Microbiology (2015), 161, 2137–2148
DOI 10.1099/mic.0.000170
Booster vaccination with safe, modified,
live-attenuated mutants of Brucella abortus
strain RB51 vaccine confers protective immunity
against virulent strains of B. abortus and Brucella
canis in BALB/c mice
Quang Lam Truong, Youngjae Cho, Kiju Kim, Bo-Kyoung Park
and Tae-Wook Hahn
Correspondence
Tae-Wook Hahn
College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University,
Chuncheon 200-701, Republic of Korea
[email protected]
Received 3 July 2015
Revised 31 August 2015
Accepted 2 September 2015
Brucella abortus attenuated strain RB51 vaccine (RB51) is widely used in prevention of bovine
brucellosis. Although vaccination with this strain has been shown to be effective in conferring
protection against bovine brucellosis, RB51 has several drawbacks, including residual virulence
for animals and humans. Therefore, a safe and efficacious vaccine is needed to overcome these
disadvantages. In this study, we constructed several gene deletion mutants (DcydC, DcydD and
DpurD single mutants, and DcydCDcydD and DcydCDpurD double mutants) of RB51 with the
aim of increasing the safety of the possible use of these mutants as vaccine candidates. The
RB51DcydC, RB51DcydD, RB51DpurD, RB51DcydCDcydD and RB51DcydCDpurD mutants
exhibited significant attenuation of virulence when assayed in murine macrophages in vitro or in
BALB/c mice. A single intraperitoneal immunization with RB51DcydC, RB51DcydD,
RB51DcydCDcydD or RB51DcydCDpurD mutants was rapidly cleared from mice within
3 weeks, whereas the RB51DpurD mutant and RB51 were detectable in spleens until 4 and
7 weeks, respectively. Vaccination with a single dose of RB51 mutants induced lower protective
immunity in mice than did parental RB51. However, a booster dose of these mutants provided
significant levels of protection in mice against challenge with either the virulent homologous
B. abortus strain 2308 or the heterologous Brucella canis strain 26. In addition, these mutants
were found to induce a mixed but T-helper-1-biased humoral and cellular immune response in
immunized mice. These data suggest that immunization with a booster dose of attenuated RB51
mutants provides an attractive strategy to protect against either bovine or canine brucellosis.
INTRODUCTION
Brucella spp. are Gram-negative, facultative and intracellular bacteria that cause a zoonotic disease that is distributed
almost worldwide. This disease is a major cause of abortions and infertility in numerous animal species (Godfroid
et al., 2005; Seleem et al., 2010). In humans, infection with
several Brucella spp. has been reported, and the most
pathogenic and invasive species for humans are Brucella
melitensis and Brucella abortus, followed in descending
order by Brucella suis and Brucella canis (Godfroid et al.,
2005; Lucero et al., 2010; Seleem et al., 2010). In Korea,
outbreaks of bovine and canine brucellosis, mainly
Abbreviations: ABC, ATP-binding cassette; BMDM, bone marrowderived macrophage; p.i., post-infection; Th1, T-helper 1; WCA, wholecell antigen.
000170 G 2015 The Authors
caused by B. abortus and B. canis, have been reported in
cattle and dogs, respectively (Bae & Lee 2009; Yoon et al.,
2010). B. abortus and B. canis infect not only their preferred
hosts, but also other domestic or wild animal species, e.g.
B. abortus infection in elk, dog and Chinese water deer or
B. canis infection in cattle (Baek et al., 2003, 2012; Her
et al., 2010; Kang et al., 2011; Truong et al., 2011) have
been reported. Of note, from 2002 to 2010, 651 human
brucellosis cases were also reported to be associated with
the livestock industry in Korea (Jang et al., 2011; Park
et al., 2005).
As a result of the economic and global public health risks,
extensive efforts have been made to control and prevent
brucellosis in animals through eradication programs
(Corbel, 2006; Yoon et al., 2010). In addition to
the implementation of a ‘test-and-slaughter’ strategy,
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2137
Q. L. Truong and others
vaccination is a critical tool for control of bovine brucellosis (Avila-Calderón et al., 2013). In the last few decades,
live-attenuated vaccines such as B. abortus strains S19
and RB51 have been developed and widely used against
bovine brucellosis (Avila-Calderón et al., 2013; Schurig
et al., 2002). The strain RB51 vaccine was first reported
in 1991 as a spontaneous mutant of B. abortus strain
2308 that developed an attenuated phenotype after
repeated in vitro passage in the presence of rifampicin
(Schurig et al., 2002). Numerous efficacy studies of this
vaccine conducted in experimental mice and in cattle
have demonstrated that the vaccinated animals, particularly cattle, are effectively protected from exposure to WT
bacteria (Schurig et al., 2002; Wang & Wu, 2013). The protective efficacy of RB51 is variable and depends on a range
of factors, including the age of the vaccinated animal and
the dose and route of the vaccination. Although RB51 typically exhibits low virulence in cattle, the vaccine may cause
abortions when administered to pregnant animals. It was
also found that after vaccination of cattle with RB51, the
bacteria possibly survived for extended periods and were
excreted in milk or vaginal exudate (Arellano-Reynoso
et al., 2004). In addition, the potential for persistent local
and systemic adverse events associated with accidental
human exposure to RB51 has also been reported (Ashford
et al., 2004).
B. abortus is known to survive and replicate in nutrient- or
oxygen-limited environments inside hosts. Therefore,
B. abortus mutants lacking the genes encoding cytochrome
terminal oxidases or required for the biosynthesis of an
essential nutrient are unlikely to survive in that harsh
environment. Using transposon mutagenesis, previous
work in our laboratory has identified the B. abortus genes
cydC (encoding the ATP-binding/permease protein) and
purD (encoding phosphoribosylamine–glycine ligase) as
being required for intracellular survival and virulence
in vitro and in vivo (Truong et al., 2014, 2015). BALB/c
mice immunized with the cydC : : Tn5 and purD : : Tn5
transposon mutants were protected against challenge
with virulent B. abortus strain 544. Although transposon
mutants are not appropriate for use as a vaccine, these
genes may merit consideration as ideal targets for future
vaccine development. In the present study, in an attempt
to diminish the virulence of the strain RB51 vaccine, we
constructed unmarked single (DcydC, DcydD and DpurD)
and double (DcydCDcydD and DcydCDpurD) gene deletion
mutants of RB51, and evaluated their safety and their
induction of protective immunity in BALB/c mice. Deletion in RB51 of cydC, cydD or purD, or both cydC and
cydD or purD, has several positive effects, including significantly reduced survival or persistence of bacteria in
cultured macrophages and in BALB/c mice, and a diminished inflammatory response associated with vaccination.
