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Journal of Clinical Microbiology, August 2000, p. 2943-2948, Vol. 38, No. 8
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Characterization of Bartonella clarridgeiae Flagellin
(FlaA) and Detection of Antiflagellin Antibodies in Patients
with Lymphadenopathy
Anna
Sander,1,*
Anja
Zagrosek,1
Wolfgang
Bredt,1
Emile
Schiltz,2
Yves
Piémont,3
Christa
Lanz,4 and
Christoph
Dehio4,5
Institute for Medical Microbiology and
Hygiene1 and Institute of Organic
Chemistry and Biochemistry,2 University of
Freiburg, Freiburg, and Max Planck Institute for Biology,
Tübingen,4 Germany; Institute
de Bactériologie de la Faculté de Médecine,
Université Louis-Pasteur, Strasbourg,
France3; and Division of Microbiology,
Biocenter of the University of Basel, Basel,
Switzerland5
Received 28 December 1999/Accepted 29 May 2000
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ABSTRACT |
Cat scratch disease (CSD) is a frequent clinical outcome of
Bartonella henselae infection in humans. Recently, two case
reports indicated Bartonella clarridgeiae as an additional
causative agent of CSD. Both pathogens have been isolated from domestic
cats, which are considered to be their natural reservoir. B. clarridgeiae and B. henselae can be
distinguished phenotypically by the presence or absence of flagella,
respectively. Separation of the protein content of purified flagella of
B. clarridgeiae by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis and immunoblot analysis indicated that the
flagellar filament is mainly composed of a polypeptide with a mass of
41 kDa. N-terminal sequencing of 20 amino acids of this protein
revealed a perfect match to the N-terminal sequence of flagellin
(FlaA) as deduced from the sequence of the flaA gene cloned
from B. clarridgeiae. The flagellin of B. clarridgeiae is closely related to flagellins of Bartonella
bacilliformis and several Bartonella-related
bacteria. Since flagellar proteins are often immunodominant antigens,
we investigated whether antibodies specific for the FlaA protein of
B. clarridgeiae are found in patients with CSD or
lymphadenopathy. Immunoblotting with 724 sera of patients suffering
from lymphadenopathy and 100 healthy controls indicated specific FlaA
antibodies in 3.9% of the patients' sera but in none of the controls.
B. clarridgeiae FlaA is thus antigenic and expressed in
vivo, providing a valuable tool for serological testing. Our results
further indicate that B. clarridgeiae might
be a possible etiologic agent of CSD or lymphadenopathy. However, it
remains to be clarified whether antibodies to the FlaA protein of
B. clarridgeiae are a useful indicator of acute infection.
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INTRODUCTION |
Cat scratch disease (CSD) caused by
Bartonella henselae is the most common Bartonella
infection worldwide. In its typical form, CSD is a self-limiting
regional lymphadenopathy, although atypical clinical courses with
severe disease manifestations can also occur (1, 3).
Recently, Bartonella clarridgeiae was suggested as an
additional causative agent of CSD. This pathogen was initially isolated
from the cat of a human immunodeficiency virus (HIV)-positive patient
with CSD. The blood culture from the patient himself, however, grew
only Bartonella henselae (7). B. clarridgeiae is closely related to the other Bartonella
species, with similarities in the 16S rRNA ranging from 97.4 to 98.5%
(17). B. clarridgeiae carries flagella
(17) as previously described for Bartonella
bacilliformis (33), but not for any other
Bartonella species described up to now (9).
Evidence for the involvement of B. clarridgeiae in causing
CSD comes from two recent case reports. Kordick et al. (15)
described a case of lymphadenopathy after a cat bite. B. clarridgeiae was isolated from a blood culture of the patient's
cat, and antibodies against this agent were found in the patient's
sera during the acute and convalescent phases by indirect
fluorescent-antibody (IFA) test. The second case of CSD suspected to be
caused by B. clarridgeiae was described in 1998 by Margileth
and Baehren (19). The symptoms of the 35-year-old patient
(fever, chills, night sweat, headaches, and somnolence) were
interpreted as possible CSD. From an abscess of the chest wall,
however, pneumococci had been isolated. Retrospective examination of
the patient's serum showed a titer of 1:128 only against B. clarridgeiae, and this species was isolated from a blood culture of his cat.
Both species, B. henselae and B. clarridgeiae,
have been isolated from domestic cats, which are considered to be
the natural reservoir of these bacteria (5, 6, 12-14, 20, 22,
30). Attempts to isolate Bartonella spp. from
immunocompetent patients suffering from CSD usually lack a positive
culture. Currently, CSD is diagnosed serologically in most cases.
Serological investigations for antibodies against
Bartonella sp. showed a cross-reactivity between
B. henselae and Bartonella quintana of 95 to
100% in many studies (10, 11, 23, 31). Similar
cross-reactions have also been observed in our laboratory with B. clarridgeiae as antigen (IFA based on infected Vero cells;
unpublished data). Therefore, a specific marker was needed for
confirmation of suspected B. clarridgeiae infections.
It was shown that B. henselae may have pili
(4), whereas B. clarridgeiae possesses multiple
unipolar flagella (17). Besides B. bacilliformis,
B. clarridgeiae is the only human pathogenic
Bartonella species which is flagellated. It was the aim of
this study to characterize the B. clarridgeiae flagellin
subunit (FlaA) and to examine its usefulness for serological detection
of B. clarridgeiae infections.
