A Salmonella typhimurium ghost vaccine induces cytokine expression in vitro and immune responses in vivo and protects rats against homologous and heterologous challenges

Salmonella enteritidis and Salmonella typhimurium are important food-borne bacterial pathogens, which are responsible for diarrhea and gastroenteritis in humans and animals. In this study, S. typhimurium bacterial ghost (STG) was generated based on minimum inhibitory concentration (MIC) of sodium hydroxide (NaOH). Experimental studies performed using in vitro and in vivo experimental model systems to characterize effects of STG as a vaccine candidate. When compared with murine macrophages (RAW 264.7) exposed to PBS buffer (98.1%), the macrophages exposed to formalin-killed inactivated cells (FKC), live wild-type bacterial cells and NaOH-induced STG at 1 × 108 CFU/mL showed 85.6%, 66.5% and 84.6% cell viability, respectively. It suggests that STG significantly reduces the cytotoxic effect of wild-type bacterial cells. Furthermore, STG is an excellent inducer for mRNAs of pro-inflammatory cytokine (TNF-α, IL-1β) and factor (iNOS), anti-inflammatory cytokine (IL-10) and dual activities (IL-6) in the stimulated macrophage cells. In vivo, STG vaccine induced humoral and cellular immune responses and protection against homologous and heterologous challenges in rats. Furthermore, the immunogenicity and protective efficacy of STG vaccine were compared with those of FKC and non-vaccinated PBS control groups. The vaccinated rats from STG group exhibited higher levels of serum IgG antibody responses, serum bactericidal antibodies, and CD4+ and CD8+ T-cell populations than those of the FKC and PBS control groups. Most importantly, after challenge with homologous and heterologous strains, the bacterial loads in the STG group were markedly lower than the FKC and PBS control groups. In conclusion, these findings suggest that the STG vaccine induces protective immunity against homologous and heterologous challenges.

Introduction Salmonella are Gram-negative foodborne zoonotic bacteria that cause salmonellosis worldwide [1,2]. S. typhimurium and S. enteritidis are mainly transmitted to humans by consumption of contaminated eggs and poultry meat products, and represent a global public health burden [3]. Salmonella species are composed of several serotypes that can cause infections in homologous vaccinated animals. Moreover, S. typhimurium strains are often resistant to multiple antibiotics, such as aminopenicillins, gentamicin, tetracycline, chloramphenicol, and sulfonamides [4][5][6][7]. Therefore, there is a need to develop Non-Typhoidal Salmonellae (NTS) vaccine that can induce both homologous and heterologous protective immunity against Salmonella strains. Meanwhile, several efforts with live attenuated vaccines have been made to induce cross-protection against NTS infections. Previous studies demonstrated that live attenuated Salmonella aroA [8][9][10], capable of reducing invasion and colonization of the gastrointestinal tract and protected animals from homologous challenge, but they lack the ability to induce heterologous protective immunity against a virulent challenge. Furthermore, the administration of attenuated aroA mutants from S. enteritidis [11] or S. typhimurium [12] has been shown to reduce fecal shedding against homologous challenge. However, vaccination with Salmonella adenylate cyclase (cya) and C-reactive protein (crp) mutant protected chickens from Salmonella serotypes challenges [13].
Bacterial ghosts (BG) are structurally intact empty bacterial cell envelopes prepared from various Gram-negative bacteria [14]. In BG preparation, bacterial cell lysis is induced by the controlled expression of the bacteriophage PhiX174 lysis gene E [15,16] or minimum inhibitory concentration (MIC) of sodium hydroxide (NaOH) [17][18][19][20][21]. Based on this lysis, protein E or MIC of NaOH produces transmembrane tunnel structures on the cell surface that result in empty non-living cell envelopes. However, there are some differences in two methods. BG produced by the former method maintains their original structure, preserves all the cell surface antigens and provides efficient protection against specific infections [15][16][17]. Especially, functional and antigenic determinants of the envelope components are not denatured during lysis. However, there are some disadvantages of the method such as limitation to Gram-negative bacteria only, potential risks because of difficulty to reach 100% lysis rate of BG strain in a short time [22], and cost expensive and time consuming, multi-step process. Alternatively, BG produced by the latter method has no limitation to both Gram-positive [18] and Gram-negative bacteria [17,19]. This method needs a short time to generate BG without any potential risks, cheap cost and time-saving, simple process. Despite some surface structures in BG may be modified or lost by NaOH [19], the NaOH-induced BG provides efficient protection against specific infections [17,18].
In the last two decades, BG is representing to be the attractive vaccine candidate, because it can elicit both humoral and cellular immune responses against specific infections in experimental animals [23,24]. In previous studies, an effective S. entritidis and S. gallinarum ghost vaccines were produced to protect animals from Salmonella infections [25,26]. In the present study, non-living S. typhimurium bacterial ghosts (STG) was successfully produced by using the chemically-induced method. The main purpose of this investigation was to assess in vitro cytotoxicity of STG and the influence of STG on mRNA induction of cytokine and nitric oxide synthase in murine macrophages and in vivo protection of STG against the homologous and heterologous challenge in a rat model. Moreover, immunogenicity and protective efficacy were compared between the STG vaccine and formalin-killed inactivated cells (FKC) vaccine. This is the first study on the cross-protective efficacy of the STG vaccine in rats.
industries and to have a job experience for a month with a partner company. Of course, graduate students should have performed R&D research to get salaries from the project. After graduation, some qualified graduate students can have a job in the company involved in the project. In this respect, the government requires principal investigators to include a commercial company in this project as a partner. The government and company support 90% and 10% of the fund, respectively. "The commercial funder provided support in the form of salaries for authors [HBN, SO, SJ, HJP], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the 'author contributions' section.

