Previous Article | Next Article 
Infection and Immunity, May 1999, p. 2633-2637, Vol. 67, No. 5
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Specific Antibodies Reactive with the 22-Kilodalton Major Outer
Surface Protein of Borrelia anserina Ni-NL Protect
Chicks from Infection
Vittorio
Sambri,1
Antonella
Marangoni,1
Andrea
Olmo,1
Elisa
Storni,1
Marco
Montagnani,1
Massimo
Fabbi,2 and
Roberto
Cevenini1,*
Sezione di Microbiologia, DMCSS, University
of Bologna, St. Orsola Hospital, 40138 Bologna,1 and Istituto
Zooprofilattico Sperimentale della Lombardia ed Emilia,
Pavia,2 Italy
Received 4 November 1998/Returned for modification 4 January
1999/Accepted 1 March 1999
 |
ABSTRACT |
An outer surface lipoprotein of 22 kDa was identified in the avian
pathogen Borrelia anserina Ni-NL by using antibody
preparations reactive with bacterial surface-exposed proteins. Amino
acid sequence analysis of the 22-kDa protein demonstrated 90% identity
with VmpA of B. turicatae, suggesting that the protein
belongs to the family of 20-kDa outer surface proteins of the genus
Borrelia. All of the 60 chicks intramuscularly treated with
antibodies specifically reacting with the 22-kDa protein and infected
with strain Ni-NL were completely protected from infection, since
no spirochetemia was detected, and from death. Control chicks were
treated with immune sera raised against apathogenic strain B. anserina Es, which expresses a prominent 20-kDa polypeptide that
is also a member of the Vmp family but does not cross-react
immunologically with the 22-kDa protein of the Ni-NL strain. These
animals, infected with B. anserina Ni-NL, showed a high
degree of spirochetemia 10 days after infection, and all died between
14 and 21 days after infection. The results showed that the 22-kDa
surface protein of B. anserina Ni-NL is a determinant of
the pathogenic potential of the strain and also confirmed that
only strain-specific antibodies are protective against B. anserina infection.
| |
TEXT |
Bacteria of the genus
Borrelia cause several human and animal diseases
(15). Many studies (3, 20, 23-25) on the
pathogenic mechanisms of these spirochetes have been carried out since
the early 1970s when Kelly (16) achieved the in vitro
cultivation of Borrelia hermsii, and especially since 1982, when the etiologic agent of Lyme disease, B. burgdorferi,
was identified (5). B. anserina, which is
responsible for avian borreliosis (10), is a worldwide
pathogen that is of economic importance for domestic poultry breeding
in defined geographic areas (10). Several studies have
demonstrated both the presence of different antigenic types (18,
26, 28, 29) and the serotype specificity of the protective immune
response (10). Attenuation of the avian pathogenicity of
B. anserina has also been obtained by serial culture
passage in vitro (18) without observation of morphological
differences between virulent and attenuated spirochetes as detected by
electron microscopy (14). The protein profile of the low-
and high-passage cultures of the strain adapted to grow in vitro showed
only one major difference: the presence of an increasingly abundant and highly represented 20-kDa polypeptide in a high-passage strain (18). However, as far as we know, specific pathogenicity
determinants in B. anserina have not yet been
identified. On the other hand, the importance of the outer surface
proteins (OSP) of B. burgdorferi in the determination
of Lyme disease is well known. Therefore, we analyzed the surface
composition of B. anserina Ni-NL, a strain pathogenic
for chickens, in comparison with B. anserina Es, a strain that has lost its avian pathogenicity, and focused on the in
vivo protective activity of antibodies reactive with the 22-kDa surface-exposed protein of pathogenic strain Ni-NL.
Bacterial strains and growth conditions.