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MATERIALS AND METHODS |
Bacterial strains and growth conditions.
The B. clarridgeiae strain used in this study was isolated from the blood
of a free-ranging cat from the city of Nancy, France (13).
Cultures were grown on Columbia agar (Difco Laboratories, Augsburg,
Germany) supplemented with 5% defibrinated sheep blood. The plates
were incubated at 37°C in a humid atmosphere containing 5% carbon
dioxide. Growth was usually observed after 3 days of incubation, and
the cultures were harvested at 6 days postinoculation. B. bacilliformis ATCC 35685T was grown on glucose-yeast
extract-cysteine-containing blood agar (Sifin GmbH, Berlin, Germany),
and plates were incubated at 26°C in a humid atmosphere.
Isolation and purification of flagella.
Early passages of
B. clarridgeiae from 15 to 20 agar plates were harvested
into 5 ml of phosphate-buffered saline (PBS). This suspension was
blended in a commercial blender (KS 10; Bühler, Tübingen,
Germany) for 30 min at room temperature to shear off the flagella.
Bacteria were sedimented by centrifugation at 4,000 × g for 40 min, and the resulting supernatant was incubated for 12 h with saturated ammonium sulfate (vol/vol). This preparation was centrifuged at 4,000 × g for 40 min, and the
resulting pellet was suspended in 1 ml of phosphate-buffered saline
(PBS) and dialyzed four times for 30 min at 4°C against 2,000 ml of
PBS to remove the ammonium sulfate. The same process was used to
obtain a crude flagella preparation from B. bacilliformis.
SDS-PAGE.
Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) was performed by the method of Laemmli
(16). The stacking and separating gels contained 4.5 and
10% acrylamide, respectively. Gel lanes were loaded with 15 to 20 µg
each of protein and run in a Mini-Protean II cell (Bio-Rad) at 100 V
for 2 h. Protein bands were stained with Coomassie brilliant blue
R-250 (Serva). The molecular masses of the proteins were calculated
from a calibration curve prepared with a low-molecular-mass calibration
kit (Amersham Pharmacia Biotech, Freiburg, Germany). Protein
concentrations were determined by the method described by Peterson
(27) with bovine serum albumin as a standard.
N-terminal amino acid sequencing of B. clarridgeiae
flagellin.
A preparative SDS-polyacrylamide gel was performed with
the isolated flagella fraction. The proteins were electrophoretically transferred overnight in a borate buffer (50 mM boric acid, 10% [vol/vol] methanol, 0.02% mercaptoethanol [pH 8.6]) to a
0.45-µm-pore-size polyvinylidene difluoride membrane (Immobilon-P;
Millipore Corporation, Bedford, Mass.) in a Transblot-Cell (Bio-Rad),
washed in distilled water, and visualized by staining with Ponceau-S.
The suspected 41-kDa flagellin band was excised from the gel, and the
first 20 N-terminal amino acids were sequenced by Edman degradation with a gas-phase protein sequencer 477A (Applied Biosystems) equipped with online analysis of the amino acid derivatives.
Cloning and sequencing of the flaA gene of B. clarridgeiae and B. bacilliformis.
The flaA
gene of B. clarridgeiae and B. bacilliformis was
PCR amplified with primer pairs prCHD127 and prCHD128 and prCHD126 and
prCHD128, respectively, which were delineated from the published sequence of the B. bacilliformis flaA gene (accession no.
L20677) (Table 1). Hotstart PCR
amplification was performed with 0.5 µg of CsCl-purified bacterial
DNA, 100 pmol of each primer, 1× reaction buffer (Stratagene), 25 µM
deoxynucleoside triphosphates (dNTPs) (Pharmacia), 2.5 U of
Taq polymerase (BRL Gibco), and 2.5 U of Taq
extender (Stratagene) in a total volume of 100 µl. The reaction
mixture was heated to 95°C for 30 s prior to addition of
Taq polymerase. Twenty-five cycles of amplification for
10 s at 95°C, 10 s at 50°C, and 7 min at 72°C were
performed, followed by a 10-min incubation at 72°C. The generated PCR
products were isolated by electrophoresis in a 1% TAE-agarose gel and
purified from an excised gel fragment by a Geneclean kit I (Bio101,
Dianova). The purified PCR fragments were cloned into the vector
pTOPO-TA by using the TOPO TA cloning kit (Invitrogen), resulting in
plasmid pCD387 for B. bacilliformis flaA and plasmid pCD388
for B. clarridgeiae flaA. DNA sequencing of the insert of
pCD387 and pCD388 was performed with an ABI 377 DNA sequencer
(Applied Biosystems). Sequencing reactions were performed by the
AmpliTaq BigDye Terminator Cycle Sequencing Ready
Reaction Kit (Perkin-Elmer) with primers prCHD126, prCHD128, prCHD151,
M13-forward, and M13-reverse for pCD387 and primers prCHD127, prCHD128,
M13-forward, and M13-reverse for pCD388 (Table 1). The sequence
obtained for pCD387 confirmed the published sequence of the
flaA gene of B. bacilliformis (accession no.