These effects lead to the increased safety of the vaccine
strain, because high doses of RB51 alone elicit splenomegaly, an undesirable side effect of vaccination (Stevens
et al., 1995; Wang & Wu, 2013). In addition, we also investigated the capacity of RB51 mutants to elicit Brucellaspecific immune responses, and to protect the mice against
challenge with virulent homologous B. abortus strain 2308
and heterologous B. canis strain HID90026.
METHODS
Bacterial strains. Bacterial strains used in this study (Table 1)
included Escherichia coli DH5a, B. abortus strain RB51 vaccine,
B. abortus strain 2308 (S2308) and B. canis strain HID90026 (B. canis
S26). The virulent B. canis S26 was isolated from a dog by our laboratory in 2010. All Brucella strains and mutants were routinely
grown in Tryptic Soy Broth (Difco) or on Tryptic Soy Agar (TSA) at
37 uC in 5 % CO2. E. coli DH5a was cultivated in Luria– Bertani
(Difco) broth or agar. Electrocompetent cells of RB51 and mutants
were prepared as described previously (McQuiston et al., 1995).
Construction of the unmarked RB51 single- and doubledeletion mutants. To construct the plasmids for deleting cydC, cydD
Table 1. Bacterial strains and plasmids
Strain or plasmid
Bacterial strains
E. coli DH5a
RB51
S2308
RB51DcydC
RB51DcydD
RB51DpurD
RB51DcydCDcydD
RB51DcydCDpurD
Plasmids
pEX18Ap
pEX18DcydD
pEX18DcydC
pEX18DpurD
2138
Relevant characteristics
Source or reference
F2 w80 lac ZDM15 D(lacZYA–argF) U169 recA1 endA1 hsdR17
(rk2, mk+) gal 2 phoA supE44 l 2 thi 21 gyrA96 relA1
Rough, B. abortus strain RB51 vaccine
Smooth, virulent B. abortus strain 2308
cydC deletion in RB51
cydD deletion in RB51
purD deletion in RB51
cydD deletion in RB51DcydC
purD deletion in RB51DcydC
Choong Ang Vaccine Laboratories
Lee et al. (2015)
This study
This study
This study
This study
This study
sacB bla, AmpR
D1F/D2R:D3F/D4R cloned into pEX18Ap for cydD deletion
C1F/C2R:C3F/C4R cloned into pEX18Ap for cydC deletion
PD1F/PD2R:PD3F/PD4R cloned into pEX18Ap for purD deletion
Wang et al. (2013)
This study
This study
This study
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Microbiology 161
Booster vaccine candidates against Brucella spp.
and purD, we designed two sets of primers to amplify from RB51
DNA fragments in the upstream and downstream regions of each
gene of interest. The sequences of each primer pair are listed in
Table 2. Using previously published methods (Kahl-McDonagh &
Ficht, 2006; Wang et al., 2013), the respective plasmids pEX18DcydC,
pEX18DcydD and pEX18DpurD were created by a two-round PCR
amplification, restricted digestion and ligation, and then introduced
into RB51 and the RB51DcydC mutant via electroporation. Transformants were selected in the presence of 100 mg carbenicillin ml21
for the first screening and 5 % sucrose for the second screening. The
deletions of cydD (D5F/D6R), cydC (C5F/C6R) and purD (PD5F/
PD6R) in RB51 or RB51DcydC were then verified by PCR amplification and DNA sequencing analysis, and the resulting mutants
referred to as RB51DcydD, RB51DcydC, RB51DpurD, RB51DcydCDcydD and RB51DcydCDpurD. Genetic complementation strains
corresponding to each single mutant were also constructed and
characterization of these mutants was performed as described previously (Truong et al., 2014, 2015).
Intracellular growth of RB51 mutants in murine macrophages.
Preparation of and the intracellular bacterial growth assays within
RAW 264.7 cells (RAW cells) and bone marrow-derived macrophages
(BMDMs) from BALB/c mice were performed as previously described
(Trant et al., 2010; Truong et al., 2015). Briefly, the RAW cells and
BMDMs were seeded at 2|105 cells per well in complete medium
[CM; RPMI 1640 (GenDepot), 10 mM HEPES and 10 % FBS] into
tissue culture plates, and infected with RB51 and its isogenic mutants
at m.o.i. 100. The plates were centrifuged at 250 g for 5 min and then
incubated at 37 uC for 30 min. After washing twice with PBS, the cells
were incubated for 1 h in the presence of 50 mg gentamicin ml21
(Sigma) to kill extracellular bacteria. The cells were then replaced by
Table 2. Primers
Primer
C1F
C2R
C3F
C4R
C5F
C6R
D1F
D2R
D3F
D4R
D5F
D6R
PD1F
PD2R
PD3F
PD4R
PD5F
PD6R
Sequence (59R39) (restriction enzyme used)*
GGAATTCCCTTCAATCGCAAGCGGTGC (Eco RI)
GCATTCACGGGATCCCGCATGAGGTGAGTCATG
CAGG (Bam HI)
CCTCATGCGGGATCCCGTGAATGCAAACCCTTC
ATTCCG (Bam HI)
GCTCTAGAGCTGTTGCTCTTCGCAAGAGA (Xba I)
AACGGTTTCCATGATGGCCA
CGACGCATGATGAAACGCTG
GGAATTCCAATGCAAACCCTTCATTCCG (Eco RI)
TGTTTCACGGGATCCCGTGTTGCTCTTCGCAAG
AGA (Bam HI)
TTGGATGCGGGATCCCGTGAAACATTGCCCCCC
GTTT (Bam HI)
GCTCTAGAGCACGATCCAACGTTTCCATG (Xba I)
CTGCCATCAGCAGTTCGGT
CGTACGGTGGGGCAGATTTA
GGAATTCCTTTACAATCGCCGTGGCTT (Eco RI)
AAAACTACGGGATCCCGCATCGACTGGCCGGA
AGGTT (Bam HI)
GTCGATGCGGGATCCCGTAGTTTTTCAAGCAG
GGGC (Bam HI)
GCTCTAGAGCTCATTATAGGTGCAGCCTGC (Xba I)
CGCACCATGTTGCGCATATT
CAGTCATAGCCGAATAGCCT
*Bold indicates restriction enzyme site.