B.
anserina Ni-NL (14), kindly provided by L. Spanjaard,
Amsterdam, The Netherlands, was maintained by intravenous passage of
infected blood in pathogen-free chicks (18), since the
strain does not grow in vitro. Briefly, 2-day-old chicks provided with antibiotic-free food and water ad libitum were intramuscularly injected
in the leg with 0.1 ml of infected blood containing approximately 2 × 105 to 3 × 105 bacteria.
Spirochetemia was evaluated daily from 3 to 20 days after infection.
Spirochetemia reaches a plateau (mean value, 2.8 × 108/ml) 10 days after infection and lasts until the death
of the animals within 15 to 21 days of infection. Examination was done by dark-field microscopy of 1 drop of blood collected from the main
wing vein as previously reported (9, 11). Ten days after infection, 40 to 50% of the animals died, whereas all of the remaining chicks died within 21 days postinfection.
Since B. anserina Es, obtained from Russell C. Johnson, Minneapolis, Minn., has lost the ability to infect chicks in
vivo (18), it was maintained in BSK II medium (2)
by serial weekly passage. The other Borrelia strains used in
this work, B. turicatae, B. parkeri,
B. coriaceae, B. hermsii, B. afzelii, B. burgdorferi sensu stricto, and
B. garinii, were similarly grown in BSK II medium as
previously described (22).
MIAFs.
Mouse immune ascitic fluids (MIAFs) to B. anserina Es and Ni-NL were obtained by the method previously
reported (9), i.e., by intraperitoneal immunization of
BALB/c mice with whole sonicated bacterial cells. Briefly, 0.8-ml
volumes of immunogen (0.05 mg of protein) emulsified 1:9 (vol/vol) with
complete Freund's adjuvant were injected intraperitoneally into 8 to
12-week-old mice on days 0, 7, 14, and 21. On day 6, 0.5 ml of pristane
(2,6,10,14-tetramethylpentadecane; Sigma, St. Louis, Mo.) was injected
intraperitoneally. Ascitic fluid was collected on day 30 by peritoneal paracentesis.
Ab-SEE.
The MIAFs were used to select antibodies reactive with
surface-exposed epitopes (Ab-SEE) on living spirochetes as previously reported (21). B. anserina Es in the
logarithmic growth phase with no more than 0.5% damaged organisms were
used. The preparations of Ab-SEE used were obtained by incubating 4 ml
of a B. anserina Es culture (108 cells/ml)
with 1 ml (diluted 1:5) of B. anserina Es MIAF
for 45 min at 37°C. The bacterial suspension was then pelleted
and washed twice with 0.15 M phosphate-buffered saline
(PBS). Antibodies bound to the spirochete surface were then
recovered by resuspending the bacteria with 0.1 ml of 0.2 M glycine-HCl
(pH 2.2) and then incubating them for 10 min at 25°C. The pH of
the suspension was then brought to neutrality by adding 120 µl of 3.75 M Tris-HCl (pH 8.8) and the suspension was centrifuged
at 13,000 × g for 15 min at 25°C. Ab-SEE
of B. anserina Es were then purified by affinity chromatography by using a HiTrap protein A column
(Pharmacia-LKB, Uppsala, Sweden) and then concentrated with Centricon
30 tubes (Amicon, Beverly, Mass.). Individual preparations were
pooled before any further use, and protein concentrations were
determined with the Bradford reagent (Bio-Rad, Richmond, Calif.). The
selection of Ab-SEE for strain Ni-NL was done by using infected blood
as follows. When chick spirochetemia, evaluated by taking 1 drop of
blood from the main wing vein, reached at least 200 microrganisms/field (×400) and spirochete viability was over 99%, the animals were sacrificed by cardiac puncture and blood was collected in vials containing heparin. Afterwards, the homologous MIAF was diluted 1:5
(vol/vol) in heparinized blood, incubated for 1 h at 37°C, and
then centrifuged at 500 × g for 10 min to separate
erythrocytes from antibody-coated bacteria. Spirochetes were then
washed with PBS, and antibodies bound to surface antigens were removed
and purified as described above.
