Introduction

Brucellae are Gram-negative, facultative intracellular pathogens that may cause severe disease in humans and animals (Bercovich 2000). Brucellosis remains endemic in many develo** countries, causing important economic losses and public health risks. At present, live attenuated vaccines are the most efficient means for prevention and control of animal brucellosis (Schurig et al. 2002). However, difficulties in differentiating vaccine immunization from natural infection limit their application (Moriyon et al. 2004). In order to circumvent this problem, several strategies for the development of alternative vaccines have been described. One is the isolation of avirulent or attenuated strains lacking the O antigen (Monreal et al. 2003). A vaccine that can differentiate infection from immunization will have extensive uses.

B. melitensis M5-90 is currently administered to sheep and goats to prevent brucellosis in China (Cosivi and Corbel 1998). It was derived from a virulent B. melitensis strain, M28, isolated from a sheep and serially passaged for 90 generations in chicken embryo fibroblasts. However, M5-90 vaccine has some disadvantages; it can induce abortion and milk excretion, and it interferes with serological tests.

Lipopolysaccharide (LPS) is one of the most important virulence factors of Brucella and dominates the antibody response of its hosts (Cardoso et al. 2006). Phosphoglucomutase (pgm, BM590_A0058) is an essential enzyme in LPS assembly in Brucella, and is responsible for the interconversion of glucose-6-phosphate to glucose-1-phosphate. B. abortus S19Δpgm could not synthesize the sugar-nucleotide UDP-glucose, and did not induce a detectable O-chain antibody response in hosts. It also exhibited extreme attenuation in BALB/c mice, but the immune protection conferred was the same as by the parental strain (Ugalde et al. 2003).

To further test the possibility of a pgm mutant as a new vaccine candidate against B. melitensis 16 M, we constructed a M5-90Δpgm deletion mutant and evaluated its virulence in HPT-8 embryonic trophoblast cells and in mice. We also used purified recombinant PGM protein to assess the antibody response.

Materials and methods

Ethics statement

The study was approved by the Institutional Committee of Post-Graduate Studies and Research at Shihezi University, China. All efforts were made to minimize animal suffering.

Bacterial strains, growth conditions and plasmids

B. melitensis strain 16 M and vaccine strain M5-90 were obtained from the Center of Chinese Disease Prevention and Control (Bei**g, China). Brucella was cultured in tryptic soy agar (TSA) or tryptic soy broth (TSB) (Sigma, St. Louis, MO, USA). Escherichia coli strains DH5α and BL21 were grown on Luria–Bertani (LB) medium. Culture medium was supplemented with appropriate antibiotics (100 μg/mL ampicillin for M5-90 or 50 μg/mL kanamycin for E. coli) when necessary. Plasmid pGEM-7Zf+ was purchased from Promega (Madison, WI, USA).

Mice

Six-week-old BALB/c female mice were obtained from the Experimental Animal Center of the Academy Military Medical Science (Bei**g, China). Animals were maintained in barrier housing with filtered inflow air in a restricted-access room in pathogen-limited conditions. They were acclimatized for a minimum of 1 week before experimentation. Water and commercial food were provided ad libitum. All experimental procedures and animal care were performed in compliance with institutional animal care regulations.

Construction of M5-90Δpgm

The M5-90Δpgm deletion mutant was constructed as described (Zhang et al. 2013) with some modifications. The sequence upstream (1016 bp) of pgm was amplified from B. melitensis M5-90 genome using the primer pair up-F and up-R (Table 1). The sequence downstream (1041 bp) of pgm was amplified from B. melitensis M5-90 genome using the primer pair dn-F and dn-R (Table 1). The two arms of M5-90 pgm were ligated to pGEM-7Zf+ via XbaI, KpnI and SacI the sites, and to generate the suicide plasmid pGEM-7Zf + -pgm. SacB-F and SacB-R primers (Table 1) were designed for amplification of the B. subtilis SacB DNA fragment (1500 bp), which is a selectable marker gene. The SacI–SacI insert from a plasmid containing the PCR amplified DNA was subcloned into plasmid pGEM-7Zf+-pgm to generate plasmid pGEM-7Zf+-pgm-SacB. Competent B. melitensis M5-90 was electroporated with pGEM-7Zf+-pgm-SacB. Potential pgm deletion mutants M5-90Δpgm were selected in the presence of 100 μg/mL ampicillin in the first screening and 5 % (w/v) sucrose in the second screening. The deletion mutant was further confirmed by PCR amplification and RT-PCR sequencing, as described previously (Wang et al. 2009). P-F and P-R primers were designed for the detection of the deletion mutant.

