in one adult stray cat and one dog. CONCLUSIONS This study shows that the B. henselae IFA test is both highly sensitive and specific for the detection of infection caused by B. henselae and for the laboratory diagnosis of CSD, and that FNA is seldom helpful in confirming the diagnosis. We further demonstrated that CSD in Hawaii is due to B. henselae and that infection is directly linked to the scratch or bite of a kitten. Older cats seldom have bacteremia but often have serologic evidence of past infection. Our study fails to implicate dogs in the epidemiology of CSD in Hawaii, and A. felis was not etiologically implicated in CSD in the human subjects and animals we studied.

Induction of a Potential Paracrine Angiogenic Loop between Human THP-1 Macrophages and Human Microvascular Endothelial Cells during Bartonella henselae Infection

ABSTRACT Bartonella henselae is responsible for various disease syndromes that loosely correlate with the immune status of the host. In the immunocompromised individual, B. henselae-induced angiogenesis, or bacillary angiomatosis, is characterized by vascular proliferative lesions similar to those in Kaposis sarcoma. We hypothesize that B. henselae-mediated interaction with immune cells, namely, macrophages, induces potential angiogenic growth factors and cytokines which contribute in a paracrine manner to the proliferation of endothelial cells. Vascular endothelial growth factor (VEGF), a direct inducer of angiogenesis, and interleukin-1β (IL-1β), a potentiator of VEGF, were detected within 12 and 6 h, respectively, in supernatants from phorbol 12-myristate 13-acetate-differentiated human THP-1 macrophages exposed to live B. henselae. Pretreatment of macrophages with cytochalasin D, a phagocytosis inhibitor, yielded comparable results, suggesting that bacterium-cell attachment is sufficient for VEGF and IL-1β induction. IL-8, an angiogenic cytokine with chemotactic properties, was induced in human microvascular endothelial cells (HMEC-1) within 6 h of infection, whereas no IL-8 induction was observed in infected THP-1 cells. In addition, conditioned medium from infected macrophages induced the proliferation of HMEC-1, thus demonstrating angiogenic potential. These data suggest that Bartonella modulation of host or target cell cytokines and growth factors, rather than a direct role of the bacterium as an endothelial cell mitogen, is the predominant mechanism responsible for angiogenesis. B. henselae induction of VEGF, IL-1β, and IL-8 outlines a broader potential paracrine angiogenic loop whereby macrophages play the predominant role as the effector cell and endothelial cells are the final target cell, resulting in their proliferation.

Disseminated cat-scratch disease: Detection ofRochalimaea henselae in affected tissue

An immunocompetent 9-year-old boy with disseminated catscratch disease involving spleen, cervical and abdominal lymph nodes, skull, and one clavicle is reported. Antibodies toRochalimaea quintana andR. henselae were detected, at increasing, then decreasing concentration. DNA extracted from the biopsied skull lesion was amplified by polymerase chain reaction and hybridized with species-specific oligonucleotides proving the presence ofR. henselae in affected tissue. Our findings suggest thatR. henselea plays a pathogenic role in cat-scratch disease.

Role of Bartonella henselae in the etiology of Henoch-Schönlein purpura.

Background. The etiology of Henoch-Schönlein purpura (HSP) has been ascribed to a variety of infectious and noninfectious agents. Because we encountered a patient with HSP who had evidence of Bartonella henselae infection and a prior report of a patient with systemic cat-scratch disease presenting as leukoclastic vasculitis, we investigated the association of B. henselae infection with HSP. Methods. We determined the antibody titers to B. henselae on the sera of 18 patients with HSP and on 57 controls. All patients presented with the characteristic leukoclastic rash of HSP. About one-half of the patients had joint or abdominal symptoms, and four had hematuria at presentation. An indirect immunofluorescent assay was used to determine serum antibody titers to B. henselae. Sera that were reactive at a dilution of 1/64 were considered positive. Results. Eight of the 57 (14%) control sera and 12 of the 18 (67%) patient sera were positive for B. henselae antibody (P < 0.0001). Conclusion. The results of this study indicate a significant association of antecedent B. henselae infection with HSP. The frequency of this association (67%) exceeds that of previously ascribed etiologic agents for this disease, such as the group A Streptococcus.

Cloning, sequencing, and expression of three Bartonella henselae genes homologous to the Agrobacterium tumefaciens VirB region.

A 17-kDa, immunodominant antigen of Bartonella henselae Houston-1 has previously been cloned, sequenced, and characterized. This clone (H13) contains the 17-kDa antigen gene plus a partial open reading frame, designated ORF1, which is 459 nucleotides long and is directly upstream of the 17-kDa gene. Comparison of the deduced partial amino acid sequence of ORF1 with that of other known genes in GenBank revealed significant identity with several other bacterial virulence genes, including VirB4 of the Agrobacterium tumefaciens virB operon (56/149 amino acids). An overlapping clone, pGB3, was recovered and shown to contain a 3.0-kb region upstream of the 17-kDa gene. Sequence analysis revealed three ORFs upstream of the gene. The deduced amino acid sequence of each ORF was compared with sequences in GenBank, and identity was found with VirB2, VirB3, and VirB4 of A. tumefaciens. In vitro transcription/translation and SDS-PAGE demonstrated that three proteins of 9 kDa, 10 kDa, and 92 kDa, corresponding to the predicted molecular weight of 10.9 kDa, 11.7 kDa, and 89.9 kDa of VirB2, VirB3, and VirB4, respectively, could be expressed from these coding regions. These results indicate that virulence-associated genes and their overall chromosomal arrangement are relatively well conserved between B. henselae and other gram-negative bacteria such as A. tumefaciens.