Gel electrophoresis, [3H]palmitate labeling, and
immunoblotting.
Single-dimension sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed by
using the buffers of Laemmli (17) as previously described
(8). The presence of fatty acid moieties linked to the
polypeptides of borrelias was determined by incubating the spirochetes
in the presence of [3H]palmitate (22); the
radiolabeled proteolipids were then detected by autoradiography as
previously described (22). Immunoblot analysis of
bacterial proteins separated by SDS-PAGE and transferred to
nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) was
performed by the method of Towbin et al. (27) as described elsewhere (21).
Amino acid sequencing.
The 22- and 24-kDa proteins of
strains Ni-NL and Es, respectively, were separated by SDS-PAGE
and then transferred to polyvinylidene difluoride
membranes (Schleicher & Schuell) by using 10 mM CAPS (3-[cyclohexylamino]-1-propanesulfonic acid; Sigma) buffer (pH 11) containing 10% methanol. Blotted proteins were stained for 1 min with 0.025% Coomassie R-250 (Sigma) in 40% methanol-60% H2O and immediately destained in 50% methanol in
H2O. The 22- and 24-kDa bands were then excised and in situ
digested with trypsin as reported by Blanco and coworkers
(4). The resulting peptides were then separated on a
capillary high-pressure liquid chromatography system (173A;
Perkin-Elmer ABI). Chemical sequencing of peptides was done with
a Perkin-Elmer ABI 476A automated sequencer by Suzanne Perry-Riehms at the NAPS Protein Service Laboratory, University of
British Columbia, Vancouver, British Columbia, Canada. Each sequence obtained was analyzed by BLASTP 1.4.11 (1)
software to perform homology searches with known protein sequences.
PCR analysis.
The flagellin gene sequence of
B. anserina (flaB gene, GenBank accession
no. X75201) was compared with those of B. turicatae (D82862), B. parkeri (D82863),
B. coriaceae (D82864), B. hermsii
(M86838 and M33839), B. afzelii (X75202), B. burgdorferi sensu stricto (X15661 and X14841), and B. garinii (L29236) to identify a species-specific region to be used
as a target of the amplification process. All of the sequences studied
were obtained from GenBank, and the accession numbers are in
parentheses. The target sequence specific for B. anserina was identified between nucleotides 581 and 1053. The
primers used to perform the PCR assay were Bafla1 (5'TAA TAC ACC AGC
ATC ACT AT3' from nucleotide 581 to nucleotide 600) and Bafla3 (5'TTG CGG ATT GTG TAA AAA TA3', complementary to the sequence from nucleotide 1053 to nucleotide 1034). The amplification product was a sequence of
473 bp. As controls for the species specificity of the PCR experiment,
target DNAs extracted as previously described (12) from
B. turicatae, B. parkeri M3001,
B. coriaceae Co53, B. hermsii HS1
B. afzelii isolate VS461, B. burgdorferi IRS sensu stricto, and B. garinii P/Bi
were used. The amplification was performed by using a Perkin-Elmer DNA
Thermal Cycler apparatus for 30 cycles, each one consisting of 45 s at 94°C, 45 s at 62°C, and 45 s at 72°C. The
reaction mixture and the detection of the amplification products were
as previously reported (12).
Infectivity neutralization assay.
To evaluate the ability of
the antibodies to neutralize the infectivity of B. anserina Ni-NL in vitro, 0.1 ml of MIAF to whole bacterial cells
and preparations of Ab-SEE were incubated with heparinized infected
blood (approximately 2 × 105 to 3 × 105 bacteria) for 60 min at 37°C and then injected into
animals as described above. Each experiment was repeated six times and
performed with 10 animals for each antibody preparation. MIAF and
preparations of Ab-SEE raised against B. anserina Es
were also tested in a similar way. In addition, MIAF raised against
whole elementary bodies of Chlamydia trachomatis
(9) was used as a control in one experiment. The outcome of
the infection was then evaluated over a period of 3 weeks, and the
number of surviving chicks was recorded.