Table 1 Primers used in this work
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Evaluation of M5-90Δpgm attenuation in HPT-8 embryonic trophoblast cells

HPT-8 embryonic trophoblast cells were seeded in six-well plate and infected with M5-90Δpgm, parental strain M5-90, or wild-type strain 16 M at a multiplicity of infection (MOI) of 100, as previously described (Hernández-Castro et al. 2008). Culture plates were centrifuged at 350×g for 5 min at room temperature and then placed at 37 °C under 5 % CO2. At 45 min post-infection, the cells were washed three times with medium without antibiotics and then incubated with 50 μg/mL of gentamicin (Invitrogen, Carlsbad, CA, USA) for 1 h to kill extracellular bacteria. Then, the culture was placed in Dulbecco’s modified Eagle’s medium (DMEM, Gibco Life Technologies, Rockville, MD, USA) containing 25 µg/mL gentamicin (defined as time zero). At different time points (0, 12, 24 and 48 h) post-infection, the supernatant was discarded and cells were lyzed by PBS containing 0.1 % (v/v) Triton X-100, and the live bacteria were enumerated by plating on TSA plates. All assays were performed in triplicate and repeated at least three times.

HPT-8 cells cytotoxicity assay

HPT-8 at 1 × 106 cells/well were cultured in six-well plate for 18 h in a 5 % CO2 atmosphere at 37 °C, and infected with 16 M, M5-90, or M5-90Δpgm at a MOI of 100. At 12 and 24 h post-infection, the supernatant was collected, and the level of lactate dehydrogenase (LDH) was determined using the CytoTox 96 nonradioactive cytotoxicity assay as previously described (Pei and Ficht 2004). Cell death was expressed as the percentage of maximum LDH release, which was calculated using the following formula: percentage of LDH release = 100× (OD490 of infected cells—OD490 of uninfected cells)/(OD490 of lysed uninfected cells—OD490 of uninfected cells). The maximum release was determined following dissolution of cell monolayers using Triton X-100 (1 % v/v). All assays were performed in triplicate wells, and the results represent the mean ± SD from at least three separate experiments.

Cytokine production assay

The procedure for IL-6, IL-10 and TNF-α detection was as previously described (Kim et al. 2006). HPT-8 at 1 × 106 cells/well were cultured in six-well plates and challenged with M5-90Δpgm, M5-90, or PBS in a total of 1 mL DMEM at a MOI of 100. At 24 h post-infection, the culture supernatants were filtered through 0.22 μm filters, centrifuged at 16,000×g for 15 min, and stored deep frozen until measurement. IL-6, IL-10 and TNF-α were determined using a Human Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. All assays were performed in triplicate and repeated at least three times.

Evaluation of M5-90Δpgm attenuation in BALB/c mice

The evaluation of Brucella survival capability in mice was carried out as previously described (Pugh et al. 1989, 1991). Briefly, 6-week-old female BALB/c mice were inoculated intraperitoneally (i.p.) with 200 μL PBS containing 1 × 106 CFU of M5-90Δpgm, M5-90, or 16 M. The negative control group was injected i.p. with 200 μL PBS. Infected mice were held in microisolator cages in biosafety level 3 facilities. Survival or persistence of the bacteria in mice was evaluated by enumerating the bacteria in spleens at different time points post infection. At 10, 20 and 30 days post-infection, mice were euthanized and spleens were removed aseptically. The spleens were collected, weighed, homogenized in 1 mL PBS containing 0.1 % (v/v) Triton X-100, serially diluted, and plated on TSA plates. Plates were incubated at 37 °C and the number of CFU was counted after 3 days to evaluate the B. melitensis survival capability in mice. All assays were performed three times with similar results.

Protection induced by M5-90Δpgm in mice

Groups of 6-week-old female BALB/c mice (n = 20 per group) were vaccinated i.p. with 1 × 106 CFU (200 μL) of M5-90Δpgm (experimental vaccine group), M5-90 (reference vaccine control group), or 200 μL PBS (unvaccinated control group). At 35 days post-vaccination, the mice were challenged i.p. with 1 × 106 CFU per mouse (200 μL) of virulent strain 16 M. Mice (n = 10 per time point per group) were euthanized by cervical dislocation 2 and 4 weeks post challenge, and bacterial CFUs in the spleens were determined as described above. A mean value for each spleen count was obtained after logarithmic conversion. Log units of protection were obtained by subtracting the mean Log CFU for the experimental group from the mean Log CFU for the control group, as previously described (Adone et al. 2005). The experiments were repeated twice.