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CM containing 10 mg gentamicin ml21, and incubated at 37 uC for 0,
6, 24 and 48 h. At different times post-infection (p.i.), the infected
cells were washed three times with PBS and lysed with 0.1 % Triton
X-100 in sterile double-distilled water. Tenfold serial dilutions of the
lysates were plated on TSA plates to determine the c.f.u. count. These
assays were performed in triplicate and repeated twice.
Clearance of RB51 mutants in mice. Female 7-week-old BALB/c
mice were purchased from ORIENTS and kept in individually ventilated cage rack systems in the Biosafety Level 2+ facilities of
Kangwon National University. All animal care and experimental
procedures were approved by Kangwon University Institutional
Animal Care and Use Committee (permit KW-150729-2). Groups of
mice were inoculated intraperitoneally with a single dose of 3.9–
4.8|108 c.f.u. RB51 or RB51 mutants in 0.2 ml PBS. Three weeks
after the first inoculation, a booster inoculation of 3.5–4.6|108 c.f.u.
RB51 mutants was given. At 1, 2, 3, 4, 5, 6, 7 and 9 weeks after the
single inoculation or 4 weeks after the booster inoculation, five mice
from each group were euthanized, and spleens were aseptically
removed, weighed and homogenized in PBS. Tenfold serial dilutions
of spleen homogenates were plated on TSA plates and incubated for
3–5 days at 37 uC. The c.f.u. counts were determined and results
presented as the mean+ SD for each group.
Evaluation of protective efficacy of RB51 mutants in mice. For
the single immunization, groups of mice were vaccinated intraperitoneally with a single dose of 3.9–4.8|108 c.f.u. RB51 and RB51
mutants in 0.2 ml PBS. For the booster immunization, groups of mice
were boosted with 3.5–4.6|108 c.f.u. RB51 mutants 3 weeks after the
first vaccination. Control groups were injected with PBS only. At 7
weeks post-vaccination, all groups were challenged intraperitoneally
with 1.7 and 2.8|105 c.f.u. per mouse of virulent B. abortus S2308
and B. canis S26, respectively. At 2 weeks post-challenge, five mice per
group were sacrificed, and the spleens were collected and weighed.
The numbers of c.f.u. recovered from spleens were determined as
described above. The degree of protection was expressed as the
mean+ SD c.f.u. of S2308 or B. canis S26 for each mouse group
obtained after challenge. Mean log c.f.u. reduction or log units of
protection (U) were obtained by subtracting the mean log c.f.u. for
the vaccinated group from the mean log c.f.u. for the PBS control
group.
Evaluation of serum antibody response. Mice were bled from the
orbital sinus during the immunization schedule and sera were stored
at 270 uC until tested. The IgG and IgG subclass antibodies against
whole-cell antigens (WCAs) of RB51 and B. canis S26 were measured
by indirect ELISA as described previously (Arenas-Gamboa et al.,
2008). Briefly, ELISA plates were coated with 25 mg per well WCAs of
each strain in carbonate buffer overnight at 4 uC. The plates were then
washed with PBS-T (PBS containing 0.05 % Tween 20) and blocked
with blocking buffer (1 % BSA in PBS). Subsequently, triplicate
samples of 1 : 200 diluted serum in blocking buffer were added and
incubated at 37 uC for 1 h. After three PBS-T washes, goat anti-mouse
IgG, IgG1 and IgG2a horseradish peroxidase conjugate (Fitzgerald) at
a dilution of 1 : 2000 was added and incubated at 37 uC for 1 h. After
extensive washing, o-phenylenediamine dihydrochloride peroxidase
substrate (Sigma) was added and incubated for 20 min, and then the
A450 was measured with an ELISA reader (Bio-Rad).
Cytokine detection in spleen cell culture supernatants. Sple-
nocytes from the mice were prepared as previously described (KahlMcDonagh & Ficht, 2006). At 7 weeks post-vaccination, individual
spleens from five mice per group were aseptically removed. Single-cell
suspensions were obtained by passing the spleens through a 40 mm
mesh cell strainer (SPL Lifesciences). The cells were then pelleted by
centrifugation at 1000 g for 10 min and then treated for 5 min with
ACK erythrocyte lysis buffer (150 mM NH4Cl, 1 mM KHCO3,
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2139
Q. L. Truong and others
and 48 h p.i., RB51 mutants showed a significantly reduced
rate of survival within RAW cells and BMDMs compared
with parental RB51 (Pv0.001): RB51 mutants exhibited
a 2–4 log reduction in the number of c.f.u. at 48 h p.i. in
both cell types. These results suggested that RB51 mutants
are more attenuated in murine macrophages than RB51,
and indicated that the deletions of cydC, cydD and purD
significantly affect the ability of RB51 to survive within
both cell types.
0.1 mM Na2EDTA, pH 7.2). After three PBS washes, splenocytes were
counted and plated at 2|106 cells per well in CM, and stimulated
with either 1|108 c.f.u. heat-killed RB51 or B. canis S26, 2 mg concanavalin A ml21 (Sigma) or media alone. After 72 h incubation at
37 uC in 5 % CO2, the culture supernatants were collected and the
concentrations of selected cytokines (IFN-c and IL-10) were determined using mouse ELISA MAX kits according to the manufacturer’s
instructions (Bio-Legend).
Statistical analysis. The data from assays for survival of RB51 and
its isogenic mutants in murine macrophages and BALB/c mice and
from efficacy studies were expressed as the mean + SD log c.f.u. for
each group. Statistical comparison between means was performed
using ANOVA. The significance of differences between the experimental groups was analysed by paired Student’s t-test and ANOVA
followed by either Dunnett’s post-test (comparison with control
group) or Tukey’s post-test (comparison between several groups).
Pv0.05 was considered to indicate significance.