Competing interests:
The commercial funder provided support in the form of salaries for authors [HBN, SO, SJ, HJP], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Two of the authors (J-SY, H-OP) are paid employees with Bioneer Corporation. This does not alter the authors' adherence to the PLOS ONE policies on sharing data and materials.

Experimental procedures
Bacterial strain and culture condition S. typhimurium KCCM40253 was kindly provided by Prof. Ki-Sung Lee, Department of Biology and Medicinal Science, Pai Chai University, Daejeon, Korea. S. enteritidis was provided from an animal health product manufacturing company (KBNP, Inc., Korea). The bacteria were grown at 37˚C in Luria-Bertani (LB) broth or on LB agar. Incubation temperature for growth and lysis was 37˚C in a shaking incubator at 200 rpm.
Production of S. typhimurium ghosts STG were produced by using the MIC of NaOH as described previously [17][18][19]. Briefly, the MIC of NaOH against S. typhimurium was determined by the two-fold broth dilution method and 72 h cultured S. typhimurium cells were harvested by centrifugation for 10 min at 10,000 × g. After washing three times with PBS (pH 7.2), the bacterial suspension was adjusted to 1 × 10 8 CFU/mL. One mL of the MIC of NaOH was added to 2 mL of the bacterial suspension and incubated at 37˚C for 1 h. After incubation, STG were collected by centrifugation and washed three times with PBS. Samples were prepared and analyzed by scanning electron microscopy (SEM) as described previously [17,18]. For FKC vaccine preparation, 0.5% formalin was added to the bacterial suspension and incubated for 24 h at 37˚C. After incubation, the bacterial suspension was washed three times with PBS, and the bacterial suspension was plated on LB agar plates to confirm the total loss of viability. FKC was stored at 4˚C until use.