Serological and cytokine assays

6-week-old female BALB/c mice were randomly divided into three groups. Group 1 and 2 were injected i.p. with 200 μL PBS containing 1 × 106 CFU of M5-90Δpgm or M5-90, respectively, and group 3 was injected i.p. with 200 μL PBS as negative control. Serum samples were obtained from peripheral blood of immunized mice at 10, 20 and 30 days post-immunization, and the serum samples were kept at −80 °C until use (Zhang et al. 2011).

IFN-γ, IL-2 and IgG levels in serum samples obtained from mice 10, 20 and 30 days after the last vaccination were determined using an Mouse Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions, as described previously (Jazani et al. 2011). All assays were performed in triplicate and the concentration of each cytokine in the serum sample was calculated using a linear regression equation obtained from the absorbance values of standards, according to the manufacturer’s procedures.

Analyses of lymphocyte subpopulations

Six-week-old female BALB/c mice were randomly divided into three groups. Groups 1 and 2 were injected i.p. with 200 μL PBS containing 1 × 106 CFU of M5-90Δpgm or M5-90, respectively, and group 3 was injected i.p. with 200 μL PBS as the negative control. Peripheral blood samples were obtained from immunized mice 10, 20 and 30 days post-immunization. The antibodies used for analysis were anti-mouse CD4-FITC monoclonal antibodies (mAb) and RPE-conjugated CD8-specific Ab. Samples were analyzed with a fluorescence flow cytometry sorting technique (FACS) can calibrator. Each assay was repeated three times.

Immunized sheep and serological and cytokine analysis

Thirty female 4-month-old lambs of the Chinese Merino breed, born in the brucellosis-free flock, were reared for the experiment. They were randomly divided into three groups (n = 10 per group). Groups 1 and 2 were subcutaneously injected (s.c.) with 200 μL PBS containing 1 × 109 CFU of M5-90Δpgm or M5-90, respectively, and group 3 was s.c. with 200 μL PBS as the negative control. Serum samples were obtained from immunized sheep at 2, 4, 6, 8 and 10 weeks post-immunization, as described above.

Serum samples were tested by RBPT and STAT (IVRI, Izatnagar) at 2, 4, 6, 8 and 10 weeks after the last vaccination, as briefly described below.

Equal volumes (20 μL) of RBPT colored antigen and the test serum were mixed on a clean glass slide with the help of a clean, sterile toothpick. The slide was observed after 1 min for the formation of clumps. The formation of clear clumps indicated a positive test, while the absence of clumps was considered a negative reaction (Edelsten 1989).

For STAT, plain antigen was used according to the standard protocol (Morgan 1967). Two-fold serial dilutions (1:25 to 1:200) of the sera were prepared in phenol saline (1150 μL of phenol saline was added to the first tube and 500 μL to the remaining tubes; then, 100 μL of serum was added to the first tube and mixed, 500 μL was transferred to the next tube, and 750 μL were discarded; further volumes of 500 μL were transferred to subsequent tubes to give a series of double dilutions. Then, 500 μL of STAT white antigen were added each tube). Half of one milliliter of plain antigen was added to each tube. After mixing, all the tubes were incubated at 37 °C for 24 h. A titer of 1:50 or above was considered positive for brucellosis.

The IFN-γ levels in the serum samples were also determined using a sheep IFN-γ ELISA Kit (Elabscience Biotechnology Co., Ltd, Wuhan, China), according to the manufacturer’s instructions. All assays were performed in triplicate.

Cloning, expression and purification of recombinant proteins

The pgm ORF was amplified by PCR from the DNA of B. melitensis M5-90. Then the amplified DNA fragment was cloned into the pET-28a vector (Novagen, Madison, WI, USA) to generate the recombinant plasmid pET-28a-pgm, and expressed in E. coli BL21 (DE3) as an N-terminally His-tagged fusion protein. The expression of the recombinant protein was analyzed by 12 % SDS-PAGE. The recombinant PGM protein was purified as described previously (Huleatt et al. 2008; Lindae et al. 2015).

Western blot analysis

Cell lysates containing recombinant PGM were analyzed by Western blotting, as previously described (Zhang et al. 2014). Briefly, the purified recombinant PGM protein was separated by 12 % SDS-PAGE. Proteins were then transferred to nitrocellulose by semi-dry Western blotting for 40 min in transfer buffer (100 mM Tris–HCl, 150 mM NaCl, 0.05 % Tween 20, pH 7.2). Membranes were incubated in blocking solution (5 % nonfat milk in TBST) for 1 h at room temperature. Then the membrane was incubated with Brucella-vaccinated sera, diluted 1:500, in 2.5 % milk/TBST, at room temperature, for 1 h. After three washes, the membrane was incubated with peroxidase conjugated rabbit anti-mouse IgG for 1 h at room temperature in 5 % milk/TBST. After three washes, bound conjugate was visualized with an enhanced HRP-DAB substrate color kit (Tiangen Biotech (Bei**g) Co. Ltd., China). Western blotting was repeated in triplicate.

