Unité des Rickettsies, CNRS UPRES-A
6020, Faculté de Médecine, Université de la
Mediterranée, 13385 Marseille Cédex, France
Received 26 October 2000/Returned for modification 22 January
2001/Accepted 3 April 2001
Monoclonal antibodies (MAbs) which react with heat-resistant
proteins with molecular masses of 32 to 33 kDa of 14 different Bartonella species were produced. These antibodies did not
react with antigens of 26 diverse bacterial strains by
microimmunofluorescence assay except MAb B3D4, which reacted with
Chlamydia psittaci and Chlamydia trachomatis at
low titers. The identification of a common Bartonella
antigenic protein will make it possible to later produce a diagnostic
antigen by cloning and expressing it in Escherichia coli.
Moreover, these MAbs allow all Bartonella species to be identified to the genus level.
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TEXT |
The genus Bartonella
currently comprises 14 species. Human infections due to
Bartonella species are widely considered emerging diseases,
although they also include long-recognized diseases such as
Carrión's disease, trench fever, and cat scratch disease (15, 17, 23). Newer clinical manifestations, such as
bacillary angiomatosis, peliosis hepatis, chronic lymphadenopathy, and
endocarditis, which are sometimes due to uncommonly encountered species
such as Bartonella elizabethae, Bartonella vinsonii subsp.
berkhoffii, or Bartonella vinsonii subsp.
arupensis, have been recently identified (1, 23, 25,
28, 32). Serologic diagnosis of Bartonella spp. is
mostly based on microimmunofluorescence (MIF) serology that detects
antibodies to B. quintana and B. henselae only
(21, 23). A serologic test that detects antibodies against
all species is not available. Such a test needs to detect an epitope
common to, but also specific to, all Bartonella spp. A
monoclonal antibody (MAb) that can recognize this epitope would be the
first step towards detecting this antigen after cloning and expressing
the Bartonella genome in Escherichia coli in
order to produce it for use in an enzyme-linked immunosorbent assay.
Bartonella spp. may be isolated from clinical samples by
using cell culture systems with endothelial cells or blood- or
hemin-containing axenic media (21, 29). When isolated,
identification of Bartonella is mostly based on molecular
methods. The availability of a MAb that could screen
Bartonella at the genus level would avoid the use of
expensive and time-consuming molecular procedures on
non-Bartonella bacteria. We thus decided to produce and
characterize Bartonella genus-specific MAbs.
The sources of Bartonella strains used to screen hybridomas
and test the specificity of MAbs are presented in Table
1. Bartonella strains were
harvested and suspended in deionized water for sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or in
phosphate-buffered saline (PBS) for the MIF assay after 5 to 7 days of
culture on blood agar plates. The procedure for the production of MAbs
has been detailed elsewhere (12, 22). Briefly,
6-week-old female BALB/c mice were inoculated with B. henselae Houston-1 suspended in 0.5 ml of PBS. The supernatants of
the hybridomas were screened for antibodies to B. henselae by MIF. Representative hybridomas were subcloned twice by limiting dilution. Isotypes of MAbs were determined with an Immuno Type mouse
monoclonal antibody isotyping kit with antisera to mouse immunoglobulin
M (IgM), IgA, IgG1, IgG2a, IgG2b, and IgG3 (Sigma). Ascitic fluids were
produced by injecting about 3 × 106 cells of
hybridoma (B2D3 and B3D4) suspended in 0.5 ml of PBS into the mice 1 week after an intraperitoneal injection of 0.5 ml of pristane
(2,6,10,14-tetramethylpentadecane; Sigma). The MIF assay
(26) was used to screen hybridoma clones and to determine the specificity of the MAbs. Blind testing of 45 bacteria by MIF with
MAbs B2D3 and B3D4 was carried out on 19 Bartonella strains, 3 Chlamydia strains, and 23 bacterial strains isolated in
our laboratory from clinical samples (Table 1). Sera from immunized mice were used as positive controls, and sera from healthy mice were
used as negative controls. SDS-PAGE and Western blotting were performed
according to a modification of the method described by Laemmli
(19, 22). Five human body lice from a laboratory colony
were infected with a B. quintana strain by feeding on a bacteremic rabbit previously infected intravenously by 108
B. quintana cells. B. quintana bacteremia at the
time the lice were fed was assessed by blood culture as previously
described for cats (3). After being crushed and smeared
onto microscope slides the lice were tested for Bartonella
by MIF as described above with ascitic fluid of hybridoma B2D3 diluted
1:1,000.
SDS-PAGE analysis of Bartonella antigens demonstrated
distinct profiles of Bartonella species. Depending on
species, 12 to 35 bands were observed. Proteins of 85, 71, 54, 44 to
47, 40, 36, 32 to 33, 30, and 18 to 19 kDa were common to all
Bartonella strains studied (Fig.