L20677), except for a deletion of two cytosines at positions 1264 and
1265. The sequence obtained for pCD388 contained most of the
flaA open reading frame (ORF) of B. clarridgeiae,
including 29 bp of 5'-untranslated sequences, while the very 3' end of
this ORF was not contained in the amplicon and was sequenced by
using chromosomal DNA as a template. Chromosomal sequencing was
performed with a LiCor automatic sequencing system (MWG Biotech) with
the infrared dye-labeled (IR 800) primer prCHD153 (Table 1). The
sequencing reactions were carried out by using 10 µg of sheared
chromosomal DNA and the Thermo Sequenase fluorescence-labeled primer
cycle sequencing kit (Amersham Life Science) according to the
manufacturer's instructions. From the resulting chromosomal
sequence, primer prCHD165 was deduced and used for the
generation of the PCR products prCHD151/165 and pcr127/165, which
encompass the 3' end of flaA (Table 1). Geneclean-purified PCR fragments were used as a template for AmpliTaq
sequencing reactions with primers prCHD151, prCHD165, prCHD168, and
prCHD169 (Table 1) to complete double-stranded
sequencing of the B. clarridgeiae flaA gene.
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TABLE 1.
Primer sequences for amplification and sequencing of the
flaA gene of B. clarridgeiae
and B. bacilliformis
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Human sera.
Sera from 724 patients with lymphadenopathy, for
which CSD was considered in the differential diagnosis, and 100 sera
from healthy controls have been investigated for detection of
antibodies to the B. clarridgeiae FlaA protein. Evidence of
infection with B. henselae was determined by IFA as
described previously (31). Ten sera from patients with
Salmonella enterica serovar Typhi infections, 9 sera from
patients with Yersinia enterocolitica infections, 19 sera
from patients with Borrelia burgdorferi infections, 15 sera
from patients with H. pylori infections, 2 sera from
patients suffering from brucellosis, and 1 serum from an HIV-positive
patient suffering from an Agrobacterium radiobacter
infection were tested for cross-reactivity to B. clarridgeiae FlaA protein. All patients' sera having FlaA
antibodies against B. clarridgeiae were additionally tested
against the FlaA protein of B. bacilliformis.
An antiserum produced in rabbit against
B. clarridgeiae (Y. Piémont, Strasbourg, France, unpublished data) served as a
positive
control, and an antiserum produced in rabbit against
Rhizobium meliloti flagellin (kindly provided by R. Schmitt,
Regensburg,
Germany) (
28) was tested for cross-reactivity
with the FlaA
protein of
B. clarridgeiae.
Western blot analysis.
For Western blot analysis, the
polyacrylamide gel was prepared as described above and the proteins
were electrophoretically transferred to a polyvinylidene difluoride
membrane (Immobilon-P; Millipore Corporation, Bedford, Mass.) as
described by Towbin et al. (35). The membrane was blocked
with 3% nonfat dry milk in PBS containing 0.05% Tween 20 (PBST),
subsequently washed, incubated with a 1:100 dilution of the test sera
in PBST-3% milk solution for 1 h at room temperature, washed
three times in PBST, and incubated with a 1:1,000 dilution of alkaline
phosphatase-conjugated antihuman immunoglobulin G (IgG) (heavy and
light chains) (Jackson ImmunoResearch Laboratories, Inc., West Grove,
Pa.) for 1 h at room temperature. After three additional washes,
antibodies were detected with BCIP/NBT (5-bromo-4-chloro-3-indolyl
phosphate-nitroblue tetrazolium; Sigma Chemical Company, St. Louis,
Mo.).
Nucleotide sequence accession number.
The nucleotide
sequence of the flaA locus of B. clarridgeiae
(bases 1 to 1260) has been assigned database accession no. AJ251711.
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RESULTS |
Isolation and identification of B. clarridgeaie
flagella.
Flagella were sheared off from the whole B. clarridgeiae cells by intensive shaking. This crude flagellum
preparation showed on a Coomassie brilliant blue-stained SDS-PAGE gel
two prominent bands at 41 and 60 kDa (Fig.
1). Some additional minor bands at approximately 98, 35, 32, and 29 kDa were seen in the flagellum preparation, which were also present as major bands in the untreated cells and in the cellular debris. Their identity, however, remains unknown. The efficiency of the method was tested by isolation of
flagella from various other flagellated bacteria, including B. bacilliformis, Proteus mirabilis, Salmonella
enterica serovar Enteritidis, and Salmonella enterica
serovar Paratyphi A (results not shown). No comparable proteins were
found in supernatants from the unflagellated species B. henselae after similar treatment (results not shown).
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FIG. 1.
Analysis of B. clarridgeiae flagellin
preparation by SDS-PAGE and Coomassie brilliant blue staining. Lanes:
MW, molecular mass marker; 1, whole-cell lysates of B. clarridgeiae; 2, cellular debris after shearing off the flagella;
3, crude flagellar protein. The arrow indicates the position of the
41-kDa band identified as the FlaA protein.
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N-terminal amino acid sequencing of B. clarridgeiae
flagellin.
The first 20 N-terminal amino acids of the flagellin
subunit were found to be GTSLLTNKSAMTALQTLXSI and were compared
with known flagellin sequences (BLAST Search). Sixteen residues were exact matches with those of B. bacilliformis
flagellin, and 4 residues were conservatively replaced. The following
sequencing of the flaA gene confirmed the amino acid
sequence of the N terminus (Fig. 2).