Statistical analysis

Bacterial survival in HPT-8 and in mice is expressed as the mean Log CFU ± the standard deviation (SD). Antibody response and cytokine production are expressed as the mean OD450 ± SD. RBPT and STAT were compared by using the Fisher test. The strain effect was further analyzed by using the non-parametric Kruskall–Wallis rank test. Results expressed as percentages were analyzed by the Fisher test. P values of <0.05 were considered statistically significant.

Results

Construction of the B. melitensis M5-90Δpgm mutant

The pgm gene deletion mutant of B. melitensis strain M5-90 (M5-90Δpgm) was successfully obtained. A 1310 bp DNA fragment was amplified from M5-90Δpgm with primers P-F and P-R, but a 2422 bp DNA fragment was amplified from M5-90 with same primers, indicating that the pgm gene was correctly knocked out (Fig. 1a). PCR products were sequenced to confirm the deletion of mutant strain. RT-PCR showed that pgm was not transcribed in M5-90Δpgm (Fig. 1b).

Fig. 1
figure 1

Identification of construction of M5-90Δpgm. a PCR identification of M5-90Δpgm. Lanes M, DNA marker; 1, the parental strain M5-90; 2 M5-90Δpgm mutant strain; 3, negative control. b RT-PCR identification of M5-90Δpgm. Lanes M, DNA marker; 1, the parental strain M5-90; 2 M5-90Δpgm mutant strain; 3, negative control

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The M5-90Δpgm mutant is attenuated in HPT-8 embryonic trophoblast cells

To assess the attenuation of B. melitensis M5-90Δpgm, HPT-8 embryonic trophoblast cells were infected with the M5-90Δpgm mutant and compared with trophoblasts infected with other B. melitensis strains to determine the bacterial ability of intracellular replication. At 0 h post-infection, there were no differences in the amount of bacteria among these three different strains (Fig. 2). By 4 h post-infection, the HPT-8 cells contained equivalent bacterial loads, which indicated no significant variation in the ability of the bacteria to invade HPT-8 cells. However, 12 h post-infection, there was a 1.0-log and 0.2-log decrease in the bacterial number of M5-90Δpgm inside the HPT-8 cells compared to 16 M and M5-90, respectively. At 24 h post-infection, this decrease was 3.2-log and 0.9-log, respectively (P < 0.01) (Fig. 2). These results show that the M5-90Δpgm mutant has a limited capability to replicate in HPT-8 cells compared with virulent 16 M or the vaccine strain M5-90, indicating that pgm is involved in Brucella chronic infection. M5-90Δpgm is attenuated in HPT-8 cells.

Fig. 2
figure 2

Intracellular replication of Brucella melitensis M5-90Δpgm in HPT-8 cells. HPT-8 cells were infected with B. melitensis M5-90Δpgm, M5-90 or 16 M at a MOI of 100. At 0, 4, 8, 12 and 24 h post-infection, infected HPT-8 cells were lyzed and bacteria were enumerated by plating serial dilutions on TSA plates. M5-90Δpgm failed to achieve the level of colonization reached by the wild-type and parental strains. Significant differences between M5-90Δpgm and M5-90 are indicated by *(P < 0.05)

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The M5-90Δpgm mutant does not induce high levels of cytotoxicity

To determine whether the cytotoxicity of M5-90Δpgm for HPT-8 cells was weakened, we used 16 M, M5-90 and M5-90Δpgm-infected HPT-8 cells at MOI of 100, and evaluated the LDH release. At 12 h post-infection, 37.58 ± 1.84 % and 25.35 ± 2.12 % of LDH was released in 16 M and M5-90-infected cells; but only 10.23 ± 1.25 % of LDH was released in M5-90Δpgm-infected cells (Fig. 3). At 24 h post-infection, 43.62 ± 1.81 % and 30.43 ± 1.02 % of LDH was released in 16 M and M5-90-infected cells; but only 12.65 ± 1.11 % of LDH was released in M5-90Δpgm-infected cells (Fig. 3). All results indicated that the M5-90Δpgm mutant did not induce high levels of cytotoxicity in HPT-8 cells at MOI of 100 by 12 and 24 h post-infection.