Safety of RB51 mutants in mice
To assess the attenuation of RB51 mutants, BALB/c mice
were inoculated intraperitoneally with a high dose
(108 c.f.u.) of RB51 or its isogenic mutants and the numbers of viable organisms in the spleens were determined
at different time points after inoculation. As shown in
Fig. 2(a), at 1 and 2 weeks post-inoculation, significantly
fewer bacteria were recovered from the spleens of mice
inoculated with a single dose of RB51 mutants than from
those of mice inoculated with the parental RB51. Recovery
of RB51 mutants was reduced by *2–3 log compared
with that of the parental RB51 (Pv0.001). By 3 weeks
post-inoculation, when the RB51DcydC, RB51DcydD,
RB51DcydCDcydD and RB51DcydCDpurD mutants had
been completely cleared from spleens of mice, RB51 and
the RB51DpurD mutant persisted in spleens at 3.8 and
1.3 log c.f.u., respectively (Fig. 2a). No viable organisms
were detected at 4 weeks post-inoculation in spleens of
mice inoculated with RB51DpurD, whereas RB51 was completely cleared by 7 weeks post-inoculation. The significantly reduced persistence of RB51 mutants in mice
correlated with reduced spleen weights compared with
those in mice inoculated with RB51 (Fig. 2b). In fact,
RESULTS
Intracellular survival of RB51 mutants in murine
macrophages
To examine the intracellular survival of bacteria in vitro,
RAW cells and BMDMs were infected with RB51 and its
isogenic RB51DcydC, RB51DcydD, RB51DpurD and
RB51DcydCDpurD mutants. As shown in Fig. 1, RB51
mutants displayed a similar rate of internalization as the
parental RB51 as measured immediately after infection,
indicating that absence of cydC, cydD or purD does not
affect internalization. However, RB51 mutants showed an
almost 1 log reduction in c.f.u. compared with RB51 at
6 h p.i., in both RAW cells and BMDMs (Fig. 1a, b,
Pv0.001), suggesting that RB51 mutants were more susceptible to macrophage killing than parental RB51. At 24
(a)
RB51 ΔpurD
RB51ΔcydCΔcydD
RB51ΔcydCΔpurD
RB51
RB51ΔcydC
RB51ΔcydD
7
(b)
7
RB51ΔpurD
RB51ΔcydCΔcydD
RB51ΔcydCΔpurD
6
Log c.f.u. per well
6
Log c.f.u. per well
RB51
RB51ΔcydC
RB51ΔcydD
5
4
3
2
1
5
4
3
2
1
0
0
0
6
24
48
0
6
24
48
Time (h p.i.)
Time (h p.i.)
Fig. 1. Intracellular survival of RB51 and its mutants in murine macrophages. (a) RAW cells and (b) BMDMs were injected
with RB51 and RB51 mutants at m.o.i. 100. At the indicated time points post-infection, both cell types were washed and
lysed, and the number of intracellular bacteria were enumerated by plating serial dilutions on TSA plates. Significant differences between the mutants and parent strain RB51 are indicated: *P,0.05; **P,0.001.
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Microbiology 161
Booster vaccine candidates against Brucella spp.
(a)
(b)
RB51
RB51ΔcydC
RB51ΔcydD
7
RB51ΔpurD
RB51ΔcydCΔcydD
RB51ΔcydCΔpurD
600
Spleen weight of mice (mg)
Log c.f.u. per spleen
6
5
4
3
2
1
RB51
RB51ΔcydC
RB51ΔcydD
RB51ΔpurD
RB51ΔcydCΔcydD
RB51ΔcydCΔpurD
500
400
300
200
100
0
0
1
2
3
6
7
4
5
Time (weeks post-inoculation)
9
1
2
3
4
5
6
Time (weeks post-inoculation)
7
9
Fig. 2. Clearance of RB51 and RB51 mutants in BALB/c mice. Groups of mice were inoculated intraperitoneally with a single
dose of ,4–56108 c.f.u. RB51, or RB51DcydC, RB51DcydD, RB51DpurD, RB51DcydCDcydD or RB51DcydCDpurD
mutants. At the indicated time points, the spleens of inoculated mice were aseptically removed, and assessed for (a) levels of
c.f.u. of strain RB51 and its mutants, and (b) spleen weight changes during infection. Data are presented as mean¡SD splenic c.f.u. and weights. **P,0.001 indicates the log numbers of c.f.u. or spleen weights are significantly different from those of
PBS-inoculated mice. *P,0.05 indicates a significant difference in c.f.u. levels between mutants.
enlarged spleens were observed only in RB51-inoculated
mice, but not in mice inoculated with RB51 mutants,
implying that there was significantly reduced splenomegaly
and inflammatory response in mice inoculated with RB51
mutants (Fig. 2b). These results demonstrated that RB51
mutants were highly attenuated in mice.
When a second inoculation of RB51DcydC, DcydD, DpurD,
DcydCDcydD or DcydCDpurD mutants was given in mice 3
weeks after the first inoculation, no mice in the experimental groups showed evidence of splenomegaly, even though
they were re-inoculated with high doses (3.5–4.6|
108 c.f.u. per mouse) of RB51 mutants (data not shown).
Moreover, these mutants were completely cleared from
spleens by 4 weeks after the second inoculation (data not
shown). The enhanced clearance and the lack of splenomegaly associated with either single or booster inoculation
suggested that from a safety perspective RB51 mutants
are attractive as live vaccine candidates.
Efficacy against B. abortus challenge of single
and booster doses of RB51 mutants
The above demonstration that RB51 mutants are highly
attenuated and have limited persistence in vitro and in
mice suggested that the rapid clearance of RB51 mutants
may enhance their safety as vaccine strains, but provide
possibly reduced levels of protection compared with
RB51. To test this assumption, the groups of vaccinated
mice were challenged at 7 weeks post-vaccination with
the virulent B. abortus S2308. The mean log c.f.u. values
of the challenge strain in spleens of mice are presented in
http://mic.microbiologyresearch.org
Table 3. At 2 weeks post-challenge, there was a significant
reduction (Pv0.001) in bacterial load in the spleens
from mice vaccinated with a single dose of RB51,
RB51DcydC, RB51DcydD, RB51DpurD, RB51DcydCDcydD
or RB51DcydCDpurD mutants relative to those in the
PBS-vaccinated mice, with 1.35, 0.83, 0.91, 1.08, 0.76 or
0.72 log reductions, respectively, in c.f.u. in the spleens.
Vaccination with a single dose of RB51 resulted in better
protection against challenge with virulent S2308 than did
a single dose of RB51 mutants.
The improved benefit of diminished splenomegaly and
respective safety post-vaccination exhibited by RB51
mutants provided us with an opportunity to evaluate
the use of a booster dose of RB51 mutants in mice.
