This study was undertaken to determine whether individual proteins encoded by the virB locus, which assemble to form a secretion apparatus at the surface of the bacterium, are immunogenic and whether serological responses to these proteins can be used for detection of brucellosis.
We purified recombinant VirB1, VirB5, VirB11, and VirB12 antigens and evaluated their potential use for the serological diagnosis of brucellosis by ELISA. Evidence of VirB12 expression (Fig. ) was detected in mice infected with B. abortus
: an IgG response specific for VirB12 was elicited after infection with B. abortus
2308 but not after infection with a mutant lacking the entire virB
locus. The cell surface localization of VirB12 may contribute to its immunogenicity, as well as the fact that it is a predicted lipoprotein. VirB1, VirB5, and VirB11 also assemble in the membrane of the bacterium (16
), but we could not find evidence of specific antibody response against these proteins in the mouse or the goat model. One possible explanation for this result that we cannot exclude is that the recombinant VirB1, VirB5, and VirB11 antigens used for indirect ELISA were not in their native conformations, which would negatively impact the performance of our detection assay. However, a lack of immunogenicity of VirB1 and VirB5 could benefit Brucella
during infection, since in the closely related bacterium Agrobacterium tumefaciens
, these two proteins have been shown to be part of a pilus structure that protrudes from the bacterial surface and is therefore exposed to the immune system (15
, and BMΔcydBA
mutants used in our study have been assessed as vaccine candidates in the goat and mouse models of infection (12
). Both the BMΔasp24
mutants have shown promise as safe vaccine candidates in mouse and goat models. BMΔasp24
was able to colonize the maternal tissues but not the fetal tissues and cause seroconversion in goats, while BMΔvirB2
was not capable of colonizing maternal or fetal tissues and did not cause seroconversion. The BMΔcydBA
mutant was as virulent in the pregnant goat model as 16M (12
Goats infected with B. melitensis
16M or the BMΔasp24
, or BMΔcydBA
mutant elicited a response specific for VirB12, but not for VirB1, VirB5, or VirB11. BMΔvirB2
is a nonpolar mutation of VirB2; thus, VirB3 to VirB12 are expected to be expressed during infection (8
). Goats infected with BMΔvirB2
did not seroconvert to whole-cell Brucella
antigen (Fig. ) (12
) but exhibited a strong VirB12 response. One of the goals for the development of vaccines and diagnostic tools for brucellosis is a test that can distinguish between vaccinated and naturally infected animals. Our results show the potential of using VirB12 for such a goal, since BMΔvirB2
was found to be a safe vaccine with no seroconversion by using the brucellosis card test and whole-cell ELISA but elicited an elevated VirB12 response when an ELISA with recombinant purified VirB12 was used to assay infection.
A second goal of the present study was to assess the potential diagnostic utility of VirB12 in natural hosts of Brucella. We obtained 145 cattle serum samples of known serology (42 seronegative [29%] and 103 seropositive [71%]). Serology status was checked by both the brucellosis card test and whole-cell Brucella antigen ELISA.
As shown in Fig. , antibodies to VirB12 were detected in 70.3% (102 of 145) of bovine serum samples tested, while 29.7% of the samples did not give a response to VirB12. One of the 145 cattle samples was serologically positive for Brucella
but negative for antibodies specific to VirB12. Serological tests for brucellosis may yield false-positive results for cattle vaccinated with B. abortus
S19 or exposed to gram-negative bacteria with LPS O-chains similar to those of Brucella
, such as Yersinia enterocolitica
O:9. The cross-reactivity between Y. enterocolitica
O:9 and Brucella
is due to a strong similarity of the LPS O-chains (21
). We do not know if this animal showed serological reactivity to Brucella
due to cross-reactivity with Y. enterocolitica
O:9 or other bacteria, such as E. coli
O157:H7, a frequent colonizer of cattle. Since we did not detect seroreactivity to VirB12, either this animal may have been infected and did not respond to VirB12 or it had a serological cross-reaction to whole cells elicited by bacteria other than Brucella
. Based on the information at hand, we are unable to distinguish between these two possibilities.
In summary, this study shows that antibodies to B. abortus
VirB12 can be identified in mice, goats, and cattle. One hundred percent of the experimentally infected mice and goats generated specific responses to VirB12, including those infected with mutants shown to be promising vaccine candidates (12
). These results encourage further investigation with patient samples to determine whether detection of humoral responses to VirB12-specific immune responses can be used for the diagnosis of human brucellosis.