Fig. 3
figure 3

M5-90Δpgm mutant does not induce high levels of cytotoxicity B. melitenesis for HPT-8 cells. HPT-8 cells were infected with 16 M, M5-90 and M5-90Δpgm at MOI of 100. Cells cytotoxicity was determined at 12 and 24 h post-infection using the lactate dehydrogenase release assay. The results are the mean ± SD deviations of a representative experiment that was repeated three times with similar results. Significant differences between M5-90Δpgm and 16 M are indicated by **(P < 0.01). Significant differences between M5-90Δpgm and M5-90 are indicated by **(P < 0.01)

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Effect of M5-90Δpgm on pro-inflammatory cytokine expression in HPT-8 cells

We examined whether M5-90Δpgm could regulate pro-inflammatory cytokines such as IL-6, TNF-α and IL-10 in HPT-8 cells. The HPT-8 cell line was challenged with M5-90Δpgm, M5-90, or PBS in a total of 1 mL DMEM at a MOI of 100 for 24 h. The culture supernatants were filtered and we measured the IL-6, TNF-α and IL-10 secretions by ELISA. The levels of cytokines IL-6, TNF-α and IL-10 were significantly higher in M5-90Δpgm infected HPT-8 cells than for M5-90 (P < 0.01), but no significant difference was observed for IL-10 (Fig. 4).

Fig. 4
figure 4

Concentrations of cytokines induced by B. melitensis M5-90Δpgm. HPT-8 were challenged with M5-90Δpgm, M5-90 or PBS in a total of 1 mL DMEM at a MOI of 100 for 24 h. The culture supernatants were collected and IL-6, TNF-α and IL-10 levels determined by ELISA. The results are mean ± SD (n = 3). Secretion of cytokines a IL-6; b TNF-α; c IL-10. Significant differences between M5-90Δpgm and PBS are indicated by **(P < 0.01)

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M5-90Δpgm is attenuated in mice

To evaluate the role of the deletion of M5-90Δpgm on B. melitensis virulence in vivo, BALB/c mice were injected i.p. with 1 × 106 CFU of strains M5-90Δpgm, M5-90 or 16 M. The number of CFU was evaluated 10, 20 and 30 days post-infection in the spleen of animals. Splenic CFU in M5-90Δpgm infected mice were significantly reduced at all times compared with the parental strain M5-90 and the wild-type strain 16 M. At 30 days post-infection, 0.3-log of M5-90Δpgm were isolated from the spleens of infected mice, while 2.2-log of M5-90 and 5.8-log of 16 M were isolated (Fig. 5a). In addition, spleens from M5-90Δpgm infected mice were significantly lighter than those from M5-90 and 16 M infected mice (Fig. 5b). Similarly, splenomegaly was observed in M5-90 infected mice, but not in M5-90Δpgm infected ones, implying reduced inflammatory response exhibited by M5-90Δpgm. The reduced virulence and inflammatory response make it advantage for M5-90Δpgm to be a vaccine candidate. All these results indicate that survival of M5-90Δpgm is attenuated in mice.

Fig. 5
figure 5

Clearance of M5-90Δpgm after infection. BALB/c mice were inoculated intraperitoneally with 1 × 106 CFU of B. melitensis M5-90Δpgm, M5-90 or 16 M. Infected mice were euthanized 10, 20 or 30 days post-inoculation. Spleens were aseptically removed and individual spleens were assessed for a colonization and b weight. Values are the means from individual mice ± SD (n = 10/time point). Significant differences between M5-90Δpgm and M5-90 are indicated by *(P < 0.05) or **(P < 0.01)

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Strain M5-90Δpgm induces immune protection against challenge with virulent B. melitensis 16 M

In order to examine the protection induced by M5-90Δpgm, a vaccine challenge experiment was performed. Mice were vaccinated i.p. with 1 × 106 CFU of M5-90Δpgm, M5-90 or PBS. Thirty-five days post-vaccination, mice were challenged with 1 × 106 CFU of virulent B. melitensis strain 16 M. Animals immunized with M5-90Δpgm exhibited significantly fewer splenic Brucella than PBS-immunized mice 2 (3.43-log) and 4 (2.83-log) weeks after challenge (P < 0.05; Table 2). M5-90 also induced significant protection at 2 (3.00-log) and 4 (2.61-log) weeks after challenge (P < 0.05; Table 2).