To determine whether a booster dose of RB51 mutants
improved protection against a challenge infection, vaccinated mice were challenged with the virulent homologous
S2308 at 4 weeks after a second vaccination. As shown
in Table 3, booster vaccination of mice with RB51
DcydC, RB51DcydD, RB51DpurD, RB51DcydCDcydD or
RB51DcydCDpurD mutants offered a 1.81, 1.86, 1.94,
1.74 or 1.68 log reduction in bacterial burden in
the spleen relative to that in the PBS control group
(Pv0.001). Mice vaccinated with a booster dose of RB51
mutants showed enhanced protection against S2308 challenge compared with those given a single dose of either
RB51 mutants or RB51 (Table 3). These results indicate
that the use of booster vaccination with RB51 mutants
could provide better protective efficacy against challenge
than single vaccination with parental RB51 or RB51
mutants.
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Q. L. Truong and others
Table 3. Protection against B. abortus S2308 and B. canis S26 provided to BALB/c mice by vaccination with RB51 and its isogenic mutants
Group (n55)
PBS
RB51
RB51DcydC
RB51DcydD
RB51DpurD
RB51DcydCDcydD
IVK15DcydCDpurD
PBS
RB51
RB51DcydC
RB51DcydD
RB51DpurD
RB51DcydCDcydD
IVK15DcydCDpurD
No. of
vaccinations
Log c.f.u. B. abortus
S2308 in spleen*
1
1
1
1
1
1
1
1
1
2
2
2
2
2
6.28¡5.97
4.93¡4.69
5.45¡5.28
5.37¡5.18
5.20¡5.11
5.52¡5.27
5.56¡5.36
6.40¡6.03
5.13¡4.87
4.59¡4.40
4.54¡4.26
4.46¡4.32
4.66¡4.50
4.72¡4.53
Log units
(U) of
protection
Log c.f.u. B. canis
S26 in spleen*
Log units
(U) of
protection
1.35D
0.83D
0.91D
1.08D
0.76Dd
0.72Dd
1.27D
1.81D
1.86D
1.94D
1.74D
1.68D
6.22¡6.08
4.66¡4.64
4.19¡4.18
4.11¡4.14
4.02¡4.11
4.34¡4.39
4.39¡4.35
1.56D
2.03D
2.11D
2.20D§
1.88D
1.83D
*Mice were vaccinated once with RB51, and once or twice at an interval of 3 weeks with RB51 mutants. Seven weeks after the first vaccination, all
groups of mice were challenged intraperitoneally with the virulent strains of B. abortus S2308 (1.76105 c.f.u. per mouse) and B. canis S26 (2.86105
c.f.u. per mouse), and mean¡SD splenic c.f.u. was counted at 2 weeks post-challenge.
DSignificant difference between the vaccinated groups and the PBS control group (P,0.001).
dSignificant difference between the mouse groups vaccinated with RB51 mutants and that vaccinated with the RB51 strain (P,0.05).
§Significant difference between the mouse groups immunized with two doses of the RB51DpurD mutant and a single dose of parental RB51
(P,0.05).
Efficacy against heterologous B. canis challenge
of a booster dose of RB51 mutants
Strain RB51 vaccine has been shown to be protective in mice
and dogs against challenge with the virulent rough strains of
Brucella ovis and B. canis, respectively (Hur & Baek, 2010;
Jiménez de Bagüés et al., 1994). Therefore, to determine
whether a booster dose of highly attenuated RB51 mutants
would be protective against rough strains of Brucella, vaccinated mice were challenged at 7 weeks post-vaccination with
the rough, virulent, heterologous B. canis S26. As shown in
Table 3, booster vaccination of mice with RB51 mutants conferred significant levels of protection at 2 weeks post-challenge.
RB51DpurD mutant provided the best protection, defined as a
2.20 log decrease in bacterial load compared with the PBS control group (Pv0.001), followed by RB51DcydD at 2.11 log,
RB51DcydC at 2.03 log, RB51DcydCDcydD at 1.88 log and
RB51DcydCDpurD at 1.83 log (Pv0.001). Notably, booster
vaccination with RB51 mutants provided better protection
than single vaccination with parental RB51 (1.56 U). These
results suggested that a booster dose of RB51 mutants could
provide effective protection in mice against challenge with
heterologous virulent B. canis.
Humoral and cell-mediated immune response
induced by RB51 mutants
To evaluate the immune response in mice vaccinated with
either single or booster vaccination with RB51 mutants, we
2142
assessed the antibody responses in serum and cytokine
responses in splenocytes from vaccinated mice at 7 weeks
post-vaccination. The antibody levels were assessed by
ELISA to reflect the humoral immune response against
WCAs of either RB51 or B. canis strains. The levels of relevant specific antibodies in the sera of the immunized mice
at 7 weeks post-vaccination are shown in Fig. 3. Mice
immunized with a single dose of RB51 mutants were
capable of producing significantly higher levels of specific
IgG antibodies against WCAs of RB51 than PBS-immunized mice (Pv0.001) (Fig. 3a). As expected, mice immunized with two doses of RB51 mutants developed
significantly higher levels of IgG specific for the WCAs of
RB51 than did those mice immunized with a single dose
of RB51 mutants (Pv0.05). In addition, assays of IgG1
and IgG2a antibody isotypes revealed that both were present at significantly higher levels in mice given a booster
dose of RB51 mutants than in mice immunized with a
single dose of RB51 mutants (Pv0.05) or in mice inoculated with PBS (Pv0.001) (Fig. 3b, c). Similarly, when
sera from mice immunized with either a single dose of
RB51 or two doses of RB51 mutants were examined for
antibody responses against WCAs from B. canis S26, high
levels of IgG, IgG1 and IgG2a antibodies to WCAs of
B. canis S26 were detected at 7 weeks post-vaccination
(Fig. 3). Notably, the antibody responses to WCAs from
B. canis S26 were slightly higher than those to WCAs
from RB51 (Fig. 3).
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Booster vaccine candidates against Brucella spp.