Animal protection test.
The protection of antibodies reactive
either with whole B. anserina Ni-NL cells (MIAF) or
with B. anserina Ni-NL surface antigens (Ab-SEE) was evaluated in chicks as follows. Chicks were given 0.1-ml doses of different antibody preparations previously adjusted to
contain the same titer of immunoglobulins (8). The animals were then challenged within 1 h by intramuscular injection into the opposite leg of an inoculum of approximately 2 × 105 to 3 × 105 borrelias in 0.1 ml of
infected blood. Each experiment was performed with 10 animals
for each antibody preparation and repeated six times over a
period of 180 days. The protective activity of MIAF and
preparations of Ab-SEE raised against B. anserina
Es was also tested in a similar way by challenging animals with
B. anserina Ni-NL. In addition, two groups of chicks
treated with MIAF to strains Ni-NL and Es were challenged with blood
freshly obtained from uninfected animals to ensure that no mortality
was due to this procedure. Spirochetemia was evaluated by dark-field
microscopic examination of six different 10-µl samples at a
magnification of ×400. Examination of the samples was performed
blindly to prevent identification of the samples. To ensure the
absence of living spirochetes, a new inoculum of each blood specimen
scored as negative for spirochetemia was done by injecting 0.1 ml
intramuscularly into a new animal. After 20 additional days, each chick
was bled and tested as described above.
Preliminary PCR typing experiments confirmed that the
two strains we were working with were indeed two
B. anserina strains, despite their substantial
difference in pathogenic potential for chicks. In fact, these strains
were the only two of the several Borrelia strains used
to be amplified by primers specific for B. anserina (Fig. 1). As expected,
SDS-PAGE analysis showed similar protein profiles of the two
B. anserina strains (Fig.
2) with a major difference in the 20 to
24-kDa region and with other, minor differences, notably, in the 46- to
66-kDa region. The identification of the OSP of B. anserina Ni-NL and Es performed by using preparations of Ab-SEE
indicated the presence of two major OSP of 22 and 24 kDa in Ni-NL and
Es, respectively, which were the proteins exclusively recognized by a
homologous antibody preparation (Fig. 3).
The fluorographs obtained after intrinsic [3H]palmitate
labeling of the borrelias showed that the only positive bands were of
22 and 24 kDa for strains Ni-NL and Es (data not shown), respectively.
This observation confirmed the previously reported presence of a major
lipoprotein of 24 kDa in B. anserina Es
(22).
View larger version (62K):
[in this window]
[in a new window]
|
FIG. 1.
Agarose gel electrophoresis analysis of the
amplification products obtained by B. anserina flaB
gene PCR assay. Lanes: 2 and 3, B. anserina Es and
Ni-NL, respectively; 4 to 10, B. afzelii VS461,
B. garinii PBi, B. burgdorferi IRS,
B. hermsii HS-1, B. parkeri M3001,
B. turicatae, and B. coriaceae Co-53,
respectively. Lane 1 contained molecular size markers. On the left,
molecular sizes are indicated in base pairs.
|
|
View larger version (25K):
[in this window]
[in a new window]
|
FIG. 2.
SDS-PAGE analysis of B. anserina
strains. Lanes: 1, pathogenic strain Ni-NL; 2, strain Es. The empty
arrowhead indicates the 22-kDa protein, and the full arrowhead
indicates the 24-kDa protein. Molecular sizes (kilodaltons) are shown
on the right.
|
|
View larger version (63K):
[in this window]
[in a new window]
|
FIG. 3.
Western blot analysis of B. anserina
strains. Lanes: a, c, e, and g, strain Es; b, d, f, and h, pathogenic
strain Ni-NL. Lanes were probed as follows: a and b with MIAF to Es, c
and d with Ab-SEE to Es, e and f with MIAF to Ni-NL, and g and h with
Ab-SEE to Ni-NL. Molecular sizes (kilodaltons) are shown on the
right.
|
|
A preliminary amino acid sequence analysis of the major OSP of
B. anserina strains was undertaken in an attempt to
identify
the NH
2-terminal sequences of both the 22- and
24-kDa proteins.