Table 2 Evaluation of the protective efficacy of B. melitensis M5-90Δpgm and M5-90 against 16 M infection in BALB/c mice
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M5-90Δpgm induces humoral responses

Heat-killed and sonicated B. melitensis M5-90 whole-cell antigens were used, and the production of Brucella-specific antibodies was measured by ELISA in sera from mice inoculated with M5-90Δpgm, M5-90 or PBS, collected at selected intervals post-immunization. Figure 6 shows that antibodies were detected in the sera of mice immunized with M5-90Δpgm and M5-90 10 days post-vaccination, and IgG levels increased with time. However, the IgG levels were low in mice immunized with PBS, indicating that M5-90Δpgm and M5-90 could induce Brucella-specific antibodies; there were no significant differences between M5-90 and M5-90Δpgm (P > 0.05).

Fig. 6
figure 6

Antibody responses following immunization with B. melitensis M5-90Δpgm. Mice were inoculated intraperitoneally with 1 × 106 CFU of B. melitensis M5-90Δpgm or M5-90. Control groups were inoculated with PBS. The mean serum level of IgG against Brucella in different groups 10, 20 and 30 days post-vaccination is shown in terms of optical density (OD). The results are mean ± SD (n = 10/time point) of the absorbance at 450 nm (OD450). Significant differences between M5-90Δpgm and PBS are indicated by *(P < 0.05). And the significant differences between M5-90 and PBS are indicated by *(P < 0.05)

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M5-90Δpgm induces cytokine responses

To characterize the cellular immune response, serum from mice as described above was monitored for cytokine IFN-γ and IL-2 levels by ELISA. As expected, M5-90Δpgm stimulation induced production of IFN-γ and IL-2 in serum, but low cytokine production was induced by PBS. Furthermore, higher cytokine production levels were observed in M5-90Δpgm immunized mice than in M5-90 mice (Fig. 7); both overall IFN-γ and IL-2 levels, and the difference between M5-90Δpgm and M5-90 mice, increased over time.

Fig. 7
figure 7

Serum production of IFN-γ and IL-2 induced by B. melitensis M5-90Δpgm. Mice were inoculated intraperitoneally with 1 × 106 CFU of M5-90Δpgm or M5-90. Control groups were inoculated with PBS. Mean serum levels (pg/mL) of a IFN-γ and b IL-2 were evaluated 10, 20 and 30 days post-vaccination. Cytokine production is expressed as the mean cytokine concentration ± SD (n = 10/time point) for each group of mice. Significant differences between M5-90Δpgm and PBS are indicated by *(P < 0.05). And the significant differences between M5-90Δpgm and M5-90 are indicated by *(P < 0.05)

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M5-90Δpgm could not produce antibodies to the O-chain of S-LPS

Thirty female 4-month-old lambs of the Chinese Merino breed were randomly divided into three groups (n = 10 per group). They were with M5-90, M5-90Δpgm or PBS, respectively. Serum samples were obtained from sheep immunized with M5-90Δpgm, M5-90 or PBS at 2, 4, 6, 8 and 10 weeks post-immunization. When subjected to serological analysis, serum from sheep (n = 10) inoculated with M5-90 was positive by RBPT and STAT, but serum from sheep (n = 10) inoculated with M5-90Δpgm was negative, and serum from sheep (n = 10) inoculated with PBS was also negative by these tests (Table 3). In M5-90 group, 10 sheep serum were positive. In M5-90Δpgm group, 10 sheep serum were negative. And in PBS group, 10 sheep serum were also negative. For M5-90 immunized sheep, the optimum experimental antibody titer at 4, 6, 8 and 10 weeks post-immunization was 1:200.

Table 3 The results of STAT and RBPT in sheep
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M5-90Δpgm elicits classical Th1 responses

The most effective vaccination strategies in animal models are those that stimulate both CD4+ and CD8+ T cells to produce Th1-associated cytokines. To determine which subpopulation of T cells plays the key role on vaccination with B. melitensis M5-90Δpgm or M5-90, we used FACS analysis to measure expression of CD4+ and CD8+ antigen-specific markers. The results show that in M5-90Δpgm inoculated mice the CD4+/CD8+ ratio was higher than for the parental strain M5-90 and this effect increased with time (Fig. 8).

Fig. 8
figure 8

FACS analysis of peripheral blood samples from mice immunized with B. melitensis (A, B, C) M5-90 or (D, E, F) M5-90Δpgm. BALB/c mice were inoculated with 1 × 106 CFU of M5-90 or M5-90Δpgm. At 10 (a, d), 20 (b, e) and 30 (c, f) days post-immunization, peripheral blood samples (n = 10/time point) were collected and determined by FACS analysis. g The mean ratio CD4+/CD8+ from 10 mice immunized with M5-90 or M5-90Δpgm and also demonstrates the ration displayed by mice inoculated with PBS (control group). The results are mean ± SD (n = 10). Significant differences between M5-90Δpgm and M5-90 are indicated by *(P < 0.05). And the significant differences between M5-90Δpgm and PBS are indicated by **(P < 0.01)

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M5-90Δpgm also induces cytokine responses in sheep

Serum samples were obtained from sheep immunized with M5-90 and M5-90Δpgm at 2, 4, 6, 8 and 10 weeks post-immunization. When subjected to serological analysis, serum from sheep inoculated with M5-90 was positive by RBPT and STAT, but serum from sheep inoculated with M5-90Δpgm was negative by these methods (Table 3). For M5-90 immunized sheep, the optimum experimental antibody titer at 4, 6, 8 and 10 weeks post-immunization was found to be 1:200.