(a) 1.6
Single dose
Booster dose
1.4
a
IgG (A450)
1.2
1.0
b
a
a
a
a
b
0.8
0.6
0.4
0.2
RB RB
5 51
RB Dcy
51 dC
RB R Dc
51 B5 ydD
1
RB Dcy Dpu
51 dC rD
Dc Dc
yd yd
C D
Dp
ur
D
PB
S
R
RB B5
1
5
RB Dcy
51 dC
D
RB R
c
51 B5 ydD
1
RB Dcy Dpu
d
51 C rD
Dc Dc
yd yd
C D
Dp
ur
D
PB
S
RB RB5
1
5
RB Dcy
51 dC
RB R Dc
51 B5 ydD
1
RB Dcy Dp
51 dC urD
Dc Dc
yd yd
C D
Dp
ur
D
PB
S
0
WCA RB51
WCA B. canis S26
(b) 1.0
Single dose
Booster dose
IgG1 (A450)
0.8
0.6
0.4
0.2
WCA RB51
Single dose
Booster dose
1.2
a
1.0
IgG2 (A450)
R
51 B5
RB Dcy 1
51 dC
RB R Dc
51 B5 ydD
1
RB Dcy Dpu
51 dC rD
Dc Dc
yd yd
C D
Dp
ur
D
PB
S
WCA B. canis S26
(c) 1.4
0.8
RB
RB
51 51
RB Dcy
51 dC
RB R Dc
51 B5 ydD
1
RB Dcy Dpu
51 dC rD
Dc Dc
yd yd
C D
Dp
ur
D
PB
S
RB
RB RB5
1
5
RB Dcy
51 dC
D
RB R
c
51 B5 ydD
1
RB Dcy Dp
51 dC urD
Dc Dc
yd yd
C D
Dp
ur
D
PB
S
0
a
a
a
a
b
0.6
0.4
0.2
WCA RB51
RB RB
5 51
RB Dcy
51 dC
RB R Dc
51 B5 ydD
1
RB Dcy Dpu
51 dC rD
Dc Dc
yd yd
C D
Dp
ur
D
PB
S
R
RB B5
1
5
RB Dcy
51 dC
RB R Dc
51 B5 ydD
1
RB Dcy Dpu
51 dC rD
Dc Dc
yd yd
C D
Dp
ur
D
PB
S
RB RB5
1
5
RB Dcy
51 dC
RB R Dcy
51 B5 dD
1
RB Dcy Dp
51 dC urD
Dc Dc
yd yd
C D
Dp
ur
D
PB
S
0
WCA B. canis S26
Fig. 3. Anti-Brucella antibodies in sera from mice vaccinated with either a single dose or two doses of RB51 mutants or a
single dose of RB51. At 7 weeks post-vaccination, the serum samples were collected from mice vaccinated with either a
single dose or two doses of RB51 mutants or a single dose of RB51. (a) IgG, (b) IgG1 and (c) IgG2a antibody levels against
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Q. L. Truong and others
WCAs of strain RB51 or B. canis S26 in sera from vaccinated mice. Results are shown as mean¡SD . Significance:
* (P,0.001) indicates significant difference in antibody levels between vaccinated and non-vaccinated mice; b (P,0.05) indicates significant difference between mutant-vaccinated and RB51-vaccinated mice; a (P,0.05) indicates significant difference between single vaccination and booster vaccination with RB51 mutants.
To evaluate the cellular immune response associated with
vaccination with RB51 and its mutants, capture ELISAs
were used to detect the levels of IFN-c and IL-10 in supernatants of splenocytes from immunized mice at 7 weeks
post-vaccination stimulated with heat-killed RB51 or
B. canis S26, concanavalin A or medium. The results
shown in Fig. 4 clearly demonstrate that there were no differences in the levels of IFN-c and IL-10 in supernatants of
splenocytes from immunized mice stimulated with either
heat-killed RB51 or heat-killed B. canis S26. Splenocytes
from mice vaccinated with RB51 or RB51 mutants produced
significantly higher amounts of IFN-c and IL-10 than those
from non-vaccinated mice (Pv0.001). The IFN-c concentration was significantly higher in supernatants of splenocytes
from mice vaccinated with a booster dose of RB51 mutants
than in those from mice vaccinated with a single dose of
RB51 mutants (Pv0.05). However, there was no significant
increase in levels of IL-10 in any of vaccinated groups.
As expected, splenocytes from all mice that were stimulated
with concanavalin A produced significant amounts of IFNc and IL-10, whereas no cytokine production was induced
by medium stimulation (data not shown).
DISCUSSION
In the campaign to eradicate bovine brucellosis, the extensive use of the strain RB51 vaccine has played an important
role in reducing the incidence of the disease in cattle in
many countries. Although the use of RB51 has virtually
eliminated interference with the serological diagnosis of
field infections, several drawbacks are associated with this
vaccine strain, including its variable efficacy and its ability
to induce human infections and sometimes abortions in
pregnant animals (Moriyón et al., 2004; Wang & Wu,
2013). In dogs, oral or intravenous inoculation with
RB51 did not result in bacterial shedding in urine or
faeces or abortion in pregnant dogs. However, it caused
infection of the oropharyngeal lymph nodes, liver, spleen
and placenta, and infected canine placental membranes
or fluids may be a source of infection for other animals
and humans (Palmer & Cheville, 1997). Therefore, questions have arisen concerning vaccine safety, which poses a
significant challenge for Brucella vaccine development.
For this reason, the development of new, safer or improved
vaccines is still a matter of great interest.
Our previous studies using transposon mutagenesis have
identified cydC and purD as being required for intramacrophage survival and virulence of Brucella in the purineor oxygen-limited environments inside hosts (Truong
et al., 2014, 2015). In particular, cydC : : Tn5 and
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purD : : Tn5 mutants were found to be attenuated for virulence and survival in both RAW and HeLa cells and in
BALB/c mice, and protected the mice against challenge
with virulent B. abortus strain 544. These data indicate
that cydC and purD can be used as the ideal candidate
genes for the generation of new or improved vaccine strains
(Truong et al., 2014, 2015). In the present study, in an
attempt to enhance the safety of RB51, cydC, cydD (ATPbinding protein) and purD were therefore selected for deletion to further attenuate strain RB51 vaccine.
The resulting RB51DcydC, RB51DcydD, RB51DpurD,
RB51DcydCDcydD and RB51DcydCDpurD mutants were
examined for survival and attenuation in RAW cells and
BMDMs. As shown in this study, the deletion of cydC,
cydD and purD did not affect bacterial internalization, but
had a marked effect on the survival of RB51 in RAW cells
and BMDMs. Notably, RB51 mutants were more attenuated
in these murine macrophages than the parental RB51. This
observation was consistent with our previous finding that
a transposon insertion in cydC of B. abortus crippled the
ability of Brucella to survive inside macrophages by diminishing its resistance to host defence mechanisms (Truong
et al., 2014). It is not surprising that a single deletion of
cydD or deletion of both cydC and cydD in RB51 had the
same attenuated phenotype in vitro as did the cydC mutant,
because both genes encode an ATP-binding cassette (ABC)type transporter that is required for the activity of cytochrome
bd and c oxidases, similar to that seen in E. coli (Goldman
et al., 1996; Poole et al., 1994). In addition, the deletion of
purD renders the RB51DcydC mutant severely attenuated.