V2 regions of 16S ribosomal RNA used as a molecular marker for the species identification of streptococci in peripheral blood and synovial fluid from patients with psoriatic arthritis

OBJECTIVE To detect the 16S ribosomal RNA (rRNA) of 3 streptococcal species in the peripheral blood and synovial fluid of patients with psoriatic arthritis (PsA). METHODS Reverse transcription-polymerase chain reaction (RT-PCR) detection targets bacterial 16S rRNA, which is present in bacteria at high copy numbers. The 3 species-specific primers for group A streptococci (GAS; Streptococcus pyogenes), group B streptococci (GBS; Streptococcus agalactiae), and Streptococcus pneumoniae were designed from the fragments of highly variable V2 regions of 16S rRNA. Total RNA was prepared from whole peripheral blood and joint fluid obtained from patients with PsA and rheumatoid arthritis (RA). All positive PCR reactions were then sequenced with a Pharmacia ALF DNA sequencing system. RESULTS Our data in 19 PsA patients showed that 7 peripheral blood samples were positive for GAS (P = 0.006 versus GAS-positive RA patients [n = 0], by Fishers exact test), and 2 were also positive for GBS. One synovial fluid sample from a PsA patient was positive for GAS. S pneumoniae was absent from all specimens. Seventeen patients with RA were PCR negative for the 3 streptococcal species. Peripheral blood from a patient with inflammatory bowel disease was positive for GAS. CONCLUSION The presence of GAS 16S rRNA in the peripheral blood and synovial fluid of patients with PsA supports the concept that PsA is a reactive arthritis to certain streptococci.

Protection of guinea-pigs from experimental Rocky Mountain spotted fever by immunization with baculovirus-expressed Rickettsia rickettsii rOmpA protein

Baculovirus recombinants that express the Rickettsia rickettsii rOmpA protein were constructed. Monoclonal antibodies (mAbs) against the rOmpA protein reacted with recombinant-infected Spodoptera frugiperda (Sf9) cells in indirect immunofluorescence assays. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of infected Sf9 cell lysates with a mAb against rOmpA showed that the recombinant-expressed rOmpA protein migrated slightly below rOmpA extracted from R. rickettsii. Guinea-pigs immunized with lysates of recombinant-infected Sf9 cells developed antibodies reactive with R. rickettsii and were protected against challenge, indicating that the baculovirus-expressed rOmpA protein could be useful in subunit vaccines and for studies of the immune response to R. rickettsii infection.

Intracellular Induction of the Bartonella henselae virB Operon by Human Endothelial Cells

ABSTRACT One of the more recently identified bacterial exportation systems is the type IV secretion mechanism, which is characterized by a multiprotein complex that spans the inner and outer bacterial membranes and contains a pilin component. The most thoroughly studied type IV secretion system is encoded by the virBoperon of Agrobacterium tumefaciens. In Bartonella henselae, 8 of the 10 virB operon genes share extensive homology and arrangement with the virBoperon of A. tumefaciens. Sequencing of the region upstream of the B. henselae virB2gene revealed a region with sequence homology to the virbox of A. tumefaciens. This possible promoter region was cloned upstream of the green fluorescent protein reporter gene in the promoterless vector pANT3 and used to transform B. henselae. Minimal reporter gene expression was seen in the transformed bacteria cultivated in the absence of host cells, but expression was strongly induced in intracellular bacteria cultivated with human microvascular endothelial cells. Deletion of an 87-bp fragment, which contained the putative vir box from the 5′ end of the promoter region, diminished intracellular induction of the reporter gene. Host cell induction of the 17-kDa antigen gene, which replaces virB5 in B. henselae, was also demonstrated at the protein level using specific antiserum. Thus, expression of the virBgenes of B. henselae is induced in bacteria, which have invaded host cells, through a mechanism that may be similar to the environment-sensing mechanism found in thevirB operon of A. tumefaciens.

Isolation, sequencing and expression of the gene encoding a major protein from the backteriophage associated with Bartonella henselae.

The gene encoding a 31-kDa major protein (Pap31) associated with the bacteriophage harbored in Bartonella henselae was cloned and sequenced. Analysis of the resulting sequence revealed an open reading frame of 837 nucleotides coding for a protein of 279 amino acids. pap31 was then subcloned downstream of the lacZ promoter in pUC19. pap31 was amplified by polymerase chain reaction, and the linear amplicon was used as template for in-vitro transcription and translation. A protein with an apparent molecular mass of approximately 31 kDa was synthesized from this reaction. Upon analysis of the deduced aa sequence, a potential signal sequence and a consensus signal peptidase cleavage site were identified, indicative that Pap31 is modified posttranslationally, and the mature protein may be targeted to the host membrane.