Serum was collected from sheep inoculated with M5-90Δpgm or M5-90 at selected intervals post-immunization to monitor the cytokine IFN-γ level by ELISA. Figure 8 shows that IFN-γ was detected in sheep immunized with either the M5-90Δpgm or the M5-90 vaccine just 2 weeks post-vaccination, indicating that M5-90Δpgm could induce cytokine responses in sheep. The IFN-γ levels due to M5-90Δpgm or M5-90 vaccine peaked 8 weeks post-vaccination (70.47 and 68.27 pg/mL, respectively) and there were no significant differences between M5-90 and M5-90Δpgm groups (P > 0.05; Fig. 9). Low IFN-γ production was induced by PBS (Fig. 9). These results indicate that immunization with B. melitensis strain M5-90Δpgm elicits a classical Th1 response.

Fig. 9
figure 9

Serum production of IFN-γ induced by B. melitensis M5-90Δpgm in sheep. Sheep were subcutaneously injected with 1 × 109 CFU of M5-90Δpgm or M5-90. Control groups were injected with PBS. Mean serum levels (pg/mL) of IFN-γ in different groups were evaluated 2, 4, 6, 8 and 10 weeks post-vaccination. Cytokine production is expressed as the mean cytokine concentration ±SD (n = 10) for each group of sheep. Significant differences between M5-90Δpgm and PBS are indicated by *(P < 0.05)

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Differentiation of M5-90Δpgm immunization from infection using PGM as the test antigen

The observation that PGM elicited antibody response in animals with brucellosis prompted us to consider whether it could be used as a diagnostic antigen for differentiation between infected and vaccinated mice and sheep. To test this, sera from mice immunized with 16 M, M5-90 or M5-90Δpgm were collected. Western blotting was performed to determine whether antibodies to PGM were induced in these sera. A single reaction band against protein PGM was observed on analysis of serum from M5-90 and 16 M vaccinated mice but not with serum from M5-90Δpgm mice (Fig. 10), and the reaction band against protein PGM was observed on analysis of serum from M5-90 vaccinated sheep but not with serum from M5-90Δpgm sheep (Fig. 10), indicating that antibodies against PGM were induced in M5-90 infected mice and sheep but not in M5-90Δpgm vaccinated animals.

Fig. 10
figure 10

Reaction of M5-90Δpgm immunization sera to PGM. Sera were collected from mice immunized with B. melitensis 16 M, M5-90 or M5-90Δpgm. Antibodies to PGM in these sera were detected by Western blotting using recombinant PGM protein. Antibodies against the PGM protein were not detected in sera from M5-90Δpgm immunized mice and sheep. Lanes M, Protein molecular weight makers; 1, the purified recombinant PGM protein on SDS-PAGE; 2, Western blot using serum from M5-90 immunized mice; 3, Western blot using serum from M5-90Δpgm immunized mice; 4, Western blot using serum from 16 M immunized mice; 5, Western blot using serum from M5-90 immunized sheep; 6, Western blot using serum from M5-90Δpgm immunized sheep

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Discussion

Brucellosis is endemic in China and the surrounding area. Brucellosis quarantine is very significant for animal husbandry, animal epidemic disease prevention and biosafety. At present, because the M5-90 vaccine has virulence, and it is difficult to distinguish between natural infection and vaccination, people are unwilling to use the vaccine in a wide range of applications, resulting in the current brucellosis rebound epidemic (Deqiu et al. 2002). Because of the limitations in the M5-90 vaccine, great efforts have been made to develop new vaccine strains. Brucellae include two phenotypes: smooth and rough. Serological interference due to classical vaccines is a significant problem. Therefore, the ideal vaccine must be protective, non-virulent for the host, carrying a genetic marker (Moriyon et al. 2004). Published reports have shown that a B. abortus 2308Δpgm mutant possesses reduced persistence in mice and that it induces protection superior to that of strains S19 and RB51; it was considered a new potential vaccine candidate against brucellosis (Ugalde et al. 2003). The data indicate that pgm gene is probably one of the candidate genes. To investigate whether B. melitensis M5-90Δpgm maintains protective efficacy similar to B. abortus 2308Δpgm, M5-90Δpgm was constructed and its virulence and protection efficacy were assessed in mice.