As expected, the sequencing failed, confirming the
previous lipidation
results suggesting that the polypeptides were
N terminally blocked.
Consequently, using the tryptic digestion and
peptide mapping
technique, several different peptides were
generated and subjected
to amino acid sequencing. In particular, a
BLAST-P analysis (
1)
of 11-base peptide E (IGANGLEADAG)
obtained by digestion of the
22-kDa protein of strain Ni-NL revealed
the highest homology (identity
90%) with the VmpA protein of
B. turicatae (accession no.
U85413)
and the second
highest homology score (identity 72%) with OspC
of
B. afzelii (accession no.
AB000348). On the other hand,
the analysis
of two different and nonoverlapping peptides of the
24-kDa protein of
B. anserina Es showed that peptide 6 (15 amino
acid
residues: IQNSDTLATEANHHG) has 73 and 60% identity with the
OspC
sequences of
B. japonica (accession no.
AB000358) and
B. burgdorferi (accession no.
L42890), respectively. The second
peptide derived from the 24-kDa
protein (peptide 3 [22 bases:
VLMGSVSTLLEEAINELTTPAP]) demonstrated 45% identity
with the VmpA
sequence of
B. turicatae (accession
no.
U85413). The tryptic
peptide analysis of these proteins,
in particular, the pairwise
comparison of peptide E from the 22-kDa
antigen that was 90% identical
to VmpA of
B. turicatae
and the very high (73 to 60%) identity
of the 24-kDa protein of strain
Es with the OspC sequence of
B. japonica and
B. burgdorferi, strongly suggested that these major
OSP
of the
B. anserina isolates used in this study belong
to the
family of 20-kDa exposed OSP described by Carter et al.
(
7)
in the genus
Borrelia.
The effects of the in vitro reaction of Ni-NL spirochetes with
different antibody preparations on the infectivity for chicks
are
reported in Table
1. All of the animals
that received borrelias
pretreated with either MIAF or Ab-SEE raised to
B. anserina Ni-NL
showed no spirochetemia during the
follow-up period, and all of
them survived after 3 weeks. On the
contrary, chicks infected
with spirochetes pretreated with antibody
preparations to strain
Es were found to be spirochetemic 5 days
postinfection with a
mean number of 2.75 × 10
8
organisms/ml at 10 days postinfection. Similar values were obtained
with control chicks pretreated both with PBS and with antibodies
to
C. trachomatis. The mortality rate in these
groups of animals
reached 100% 3 weeks after infection (Table
1). The results obtained
by infectivity neutralization in vitro were
confirmed when animal
protection tests were performed in vivo by
treating chicks with
anti-Ni-NL antibodies before infection. The
results are reported
in Table
2. Two
groups of animals treated with antibodies (either
MIAF or Ab-SEE) to
pathogenic strain Ni-NL were completely protected
from infection; all
of the chicks were alive 3 weeks after infection
and showed no
spirochetemia during this period (Fig.
4A). On the
contrary, animals treated
with MIAF and Ab-SEE to nonpathogenic
strain Es were not protected, and
all died by 3 weeks after infection,
with a high level of spirochetemia
starting 3 and 5 days postinfection,
respectively (Fig.
4B).
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Results of in vitro B. anserina Ni-NL
infectivity neutralization assay using specific antibodies reacting
with B. anserina Ni-NL or Es
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Results of in vivo experiments testing protection of
chicks from B. anserina Ni-NL challenge by use of
specific antibodies reacting with B. anserina Ni-NL
or Es
|
|
View larger version (79K):
[in this window]
[in a new window]
|
FIG. 4.