In this case, the attenuation of RB51DcydCDpurD is based
on two altered behaviours: (1) the phenotype observed in
the RB51DcydC mutant and (2) the purine auxotrophy
caused by deletion of purD, which has previously been
demonstrated to be required for de novo purine biosynthesis
and survival of Brucella in macrophages (Truong et al., 2015).
In BALB/c mice, we found that RB51 mutants were cleared
faster than the parental RB51. Notably, the RB51DcydC,
RB51DcydD, RB51DcydCDcydD and RB51DcydCDpurD
mutants were rapidly cleared from spleens of mice by 3
weeks post-inoculation, whereas the RB51DpurD mutant
and its parent RB51 were detectable in spleens up to 4 and
7 weeks post-inoculation, respectively. Moreover, no splenomegaly was observed in mice after intraperitoneal inoculation
of RB51 mutants in mice, compared with the splenomegaly
induced by the parental RB51 (Fig. 2b). The results presented
here are consistent with our previous findings (Truong et al.,
2014, 2015) and the work of other investigators (Alcantara
et al., 2004; Yang et al., 2010) demonstrating the important
roles of purine biosynthesis (purD, purE) and ABC-type
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Booster vaccine candidates against Brucella spp.
(a)
3500
Single dose
3000
a
IFN-g (pg ml–1)
2500
b
a
a
Booster dose
a
a
b
2000
1500
1000
500
R
51 B51
RB Dc
5 yd
RB R 1Dc C
51 B5 yd
1 D
RB Dcy Dp
51 dC urD
Dc Dc
yd yd
C D
Dp
ur
D
PB
S
RB
RB
RB RB
5 51
RB Dc
51 ydC
RB R D
51 B5 cyd
RB Dc 1Dp D
51 ydC urD
Dc Dc
yd yd
C D
Dp
ur
D
PB
S
R
51 B5
1
RB Dc
51 ydC
RB R Dc
51 B5 yd
1 D
RB Dcy Dp
51 dC urD
Dc Dc
yd yd
C D
Dp
ur
D
PB
S
0
HK RB51
(b)
HK B. canis S26
500
Single dose
Booster dose
IL-10 (pg ml–1)
400
300
200
100
RB RB
5 51
RB Dc
51 ydC
RB R Dc
51 B5 yd
1 D
RB Dcy Dp
51 dC urD
Dc Dc
yd yd
C D
Dp
ur
D
PB
S
RB RB
5 51
RB Dc
51 ydC
RB R Dc
51 B5 yd
1 D
RB Dcy Dp
51 dC urD
Dc Dc
yd yd
C D
Dp
ur
D
PB
S
RB RB
5 51
RB Dc
51 ydC
RB R D
51 B5 cyd
RB Dc 1Dp D
51 ydC urD
Dc Dc
yd yd
C D
Dp
ur
D
PB
S
0
HK RB51
HK B. canis S26
Fig. 4. Cytokine production by stimulated splenocytes from mice vaccinated with either a single dose or two doses of RB51
mutants or a single dose of RB51. At 7 weeks post-vaccination, mice were euthanized and splenocytes were harvested and
stimulated with heat-killed (HK) RB51 and HK B. canis S26 or with concanavalin A or medium alone as controls. IFN-c (a)
and IL-10 (b) production after 72 h of stimulation was detected using ELISA. Cytokine production is expressed as mean¡SD
cytokine concentration for each group of mice. Significance: *(P,0.001) indicates significant difference between vaccinated
and non-vaccinated mice; b (P,0.05) indicates significant difference between mutant-vaccinated and RB51-vaccinated
mice; a (P,0.05) indicates significant difference between single vaccination and booster vaccination with RB51 mutants.
transporter (cydC) in the virulence of B. abortus in mice.
These results suggest that the rapid clearance of RB51
mutants from mice may provide a measure of safety for the
host species. These properties make these novel mutant
strains attractive candidates as safe vaccines.
http://mic.microbiologyresearch.org
To investigate whether RB51 mutants induce protection,
the protective efficacy of these mutants was assessed in
mice. The protection study showed that a single dose of
RB51 and its RB51DcydC, RB51DcydD, RB51DpurD,
RB51DcydCDcydD and RB51DcydCDpurD mutants could
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Q. L. Truong and others
protect the mice against homologous B. abortus S2308
challenge, but vaccine efficacy was reduced compared
with that induced by RB51 in the mice vaccinated with a
single dose of RB51 mutants. The lower protection levels
observed in RB51 mutant-vaccinated mice probably
resulted from the reduced persistence and lower numbers
of bacteria following vaccination with mutants than with
parental RB51, which was induced by the additional deletion of cydC, cydD and purD in RB51 or cydD and purD
in RB51DcydC. When a booster dose of attenuated RB51
mutants was administered 3 weeks after the first vaccination, these mutants were also rapidly cleared from
mice without causing a marked inflammatory response.
Therefore, we next investigated whether prime–boost vaccination with highly attenuated RB51 mutants could
enhance their protective efficacy in mice. As shown here,
booster vaccination with RB51 mutants was significantly
more effective than single vaccination with either RB51
or RB51 mutants. Mice vaccinated with two doses of
RB51DcydC, RB51DcydD, RB51DpurD, RB51DcydCDcydD
or RB51DcydCDpurD mutants produced significantly
higher log units of protection against challenge with virulent B. abortus S2308 than mice vaccinated with a single
dose of these mutants. To the best of our knowledge, this
trial represents the first time that the use of booster vaccination with RB51 mutants can increase protection against
B. abortus infection in mice. Also, the use of this strategy
is in agreement with previous studies that have shown
that RB51 booster vaccination is an effective vaccination
strategy for enhancing protection against B. abortus infection in bison, water buffalo and cattle (Caporale et al.,
2010; Leal-Hernandez et al., 2005; Olsen & Johnson, 2012).