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FIG. 2.
DNA sequence and protein translation of the B. clarridgeiae flagellin locus (flaA). The DNA sequence
and translation of the flaA ORF are shown. The putative
ribosome binding site (RBS) is underlined, and the N-terminal amino
acids determined by protein sequencing are in boldface.
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Cloning and sequencing of the B. clarridgeiae flagellin
gene.
Heterologous primers deduced from the published sequence of
B. bacilliformis flagellin (flaA) locus were used
to amplify a major part of the flaA gene of B. clarridgeiae. The amplicon was cloned and sequenced, and the
missing part at the very 3' end of this gene was determined by
chromosomal sequencing and confirmed by sequencing of longer-range PCR
products generated with primers deduced from the chromosomal sequence.
A total of 1,260 bp of double-stranded sequence was determined,
encompassing 29 bp of 5'-untranslated sequence, the ORF, and 37 bp of
3'-untranslated sequence (Fig. 2). The ORF of 1,197 bp encodes a
protein of 399 amino acids. The 20 N-terminal amino acids of the major
flagellin subunit determined by protein sequencing are perfectly
matched, suggesting that the cloned flaA gene of B. clarridgeiae encodes the major flagellar subunit characterized
before (Fig. 2). The deduced amino acid sequence of B. clarridgeiae is most closely related to flagellin encoded by
B. bacilliformis (accession no. L20677) (Fig.
3A) with 68% identity and 80%
similarity. Resequencing of the B. bacilliformis flaA gene
revealed an error in the published sequence, resulting in a reading
frame shift close to the stop codon (see Materials and Methods). An
average distance tree generated after alignment of flagellin
protein sequences of various bacterial species is illustrated in Fig.
3B. Related bacteria of the 
2-subgroup of proteobacteria, such
as B. clarridgeiae and B. bacilliformis, Brucella abortus, Caulobacter crescentus,
Rhizobium meliloti, Agrobacterium tumefaciens,
and Azospirillum brasiliense are clustered in one
clade, while the majority of flagellated bacterial pathogens, such as
Legionella pneumophila, Pseudomonas aeruginosa,
Vibrio cholerae, Serratia marcescens,
Yersinia enterocolitica, S. enterica serovar
Typhimurium, Escherichia coli, and Borrelia
burgdorferi, fall into a separate clade, and Campylobacter
jejuni and Helicobacter pylori are even less related
(Fig. 3B).
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FIG. 3.
Comparison of the protein sequence of B. clarridgeiae FlaA with those of other known flagellins. (A)
Protein sequence comparison of FlaA from B. clarridgeiae
(this work) and B. bacilliformis (accession no. L20677
[sequence corrected by resequencing]) by BLAST search. Identities and
similarities are indicated by vertical bars and colons, respectively.
(B) Average distance tree of a protein sequence alignment (CLUSTAL W)
of flagellins from various bacteria. Abr, Azospirillum
brasiliense Laf1 (accession no. U26679); Atu, Agrobacterium
tumefaciens FlaD (accession no. U95165); Bba, Bartonella
bacilliformis flagellin (accession no. L20677); Bcl,
Bartonella clarridgeiae (this work); Bbu, Borrelia
burgdorferi flagellar filament 41-kDa core protein (flagellin)
(accession no. P11089), Bab, Brucella abortus FliC
(accession no. AF019251); Ccr, Caulobacter crescentus
flagellin Fljm (accession no. 052529); Cje, Campylobacter
jejuni flagellin A (accession no. AAC25643); Eco,
Escherichia coli flagellin (accession no. AB028473); Hpy,
Helicobacter pylori flagellin B (accession no. AE001449);
Lpn, Legionella pneumophila flagellin (accession no.
X83232); Pae, Pseudomonas aeruginosa flagellin (accession
no. AF034764); Rme, Rhizobium meliloti flagellin
flaA (accession no. A39436); Sma, Serratia
marcescens flagellin (accession no. D32256); Sty, Salmonella
enterica serovar Typhimurium flagellin (accession no. AAB33952);
Vch, Vibrio cholerae flagellin (accession no. AF007121);
Yen, Yersinia enterocolitica thermoregulated motility
protein (accession no. L33468).
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Altogether, the cloned
flaA locus of
B. clarridgeiae encodes a major flagellar subunit, which is well
conserved among the
2 proteobacteria, but clearly different from
flagellins of other
bacteria.
Western blot analysis.
Immunoblot analysis with rabbit
anti-B. clarridgeiae antiserum demonstrated the
immunogenicity of the 41-kDa protein. The FlaA protein was recognized
by the antiserum against B. clarridgeiae in both the
whole-cell lysate of B. clarridgeiae and the flagellar preparation. This flagellin subunit of B. clarridgeiae was
further recognized by positive immunoblot reactions with an antiserum against both Agrobacterium radiobacter and Rhizobium
meliloti, two bacteria closely related to Bartonella
spp. (21). These results indicated that FlaA might be an
immunogenic protein during B. clarridgeiae infections.