In this work, a deletion mutant of pgm was constructed to confirm that the reduced survival capability of the mutant was directly related to the deleted gene pgm. B. melitensis M5-90Δpgm mutant was confirmed by PCR. Our findings show that M5-90Δpgm was defective for survival in BALB/c mice, and it was cleared faster than M5-90. The lack of splenomegaly in inoculated mice increased the safety of M5-90Δpgm. This indicated that M5-90Δpgm elicit a significantly reduced virulence and inflammatory response observed with the mutant. This is consistent with previous results and substantiates that pgm is involved in the virulence of Brucella. In conclusion, wild-type strain 16 M and the mutant’s parental strain M5-90 showed similar intracellular replication, whereas the pgm mutant failed to replicate in mouse models.

The ideal live Brucella vaccine must induce high protection. Therefore, we performed protection experiments in BALB/c mice. Immunization with M5-90Δpgm could provide slightly better protection than M5-90.

The cytokine and antibody responses were also evaluated following M5-90Δpgm immunization. We detected antigen-specific production of Th1 cytokines (IFN-γ and IL-2) in serum samples from M5-90Δpgm-immunized mice and sheep. Th1 immune responses characterized by the production of IFN-γ and IL-2 are associated with protective immunity against Brucella (Golding et al. 2001; Wu et al. 2009). IFN-γ and IL-2 are critical cytokines in Brucella immunity (Sathiyaseelan et al. 2006). Our results indicate that M5-90Δpgm induced higher levels of IFN-γ and IL-2 production than M5-90. Moreover, we also found that IFN-γ levels in M5-90Δpgm vaccinated sheep peaked 8 weeks post-vaccination. Humoral immunity results showed that M5-90Δpgm immunized mice could produce anti-Brucella IgG, and M5-90Δpgm conferred a level of IgG production similar to that due to M5-90. These results indicate that immunization with strain Δpgm elicits Th1 and humoral immunity responses.

T lymphocytes play a key role in immune response to Brucella. Brucella infected host macrophages and the induced immunity is mainly cellular immunity (Mosmann and Sad 1996). Studies demonstrate the important role of T lymphocytes in cellular immunity. Mature T lymphocytes can divide into CD4+ T cells and CD8+ T cells (Ko and Splitter 2003). Activated CD4+ T lymphocytes are differentiated Th cells that can enhance the killing of intracellular pathogens (Oliveira and Splitter 1995). Activated CD8+ T cells are differentiated CTL cells that can effectively kill infected cells and release intracellular bacteria (Murphy et al. 2001). IFN-γ and IL-2 are associated with protective immunity against Brucella by production of T lymphocytes. Therefore, we detected the CD4+ and CD8+ T lymphocyte, and cytokine IFN-γ and IL-2 levels. M5-90Δpgm induced slightly higher levels of IFN-γ and IL-2 than M5-90.

Current serological diagnostic tests such as RBPT, STAT and iELISA use smooth LPS antigens. The LPS of smooth Brucella is by far the strongest antigen in hosts (Wang et al. 2011). However, it is difficult to differentiate between the sera of vaccinated animals and infected animals using these serological tests. We found that serum from mice inoculated with M5-90 was positive by RBPT and STAT, but serum from mice inoculated with M5-90Δpgm was negative in these tests. These results suggest that the PGM protein may be used for the development of serological tests to differentiate the two strains, but still needs to be tested on small ruminant sera immunized with M5-90 and M5-90Δpgm and also experimentally or naturally infected ruminants with B. melitensis 16 M or other virulent strain to claim to be able to differentiate between infected and vaccinated animals.

In conclusion, our results indicate that B. melitensis M5-90Δpgm may be a suitable live vaccine against B. melitensis because it had low virulence in BALB/c mice and it provided a level of protection similar to that provided by the M5-90 vaccine strain against virulent strain 16 M challenge. The humoral responses indicated that M5-90Δpgm elicited an anti-Brucella-specific IgG response, which provided an ideal diagnostic PGM antigen for the differentiation of immunization from infection. Therefore, B. melitensis M5-90Δpgm should be considered a potential new vaccine candidate against brucellosis. In future studies, the mechanisms that contribute to the humoral immune response in mice will be evaluated, and further testing in animals will determine whether M5-90Δpgm is indeed a good live vaccine candidate.