vaccinated mice were also investigated. The data showed
that mice immunized with a single dose of highly attenuated RB51 mutants produced significantly higher levels of
specific IgG, IgG1 and IgG2a antibodies and IFN-c compared with PBS-immunized mice; however, these levels
were lower than those from mice immunized with parental
RB51. This can be explained by the rapid clearance of RB51
mutants, because these mutants do not persist long enough
in the host to elicit a strong immune response. As expected,
further parenteral boosting with RB51 mutants administered 3 weeks after the first dose was observed to induce
a significant enhancement of antibody and cellular
immune responses, as shown by higher levels of IgG,
IgG1 and IgG2a antibodies and IFN-c compared with
those produced by a single dose of RB51 mutants. Furthermore, the presence in the serum of vaccinated mice of predominantly IgG2a rather than IgG1 antibodies specific for
RB51 antigens indicates the preferential development of a
T-helper 1 (Th1)-type immune response. This is further
corroborated by the elevated levels of IFN-c compared
with IL-10. As protective immunity induced by RB51 vaccination against smooth Brucella strains generally depends
on the generation of a Th1-type cellular immune response
characterized by secretion of IFN-c from antigen-specific
T cells (Pasquali et al., 2001; Stevens et al., 1995), we therefore speculate that the high level of IFN-c induced by a
booster dose of RB51 mutants may offer an explanation
for the better protection provided by these mutants against
homologous B. abortus S2308 challenge.
In terms of protection, vaccination with RB51 has been
shown previously to provide effective protection against
infections caused by B. abortus, B. melitensis and B. ovis in
mice (Jiménez de Bagüés et al., 1994; Moriyón et al., 2004)
or B. canis in dogs (Hur & Baek, 2010). Therefore, it is to
be expected that attenuated mutants of RB51 would also
be the best choice for a vaccine targeting B. canis infection.
As described herein, a single vaccination with RB51 or
a booster vaccination with RB51 mutants not only provided
protection against challenge with the homologous B. abortus
S2308, but also conferred high levels of protection in
mice against challenge with the heterologous B. canis S26.
Remarkably, vaccination with RB51 or its isogenic RB51
mutants afforded even better protection against B. canis challenge than against B. abortus challenge. In dogs, brucellosis
has been shown to be caused mainly by B. canis and
occasionally by B. abortus (Baek et al., 2003, 2012; Forbes,
1990; Wanke, 2004); however, there is no vaccine available
at the current time that protects dogs against these bacterial
strains. Therefore, highly attenuated mutants such as RB51
mutants could be ideal safe vaccine candidates to protect
against canine brucellosis caused by B. abortus and B. canis.
As RB51 has the same rough phenotype as the experimental
B. canis strain used in this study, several factors can be
invoked to explain the high level of protective immunity
to B. canis in BALB/c mice. First, it is likely that the antibodies induced by RB51 and its mutants, probably directed
against the outer membrane proteins of B. canis, could also
protect against rough B. canis infection. In fact, when the
WCAs from B. canis were used for testing the humoral
immune response induced by RB51 and its mutants, high
levels of specific IgG antibodies were observed in immunized mice. These antibody responses were similar to
those determined by assays using WCAs from RB51. This
observation suggests that immunization with RB51 and
its mutants offered cross-reactive immunity to B. canis
antigens, and that this antibody response may have contributed to protection against challenge with B. canis.
Second, we showed here that the protective properties of
RB51 and its mutants may be the result of their ability to
induce a strong Th1-type cellular immune response, as
shown by the high levels of serum IgG2a antibodies and
of IFN-c secretion by splenocytes from RB51- and RB51
mutant-immunized mice in response to exposure to
B. canis whole cells – an immune response that is considered to play a crucial role in protection against Brucella
infection (Baldwin & Parent, 2002).
To ascertain the protective mechanisms induced by vaccination with RB51 and its mutants, serum antibody
responses and the cytokines produced by splenocytes of
In conclusion, to the best of our knowledge, this is the first
study to investigate the protective immunity against
B. canis infection conferred by vaccination of mice with
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Microbiology 161
Booster vaccine candidates against Brucella spp.
RB51 or its isogenic mutants. Although a single dose of
genetically modified, live-attenuated mutants of RB51
may not be an ideal vaccine candidate for use in animals,
our results demonstrate that the use of two doses of
RB51 mutants is superior to a single dose of RB51 or
RB51 mutants, not only in inducing a specific antibody
response, but also in eliciting specific cytokine secretion,
resulting in significant protection of mice against challenge
with virulent strains of B. abortus and B. canis. The
enhanced safety of RB51 mutants and the promising efficacies achieved by booster vaccination with these mutants
suggest that RB51 mutants, such as RB51DcydD and
RB51DpurD, which provide the best protective immunity
in mice, could be suitable as live-attenuated booster vaccine candidates against brucellosis caused by B. abortus
and B. canis, and deserve further investigation in domestic
animals such as cattle or dogs.
Caporale, V., Bonfini, B., Di Giannatale, E., Di Provvido, A., Forcella,
S., Giovannini, A., Tittarelli, M. & Scacchia, M. (2010). Efficacy of
Brucella abortus vaccine strain RB51 compared to the reference
vaccine Brucella abortus strain 19 in water buffalo. Vet Ital 46 (1319), 5–11.
Corbel, M. J. (2006). Brucellosis in Humans and Animals. Geneva:
World Health Organization.
Forbes, L. B. (1990). Brucella abortus infection in 14 farm dogs. J Am
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Godfroid, J., Cloeckaert, A., Liautard, J. P., Kohler, S., Fretin, D.,
Walravens, K., Garin-Bastuji, B. & Letesson, J. J. (2005). From the
discovery of the Malta fever’s agent to the discovery of a marine
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ACKNOWLEDGEMENTS
The B. abortus strain RB51 vaccine was kindly provided by Choong
Ang Vaccine Laboratories, Korea. This research was supported by
the Basic Science Research Program through the National Research
Foundation of South Korea funded by the Ministry of Education,
Science and Technology (grant 2012R1A1A4A01015303).
domestic elk in Korea. Zoonoses Public Health 57, 155–161.
Hur, J. & Baek, B. K. (2010). Efficacy of Brucella abortus strain RB51
vaccine in Korean mongrel dogs against virulent strains of B. abortus
biotype 1 and B. canis. Korean J Vet Serv 33, 29–35.
Jang, Y., Kim, H., Bang, H. A., Lee, M. J., Che, N. H. & Lee, W. C.
(2011). Epidemiological aspects of human brucellosis and
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