However, it must be considered that sera containing antibodies to
B. bacilliformis, A. radiobacter, or R. meliloti may cross-react with this protein.
The potential applicability of the FlaA protein as an antigen for
detection of
B. clarridgeiae infections in humans was
examined
by Western blot analysis of 724 sera of patients with
lymphadenopathy
and suspected CSD. Based on an IFA titer against
B. henselae of
1:
512, CSD was diagnosed serologically in
156 patients. Nearly
one-third (229) of all patients had low antibody
titers of 1:64
to 1:256, indicating the onset or the end of CSD or
simply contact
with
Bartonella species in the past.
B. clarridgeiae antiflagellin
antibodies were present in 3.9% (28 of
724) of the serum samples
(Table
2).
Typical reactions of some of these sera with the
B. clarridgeiae FlaA protein are shown in Fig.
4. The 60-kDa protein
seems not to be of
immunologic importance in all these sera. All
serological results,
including the detection of antiflagellin
antibodies to
B. clarridgeiae by Western blot analysis and the
antibody titers
against
B. henselae measured in the IFA assay
are
given in Table
2. There was no correlation between the sera
containing
antiflagellin antibodies and the IFA titer against
B. henselae. Seven patients with antibodies against the FlaA protein
had clinically and serologically (
B. henselae titer of
1:512
in the IFA) confirmed CSD, 12 had low antibody titers, and 9 had
no antibodies to
B. henselae in the IFA. No
antibodies against
the 41-kDa protein were found in the 100 sera of the
healthy control
group. Sera of patients with antibodies against
flagellated bacteria
like
Salmonella enterica serovar Typhi
(10 sera),
Brucella spp.
(2 sera),
Yersinia
enterocolitica (9 sera),
Helicobacter pylori (15 sera),
and
Borrelia burgdorferi (19 sera) were tested for
cross-reactivity to the FlaA protein of
B. clarridgeiae.
None
of these 55 sera reacted with the 41-kDa protein. In contrast,
the
FlaA protein band was recognized by the human serum with an
A. radiobacter infection and by the rabbit
R. meliloti antiserum.
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TABLE 2.
IFA titers (IgG) of anti-B. henselae
antibodies and antibody recognition of the 41-kDa (FlaA) band in sera
of 724 patients with lymphadenopathy and 100 healthy controls
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FIG. 4.
Examination of human sera for antibodies to B. clarridgeiae FlaA protein by immunoblot analysis. Lanes: MW,
molecular mass marker; Bc and Fla, amido black-stained B. clarridgeiae whole-cell lysate and flagellin preparation,
respectively; 1, positive control (rabbit antiserum); N, negative
control without serum; 2, 3, 4, 8, 9, and 10, negative sera; 5, 6, and
7, positive reactive sera containing antiflagellin antibodies. The
arrow indicates the position of the 41-kDa FlaA protein.
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The positive reaction of the 28 sera against the 41-kDa
protein of
B. clarridgeiae was not changed by absorption
of these
sera with
B. henselae cells. However, it was
considerably removed
in all cases by absorbing with
B. clarridgeiae whole-cell antigen,
indicating the specificity of the
antiflagellin antibodies (Fig.
5).
Absorption of the sera with
B. henselae whole cells resulted
in a strongly diminished reaction of some other protein bands
(with
exception of the 41-kDa band) in the immunoblot, which confirms
the cross-reactivity between
B. henselae and
B. clarridgeiae,
as already seen in the IFA. Additionally, 7 of the 28 anti-
B. clarridgeiae FlaA-positive sera did
recognize the FlaA protein
of
B. bacilliformis, indicating
a cross-reactivity between the
two
Bartonella species.
The FlaA protein band of
B. bacilliformis migrated faster
than that of
B. clarridgeiae in each of our flagellin
preparations as well as in the protein profile of the whole bacteria
(data not shown). We determined an apparent molecular mass of
40 kDa
for the
B. bacilliformis flagellin as opposed to 42 kDa
reported previously (
33).
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FIG. 5.
Immunoblot analysis of a representative anti-FlaA
antibody-positive serum showing the specificity of the anti-B.
clarridgeiae-flagellin antibodies. Lanes: MW, molecular mass
marker; Bh and Bc, amido black-stained B. henselae and
B. clarridgeiae whole-cell antigen, respectively; 1, unabsorbed serum; 2, serum absorbed with whole cells of B. henselae; 3, serum absorbed with whole cells of B. clarridgeiae.
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DISCUSSION |
Some Bartonella spp. cause identical clinical symptoms
(e.g., B. quintana or B. henselae in bacillary
angiomatosis), and in these cases, differential diagnosis by serology
proves difficult because of considerable antigenic cross-reactions
(10, 11, 23, 31). Similar problems certainly arise with
B. clarridgeiae as an additional causative agent of CSD.
This problem will be even more complicated by the fact that both
B. henselae and B. clarridgeiae have their
reservoirs in cats, and humans injured by cats can be infected either
by only one of the two agents (7) or by both. In such a
situation, the presence of a specific antigen in one of the related
species could provide a valuable tool for a specific test.