Dark-field (×400) photomicrographs of blood samples
from chicks. (A) Blood from a chick protected with Ab-SEE to Ni-NL and
infected with pathogenic strain Ni-NL (no spirochetemia). (B) Blood
from a chick protected with Ab-SEE to Es and inoculated with pathogenic
strain Ni-NL (high spirochetemia).
|
|
Animals injected with uninfected blood from pathogen-free chicks and
treated with MIAF to Ni-NL and Es showed 100% survival,
and no
spirochetes were detected in their blood samples at the
end of the
follow-up period of 3
weeks.
In conclusion, the results of the present study confirm the lack of
protective cross-immunity between different isolates of
B. anserina and demonstrate that the principal
difference in the
polypeptide profile between pathogenic
strain Ni-NL and apathogenic,
culture-adapted strain Es is due to the
presence of two immunogenic,
surface-exposed proteins of the family of
20-kDa OSP of
Borrelia,
of 22 and 24 kDa, respectively, for
strains Ni-NL and
Es.
The outstanding role of the family of 20-kDa exposed OSP as
immunodominant antigens in
Borrelia infections has
emerged from
results of in vivo studies with both gerbils
(
19) and mice (
13)
for Lyme disease and with mice
(
6) for relapsing fever infection
with
B. turicatae. The results reported here add consistency to
these data
by suggesting that the 22-kDa OSP is a determinant
of the pathogenic
potential of
B. anserina Ni-NL and demonstrating
the
protective role of strain-specific antibodies in a chick infection
model.
| |
ACKNOWLEDGMENTS |
This work was supported in part by MURST (co-finaziamento 1997 grant) and by the University of Bologna (ex-quota 60% grant).
We thank Suzanne Perry-Riehm, NAPS Protein Service Laboratory,
Biotechnology Department, University of British Columbia, Vancouver, British Columbia, Canada, for performing amino acid analysis and for
helpful discussion of the sequence data. We also thank Aldo Farencena
for his early contribution to this study.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Section of
Microbiology, DMCSS, University of Bologna, St. Orsola Hospital, via
Massarenti 9, 40138 Bologna, Italy. Phone: 39/051/4290913. Fax:
39/051/341632. E-mail: cevenini{at}med.unibo.it.
Editor:
R. N. Moore
| |
REFERENCES |
| 1.
|
Altschul, S. F.,
W. Gish,
W. Miller,
E. W. Myers, and D. J. Lipman.
1990.
Basic local alignment search tool.
J. Mol. Biol.
215:403-410[Medline].
|
| 2.
|
Barbour, A. G.
1984.
Isolation and cultivation of Lyme disease spirochete.
Yale J. Biol. Med.
57:521-525[Medline].
|
| 3.
|
Barbour, A. G., and S. F. Hayes.
1986.
Biology of Borrelia species.
Microbiol. Rev.
50:381-400[Free Full Text].
|
| 4.
|
Blanco, D. R.,
C. I. Champion,
M. M. Exner,
H. Edrjument-Bromage,
R. E. W. Hancock,
P. Tempst,
J. N. Miller, and M. A. Lovett.
1995.
Porin activity and sequence analysis of a 31-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp1).
J. Bacteriol.
177:3556-3562[Abstract/Free Full Text].
|
| 5.
|
Burgdorfer, W.,
A. G. Barbour,
S. F. Hayes,
J. L. Benach,
E. Grunwaldt, and J. P. Davis.
1982.
Lyme disease a tick borne spirochetosis?
Science
216:1317-1319[Abstract/Free Full Text].
|
| 6.
|
Cadavid, D.,
P. M. Pennington,
T. A. Kerentseva,
S. Bergström, and A. G. Barbour.
1997.
Immunologic and genetic analyses of VmpA of a neurotropic strain of Borrelia turicatae.
Infect. Immun.
65:3352-3360[Abstract].
|
| 7.
|
Carter, C. J.,
S. Bergström,
S. J. Norris, and A. G. Barbour.
1994.
A family of surface-exposed proteins of 20 kilodaltons in the genus Borrelia.