1a). Both MAbs reacted with all tested
Bartonella species. The immunofluorescence assay titers of
MAbs with different Bartonella bacteria showed obvious
differences. Titers from the homologous strain Houston-1 were the
highest. The isotypes of B2D3 and B3D4 were identified as subclass
IgG1. MAbs B2D3 and B3D4 showed reactivity with 32- or 33-kDa protein
bands (Fig. 1b). The MAbs were directed against heat-resistant proteins
because digestion with proteinase K completely destroyed the antigen's reactivities and heat treatment at 100°C for 10 min did not. The ascitic fluid from hybridomas B2D3 and B3D4 reacted with all of the
Bartonella strains tested, but it did not react with the 23 other bacteria tested. Cross-reactivity was observed with
Chlamydia psittaci and Chlamydia trachomatis.
Nevertheless, the immunofluorescence assay titers of MAbs to
Chlamydia spp. were much lower (Table 1).
Bartonella spp. were demonstrated in four of the five
infected lice by MIF with MAbs B2D3 and B3D4.
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FIG. 1.
(a) SDS-PAGE analysis of different Bartonella
species. Lanes: 1, B. bacilliformis; 2, B. henselae Houston-1; 3, B. henselae URBHLLY8; 4, B. clarridgeiae; 5, B. quintana; 6, B. elizabethae; 7, B. grahamii; 8, B. taylorii;
9, B. doshiae; 10, B. vinsonii subsp.
vinsonii; 11, B. tribocorum; 12, B. koehlerae; 13, B. alsatica. (b) Western immunoblotting
of MAb B2D3 with Bartonella antigens. Lanes: 1, B. bacilliformis; 2, B. henselae Houston-1; 3, B. henselae URBHLLY8; 4, B. clarridgeiae; 5, B. quintana; 6, B. elizabethae; 7, B. grahamii;
8, B. taylorii; 9, B. doshiae; 10, B. vinsonii subsp. vinsonii; 11, B. vinsonii
subsp. berkhoffii; 12, B. vinsonii subsp.
arupensis; 13, B. tribocorum; 14, B. koehlerae; 15, B. alsatica. Molecular masses (in
kilodaltons) are shown at left.
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The clinical manifestations of infections due to Bartonella,
Coxiella, and Chlamydia can often be confused,
especially in cases of infectious endocarditis. However, differential
diagnosis of the diseases is important because their treatments are
different. As Chlamydia spp. and Coxiella
burnetii are strictly intracellular bacteria and
Bartonella spp. are fastidious slowly growing organisms, they are difficult to isolate. Therefore, diagnosis of these infections continues to rely mainly on serology in spite of the serological cross-reactions among members of these genera that have been described (11, 20, 24). Moreover, because recently described species such as B. elizabethae, B. vinsonii subsp. berkhoffii,
B. vinsonii subsp. arupensis, and B. clarridgeiae may be encountered in humans, a specific serologic
test that recognizes all Bartonella infections is needed. In
our work, we have obtained, as a first step, MAbs that recognize a
protein antigen common to all Bartonella species. After
cloning the Bartonella sp. genome in E. coli in
order to obtain an expression bank, these MAbs could be used for
screening products of clones in order to obtain a protein antigen
common to all Bartonella spp. that could be used in an
enzyme-linked immunosorbent assay for the detection of antibodies to
all Bartonella spp. The anti-Bartonella
genus-specific MAbs obtained in this study are highly specific, as they
did not cross-react with 26 other bacterial species, except that MAb
B3D4 cross-reacted at low titers with C. trachomatis and
C. psittaci. Interestingly, none of the MAbs obtained
reacted with Chlamydia pneumoniae or Coxiella
burnetii, in spite of the cross-reactivity of these two genera and
Bartonella spp. which has been previously described (11, 20, 24). Serological cross-reactivity between
B. bacilliformis and C. psittaci antigens has
been demonstrated previously (16). It was associated with
a cross-reacting lipopolysaccharide antigen. Cross-reactivity between
B. quintana, C. psittaci, and C. pneumoniae was
later demonstrated in patients with B. quintana endocarditis (8). The serological cross-reactivity of
Bartonella sp. and Chlamydia sp. antigens in the
sera of patients infected by a member of these genera was also
demonstrated to be due to cross-reacting protein antigens with
molecular masses ranging from 30 to 90 kDa (24). Thus, our
MAb B3D4 could also be used to investigate cross-reacting epitopes
between Bartonella spp. and Chlamydia spp.
In conclusion, our Bartonella genus-specific MAbs recognized
specifically all the tested species of Bartonella, and they
successfully detected B. quintana in body lice. Thus, our
MAbs may provide a tool to identify, at the genus level, isolated
bacteria for which presumptive identification is compatible with
Bartonella spp. or to detect such bacteria within
arthropods, avoiding the use of molecular techniques for screening
(4, 21).
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