Although the flagellins of B. bacilliformis and B. clarridgeiae are, according to our results, closely related,
cross-reactions are less likely to occur because of the geographical
restriction of B. bacilliformis to the Andean regions of
South America. However, in 25% (7 of 28) of the B. clarridgeiae anti-FlaA-positive sera, a cross-reaction was seen
with the FlaA protein of B. bacilliformis. This must be
considered when sera from patients living in areas in which B. bacilliformis is endemic are investigated. Other closely Bartonella-related bacteria, like
Azospirillum, Caulobacter crescentus, or
Rhizobium meliloti, are not pathogenic for humans, except
for some Brucella species and also Agrobacterium
radiobacter (tumefaciens), which has been shown to be
an opportunistic pathogen in immunocompromised individuals (29,
34). We could demonstrate that an antiserum against
Agrobacterium radiobacter and against Rhizobium
meliloti strongly cross-reacted with the 41-kDa FlaA protein
of B. clarridgeiae. However, such a cross-reactivity was
not observed with human sera from patients suffering from brucellosis.
Dendrogram analysis of several flagellin sequences has shown
that the Bartonella flagella together with
Azospirillum, R. meliloti, A. tumefaciens, and C. crescentus flagellins form a
cluster that is separated from all other flagellins, including that of
E. coli (Fig. 3B) (24). Flagellar proteins
of other pathogenic bacteria are clearly different, and with sera of
patients suffering from salmonellosis or Lyme borreliosis, for example,
we could not detect cross-reactions to the 41-kDa protein. In a
comparison of the protein profiles of the human pathogenic
Bartonella species like B. henselae, B. quintana, B. elizabethae, B. bacilliformis,
and B. clarridgeiae, a 41-kDa protein band is found only for
the flagellated species B. clarridgeiae
(32; data not shown). In our investigations, the
FlaA protein band of B. bacilliformis was found to migrate with an apparent molecular mass of 40 kDa as opposed to 42 kDa reported
previously (33).
The simultaneous occurrence of antibodies against both B. henselae and B. clarridgeiae provides a diagnostic
problem, which only can be solved by careful diagnostic observation of
patients with acute CSD. This could be done by serologic follow-up in
the acute and convalescent phases of disease or identification of the
infecting agents by other methods, like culture or PCR. Unfortunately only one lymph node was available from our 28 patients with antibodies against the 41-kDa protein, and the PCR in this case indicated an
infection with B. henselae rather than with B. clarridgeiae (results not shown). Similar observations were made
by Lawson and Collins (17) when they first isolated B. clarridgeiae from a patient's cat, although the patient himself,
however, suffered from a B. henselae septicemia. It
cannot be excluded that either the infection with B. clarridgeiae is less symptomatic than the typical B. henselae infection or a simultaneous infection with both
agents from the same source may occur. Alternatively, since B. clarridgeiae is also found in cats (12, 13, 14, 20), patients injured by cat bites or scratches in particular may suffer from double infections with these Bartonella species.
A large number of studies have been carried out with various
flagellated human pathogens, demonstrating the immunogenic effect of
the flagellins (2, 8, 18, 25, 26, 33, 36). For some
infections, FlaA is a major antigen and anti-FlaA antibodies can be
detected in the serum of infected hosts (18, 26). In contrast, for Borrelia burgdorferi, FlaA was found not to be
an immunodominant antigen in mammalian hosts (mouse, rabbit, and rhesus
monkey), but it was expressed in some patients with Lyme disease
(36). Anti-FlaB is usually the first detectable antibody found in the acute stage of Lyme disease and is consistently present during the entire infection (8, 36). A study with sera of patients with culture-proven Legionella pneumophila
serogroup 1 pneumonia demonstrated that antibodies against flagella
appeared later in the course of infection (2), a situation
contrary to infections with Borrelia burgdorferi
(8).
Up to now, no culture-proven or at least PCR-proven
B. clarridgeiae infection in humans has been
reported. Since the culturing of Bartonella species from
human specimens is very difficult, serologic testing for antibodies
will remain the main diagnostic procedure for Bartonella
infections. Our results suggest that B. clarridgeiae indeed
might be a possible, but rare, agent of CSD. However, whether
antibodies to the FlaA protein of B. clarridgeiae are a
useful indicator of acute infection remains to be clarified. Because of
the lack of a "gold standard" (e.g., culture), serologic results
should be interpreted critically until the role of B. clarridgeiae and the detection of anti-FlaA antibodies in CSD have
been sufficiently confirmed.
| |
ACKNOWLEDGMENTS |
We greatly thank Stefan Bereswill for helpful suggestions, Tanja
Schülin and Daniela Huzly for providing the Agrobacterium radiobacter antiserum, and Rüdiger Schmitt for kindly
providing the Rhizobium meliloti antiserum. We also thank
Karin Oberle, Ina Wagner, and Vera Augsburger for excellent technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Hygiene, Institute for Medical Microbiology and
Hygiene, University of Freiburg, Hermann-Herder-Str. 11, D-79104
Freiburg, Germany. Phone: (49) 761-203 6529. Fax: (49) 761-203 6562. E-mail: sander{at}ukl.uni-freiburg.de.
| |
REFERENCES |
| 1.
|
Anderson, B. E., and M. A. Neuman.
1997.
Bartonella spp. as emerging human pathogens.
Clin. Microbiol. Rev.
10:203-219[Abstract].
|
| 2.
|
Bangsborg, J. M.,
G. Shand, and N. Høiby.
1993.
Antibody response to major cross-reactive Legionella antigens during infection, p. 26-29.