Infect. Immun.
62:2792-2799[Abstract/Free Full Text].
|
| 8.
|
Cevenini, R.,
V. Sambri,
F. Massaria,
M. La Placa, Jr.,
E. Brocchi, and F. De Simone.
1992.
Complement mediated in vitro bactericidal activity of monoclonal antibodies reactive with outer-surface protein OspB of Borrelia burgdorferi.
FEMS Microbiol. Lett.
90:147-152.
|
| 9.
|
Cevenini, R.,
V. Sambri,
S. Pileri,
G. Ratti, and M. La Placa.
1991.
Development of transplantable ascites tumours which continuously produce polyclonal antibodies in pristane primed Balb/C mouse immunized with bacterial antigens and complete Freund's adjuvant.
J. Immunol. Methods
140:111-118[Medline].
|
| 10.
|
DaMassa, A. J., and H. E. Adler.
1979.
Avian spirochetosis: natural transmission by Argas (Persicargas) sanchezii (Ixodoidea: Argasidae) and existence of different serologic and immunologic types of Borrelia anserina in the United States.
Am. J. Vet. Res.
40:154-157[Medline].
|
| 11.
|
Fabbi, M.,
V. Sambri,
A. Marangoni,
S. Magnino,
F. Solari-Basano,
R. Cevenini, and C. Genchi.
1995.
Borrelia in pigeons: no serological evidence of Borrelia burgdorferi infection.
J. Vet. Med. B:
42:503-507.
|
| 12.
|
Farencena, A.,
M. Comanducci,
M. Donati,
G. Ratti, and R. Cevenini.
1997.
Characterization of a new isolate of Chlamydia trachomatis which lacks the common plasmid and has properties of biovar trachoma.
Infect. Immun.
65:2965-2969[Abstract].
|
| 13.
|
Gilmore, R. D., Jr.,
K. J. Kappel,
M. C. Dolan,
T. R. Burkot, and B. J. Johnson.
1996.
Outer surface protein C (OspC), but not P39, is a protective immunogen against a tick-transmitted Borrelia burgdorferi challenge: evidence for a conformational protective epitope in OspC.
Infect. Immun.
64:2234-2239[Abstract].
|
| 14.
|
Hovind-Hougen, K.
1995.
A morphological characterization of Borrelia anserina.
Microbiology
141:79-83[Abstract/Free Full Text].
|
| 15.
|
Johnson, R. C.
1977.
The spirochetes.
Annu. Rev. Microbiol.
31:89-106[Medline].
|
| 16.
|
Kelly, R.
1971.
Cultivation of Borrelia hermsii.
Science
173:443-444[Abstract/Free Full Text].
|
| 17.
|
Laemmli, U. K.
1970.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature (London)
227:680-685[Medline].
|
| 18.
|
Levine, J. F.,
M. Y. Dykstra,
W. L. Nicholson,
R. L. Walker,
G. Massey, and H. J. Barnes.
1990.
Attenuation of Borrelia anserina by serial passage in liquid medium.
Res. Vet. Sci.
48:64-69[Medline].
|
| 19.
|
Preac-Mursic, V.,
B. Wilske,
E. Patsouris,
S. Jauris,
G. Will,
E. Soutscheck,
S. Reinhardt,
G. Lehnert,
E. Klockmann, and P. Mehraein.
1992.
Active immunization with pC protein of Borrelia burgdorferi protects gerbils against Borrelia burgdorferi infection.
Infection
20:342-349[Medline].
|
| 20.
|
Radolf, J. D.
1994.
Role of outer membrane architecture in immune evasion by Treponema palidum and Borrelia burgdorferi.
Trends Microbiol.
2:307-311[Medline].
|
| 21.
|
Sambri, V.,
A. Marangoni,
F. Massaria,
A. Farencena,
M. La Placa, Jr., and R. Cevenini.
1995.