In
J. M. Barbaree, R. F. Breiman, and H. P. Dufour (ed.), Legionella current status and emergent prospectives. American Society for Microbiology, Washington, D.C.
|
| 3.
|
Bass, J. W.,
J. M. Vincent, and D. A. Person.
1997.
The expanding spectrum of Bartonella infections. II. Cat-scratch disease.
Pediatr. Infect. Dis. J.
16:163-179[CrossRef][Medline].
|
| 4.
|
Batterman, H. J.,
J. A. Peek,
J. S. Loutit,
S. Falkow, and L. S. Tompkins.
1995.
Bartonella henselae and Bartonella quintana adherence to and entry into cultured human epithelial cells.
Infect. Immun.
63:4553-4556[Abstract].
|
| 5.
|
Bergmans, A. M. C.,
C. M. A. de Jong,
G. van Amerongen,
C. S. Schot, and L. M. Schouls.
1997.
Prevalence of Bartonella species in domestic cats in The Netherlands.
J. Clin. Microbiol.
35:2256-2261[Abstract].
|
| 6.
|
Chomel, B. B.,
E. T. Carlos,
R. W. Kasten,
K. Yamamoto,
C.-C. Chang,
R. S. Carlos,
M. V. Abenes, and C. M. Pajares.
1999.
Bartonella henselae and Bartonella clarridgeiae infection in domestic cats from the Philippines.
Am. J. Trop. Med. Hyg.
60:593-597[Abstract].
|
| 7.
|
Clarridge, J. E., III,
T. J. Raich,
D. Pirwani,
B. Simon,
L. Tsai,
M. C. Rodriguez-Barradas,
R. Regnery,
A. Zollo,
D. C. Jones, and C. Rambo.
1995.
Strategy to detect and identify Bartonella species in routine clinical laboratory yields Bartonella henselae from human immunodeficiency virus-positive patient and unique Bartonella strain from his cat.
J. Clin. Microbiol.
33:2107-2113[Abstract].
|
| 8.
|
Craft, J. E.,
D. K. Fischer, and G. T. Shimamoto.
1986.
Antigens of Borrelia burgdorferi recognized during Lyme disease.
J. Clin. Investig.
78:934-939.
|
| 9.
|
Dehio, C., and A. Sander.
1999.
Bartonella as emerging pathogens.
Trends Microbiol.
7:226-228[CrossRef][Medline].
|
| 10.
|
Dupon, M.,
A. M. S. Delarclause,
P. Brouqui,
M. Drancourt,
D. Raoult,
A. Demascarel, and J. Y. Lacut.
1996.
Evaluation of serological response to Bartonella henselae, Bartonella quintana and Afipia felis antigens in 64 patients with suspected cat scratch disease.
Scand. J. Infect. Dis.
28:361-366[Medline].
|
| 11.
|
Engbaek, K., and C. Koch.
1994.
Immunoelectrophoretic characterization and cross-reactivity of Rochalimaea henselae, Rochalimaea quintana and Afipia felis.
APMIS
102:931-942[Medline].
|
| 12.
|
Gurfield, A. N.,
H.-J. Boulouis,
B. B. Chomel,
R. Heller,
R. W. Kasten,
K. Yamamoto, and Y. Piemont.
1997.
Coinfection with Bartonella clarridgeiae and Bartonella henselae and different Bartonella henselae strains in domestic cats.
J. Clin. Microbiol.
35:2120-2123[Abstract].
|
| 13.
|
Heller, R.,
M. Artois,
V. Xemar,
D. De Briel,
H. Gehin,
B. Jaulhac,
H. Monteil, and Y. Piemont.
1997.
Prevalence of Bartonella henselae and Bartonella clarridgeiae in stray cats.
J. Clin. Microbiol.
35:1327-1331[Abstract].
|
| 14.
|
Kordick, D. L., and E. B. Breitschwerdt.
1998.
Persistent infection of pets within a household with three Bartonella species.
Emerg. Infect. Dis.
4:325-328[Medline].
|
| 15.
|
Kordick, D. L.,
E. J. Hilyard,
T. L. Hadfield,
K. H. Wilson,
A. G. Steigerwalt,
D. J. Brenner, and E. B. Breitschwerdt.
1997.
Bartonella clarridgeiae, a newly recognized zoonotic pathogen causing inoculation papules, fever, and lymphadenopathy (cat scratch disease).
J. Clin. Microbiol.
35:1813-1818[Abstract].
|
| 16.
|
Laemmli, U. K.
1970.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature (London)
227:680-685[CrossRef][Medline].
|
| 17.
|
Lawson, P. A., and M. D. Collins.
1996.
Description of Bartonella clarridgeiae sp. nov. isolated from the cat of a patient with Bartonella henselae septicemia.
Med. Microbiol. Lett.
5:64-73.
|
| 18.
|
Li, Z.,
F. Dumas,
D. Dubreuil, and M. Jacques.
1993.
A species-specific periplasmatic flagellar protein of Serpulina (Treponema) hyodysenteriae.
J. Bacteriol.
175:8000-8007[Abstract/Free Full Text].
|
| 19.
|
Margileth, A. M., and D. F. Baehren.
1998.
Chest-wall abscess due to cat-scratch disease (CSD) in an adult with antibodies to Bartonella clarridgeiae: case report and review of the thoracopulmonary manifestations of CSD.
Clin. Infect. Dis.
27:353-357[Medline].
|
| 20.
|
Marston, E. L.,
B. Finkel,
R. L. Regnery,
I. L. Winoto,
R. Ross Graham,
S. Wignal,
G. Simanjuntak, and J. G. Olson.
1999.
Prevalence of Bartonella henselae and Bartonella clarridgeiae in an urban Indonesian cat population.
Clin. Diagn. Lab. Immunol.
6:41-44[Abstract/Free Full Text].
|
| 21.
|
Marston, E. L.,
J. W. Sumner, and R. L. Regnery.
1999.
Evaluation of intraspecies genetic variation within the 60 kDa heat-shock protein gene (groEL) of Bartonella species.
Int. J. Syst. Bacteriol.
49:1015-1023[Abstract/Free Full Text].
|
| 22.
|
Maruyama, S.,
S. Tanaka,
T. Sakai, and Y. Katsube.
1999.
Prevalence of Bartonella species among pet cats in Japan. 1st International Conference on Bartonella as Emerging Pathogens. Tubingen, Germany, March 5-7.
J. Microbiol. Methods
37:284-285. (Abstract.)
|
| 23.
|
McGill, S. L.,
R. L. Regnery, and K. L. Karem.
1998.
Characterization of human immunoglobulin (Ig) isotype and IgG subclass response to Bartonella henselae infection.
Infect. Immun.
66:5915-5920[Abstract/Free Full Text].
|
| 24.
|
Moens, S.,
K. Michiels,
V. Keijers,
F. Van Leuven, and J. Vanderleyden.
1995.
Cloning, sequencing, and phenotypic analysis of laf1, encoding the flagellin of the lateral flagella of Azospirillum brasilense Sp7.
J. Bacteriol.
177:5419-5426[Abstract/Free Full Text].
|
| 25.
|
Neumeister, B.,
M. Susa,
B. Nowak,
E. Straube,
G. Ruckdeschel,
J. Hacker, and R. Marre.
1995.
Enzyme immunoassay for detection of antibodies against isolated flagella of Legionella pneumophila.
Eur. J. Clin. Microbiol. Infect. Dis.
14:764-767[CrossRef][Medline].
|
| 26.
|
Norris, S. J., and Treponema pallidum Polypeptide Research Group.
1993.
Polypeptides of Treponema pallidum: progress toward understanding their structural, functional, and immunologic roles.
Microbiol. Rev.
57:750-779[Abstract/Free Full Text].
|
| 27.
|
Peterson, G. L.
1977.
A simplification of the protein assay method of Lowry et al. which is more generally applicable.
Anal. Biochem.
83:346-356[CrossRef][Medline].
|
| 28.
|
Pleier, E., and R. Schmitt.
1991.
Expression of two Rhizobium meliloti flagellin genes and their contribution to the complex filament structure.
J. Bacteriol.
173:2077-2085[Abstract/Free Full Text].
|
| 29.
|
Salavert, M.,
J. R. Breton,
D. Perez-Tamarit,
C. Perez-Belles, and M. Gobernado.
1997.
Persistent bacteremia and infection of an intravascular device by Agrobacterium radiobacter (tumefaciens) in a boy with AIDS.
Enferm. Infect. Microbiol. Clin.
15:496-497[Medline].
|
| 30.
|
Sander, A.,
C. Bühler,
K. Pelz,
E. von Cramm, and W. Bredt.
1997.
Detection and identification of two Bartonella henselae variants in domestic cats in Germany.
J. Clin. Microbiol.
35:584-587[Abstract].
|
| 31.
|
Sander, A.,
M. Posselt,
K. Oberle, and W. Bredt.
1998.
Seroprevalence to Bartonella henselae in patients with cat scratch disease and in healthy controls: evaluation and comparison of two commercial serological tests.
Clin. Diagn. Lab. Immunol.
5:486-490[Abstract/Free Full Text].
|
| 32.
|
Sander, A.
1998.
Microbiological diagnosis of Bartonella species and Afipia felis, p. 98-112.
In
A. Schmidt (ed.), Bartonella and Afipia species emphasizing Bartonella henselae, vol. 1. Karger, Basel, Switzerland.
|
| 33.
|
Scherer, D. C.,
I. DeBuron-Connors, and M. F. Minnick.
1993.
Characterization of Bartonella bacilliformis flagella and effect of antiflagellin antibodies on invasion of human erythrocytes.
Infect. Immun.
61:4962-4971[Abstract/Free Full Text].
|
| 34.
|
Southern, P. M., Jr.
1996.
Bacteremia due to Agrobacterium tumefaciens (radiobacter). Report of infection in a pregnant woman and her stillborn fetus.
Diagn. Microbiol. Infect. Dis.
24:43-45[CrossRef][Medline].
|
| 35.
|
Towbin, H.,
T. Staehelin, and J. Gordon.
1979.
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc. Nat. Acad. Sci. USA
76:4350-4354[Abstract/Free Full Text].
|
| 36.
|
Yigong, G. E., and N. W. Charon.
1997.
FlaA, a putative flagellar outer sheath protein, is not an immunodominant antigen associated with Lyme disease.
Infect. Immun.
65:2992-2995[Abstract].
|
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Abstract
Introduction