Functional activities of antibodies directed against surface lipoproteins of Borrelia hermsii.
Microbiol. Immunol.
39:623-627[Medline].
|
| 22.
|
Sambri, V.,
C. Stefanelli,
C. Rossoni,
M. La Placa, and R. Cevenini.
1993.
Acylated proteins in Borrelia hermsii, Borrelia parkeri, Borrelia anserina, and Borrelia coriaceae.
Appl. Environ. Microbiol.
59:3938-3940[Abstract/Free Full Text].
|
| 23.
|
Sambri, V.,
R. Aldini,
F. Massaria,
M. Montagnani,
S. Casanova, and R. Cevenini.
1996.
Uptake and killing of Lyme disease and relapsing fever borreliae in the perfused rat liver and by isolated Kupffer cells.
Infect. Immun.
64:1858-1861[Abstract].
|
| 24.
|
Sambri, V.,
S. Armati, and R. Cevenini.
1993.
Animal and human antibody reactive with the outer surface protein A and B of Borrelia burgdorferi are borreliacidal.
FEMS Immunol. Med. Microbiol.
7:67-72[Medline].
|
| 25.
|
Schaible, U. E.,
R. Wallich,
M. D. Kramer,
C. Museteanu,
M. Rittig,
S. Moter, and M. M. Simon.
1992.
Role of the immune response in Lyme disease: lessons from the mouse model, p. 243-262.
In
S. E. Schutzer (ed.), Lyme diseasemolecular and immunological approaches. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 26.
|
Soni, J. L., and A. G. Joshi.
1980.
A note on strain variation in Akola and Jabalpur strains of Borrelia anserina.
Zentrabl. Veterinaermed. Reihe B
27:70-72.
|
| 27.
|
Towbin, H.,
T. Staehelin, and J. Gordon.
1979.
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc. Natl. Acad. Sci. USA
76:4350-4354[Abstract/Free Full Text].
|
| 28.
|
Walker, R. L.,
R. T. Greene,
W. L. Nicholson, and J. F. Levine.
1989.
Shared flagellar epitopes of Borrelia burgdorferi and Borrelia anserina.
Vet. Microbiol.
19:361-371[Medline].
|
| 29.
|
Wouda, W.,
T. W. S. van Veen, and H. J. Barnes.
1974.
Borrelia anserina in chickens previously exposed to Borrelia theileri.
Avian Dis.
19:209-210.
|
Infection and Immunity, May 1999, p. 2633-2637, Vol. 67, No. 5
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Giacani, L., Sambri, V., Marangoni, A., Cavrini, F., Storni, E., Donati, M., Corona, S., Lanzarini, P., Cevenini, R.
(2005). Immunological Evaluation and Cellular Location Analysis of the TprI Antigen of Treponema pallidum subsp. pallidum. Infect. Immun.
73: 3817-3822
[Abstract]
[Full Text]
-
Marangoni, A., Sparacino, M., Cavrini, F., Storni, E., Mondardini, V., Sambri, V., Cevenini, R.
(2005). Comparative evaluation of three different ELISA methods for the diagnosis of early culture-confirmed Lyme disease in Italy. J Med Microbiol
54: 361-367
[Abstract]
[Full Text]
-
Sambri, V., Marangoni, A., Giacani, L., Gennaro, R., Murgia, R., Cevenini, R., Cinco, M.
(2002). Comparative in vitro activity of five cathelicidin-derived synthetic peptides against Leptospira, Borrelia and Treponema pallidum. J Antimicrob Chemother
50: 895-902
[Abstract]
[Full Text]
-
Sambri, V., Marangoni, A., Eyer, C., Reichhuber, C., Soutschek, E., Negosanti, M., D'Antuono, A., Cevenini, R.
(2001). Western Immunoblotting with Five Treponema pallidum Recombinant Antigens for Serologic Diagnosis of Syphilis. CVI
8: 534-539
[Abstract]
[Full Text]
Top
Abstract
Text