Assessment of macrophage-mediated cytotoxicity
Murine macrophage (KCLB:40071, RAW 264.7) cells were purchased from Korean Cell Line Bank (Seoul, Korea) and cultured in 96-well plates (BD Falcon; BD Bioscience Discovery Labware, Bedford, MA, USA) for 24 h at 37˚C, in humidified 5% CO 2 , 95% air. The cells (1.0 × 10 4 cells/well) were then treated with 1.0 × 10 8 CFU/mL of the FKC, wild-type bacterial cells and NaOH-induced STG, respectively, in culture medium, and incubated for a further 24 h. PBS treated-and LPS-treated macrophages were used as negative and positive controls, respectively. LPS was extracted from S. typhimurium (1 × 10 8 CFU/mL) using an LPS extraction kit (iNtRON Biotechnology Inc., Seongnam-si, Gyeonggi-do, Korea) according to the manufacturer's protocol. Briefly, 5 mL culture was centrifuged at 13,000 rpm for 5 min. The bacterial pellet was treated with 1 mL of supplied lysis buffer and vortexed vigorously. After adding 200 μL of chloroform, the mixture was centrifuged at 13,000 rpm for 10 min at 4˚C. Then, the supernatant (400 μL) was mixed well with a supplied purification buffer and incubated for 10 min at −20˚C. After centrifuging the mixture solution at 13,000 rpm for 15 min at 4˚C, the upper layer was removed to obtain the LPS pellet. The pellet was washed with 70% ethanol and centrifuged at 13,000 rpm for 3 min at 4˚C. After discarding the resulting upper layer, the pellet was dried at room temperature and dissolved in 10 mM Tris-HCl (pH 8.0) by boiling for 2 min. To get the pure LPS, proteinase K (2.5 μg/ LPS 1 μg) as added to the dissolved LPS and the mixture was incubated at 4˚C for 30 min. The cell density was then assessed by using Cell Counting Kit-8 (CCK-8, Sigma-Aldrich, St. Louis, MO, USA). Absorbance was measured at 450 nm and all experiments were performed in triplicate. Cytotoxic activity is expressed as the percentage of cell viability by the following formula: % Cytotoxicity = (1 − A 450nm of target cells/A 450nm of control cells) × 100.
Quantitative analysis of cytokine mRNA by reverse transcription (RT)-qPCR RAW 264.7 cells (1.0 × 10 4 cells/well) were cultured in 24-well flat-bottom plates and treated with the FKC, wild-type bacterial cells and NaOH-induced STG, respectively, at a concentration of 1.0 × 10 8 CFU/mL. After 1-12 h stimulation, total RNA was isolated using RNAiso (Takara Bio, Shiga, Japan), according to the manufacturer's instructions. Tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-10 and inducible nitric oxide synthase (iNOS) mRNA levels were quantified by RT-qPCR amplification. Sequences for the primers of target genes are listed in S1 Table. RT reaction was performed in a 20 μL reaction mixture containing 300 ng of total RNA, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 8 mM MgCl 2 , 10 mM DTT, 0.1% NP-40, 40 mM dNTP, 2 pM of respective primer set, 20 U of RNase inhibitor (Takara Bio), and 200 U PrimeScript Reverse Transcriptase (Takara Bio). The thermal cycler was programmed for 1 RT cycle at 50˚C for 30 min and 70˚C for 15 min. cDNA was amplified in a 20 μL reaction mixture containing 10 μL 2X SYBR 1 Premix Ex Taq™ II (Tli RNaseH Plus, Takara Bio, Shiga, Japan), 0.2 μL ROX reference dye II, 0.4 μL of 10 μM of both forward and reverse primer and 1 ng of cDNA, using Stratagene Mx3005P cycler (1 cycle at 95˚C for 30 s, 30 cycles of denaturation at 95˚C for 5 s, and primer annealing and extension at 60˚C for 34 s). Each gene was amplified in triplicate and cDNA concentration differences were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Experimental animals
Ten-week-old male Sprague-Dawley rats were housed under standard conditions of temperature and relative humidity with a 12 h light/dark cycle. Food and water were available ad libitum. All animal experiments were approved by institutional animal care and use committee at Pai Chai University.

Detection of antibody levels by ELISA
Serum Immunoglobulin (Ig) G antibodies were determined by indirect enzyme-linked immunosorbent assay (ELISA) as previously described [18,19]. Microtiter plates were coated with 100 μL of S. typhpimurium or S. enteritidis (1 × 10 8 cells/mL) in coating buffer (pH 9.6). ELISA was carried out with sera from non-vaccinated and vaccinated rats. Goat anti-rat IgG conjugated alkaline phosphatase (1:30,000; Sigma-Aldrich) was used as the secondary antibody. The plates were developed with 100 μL of the substrate p-nitrophenyl phosphate (Sigma-Aldrich, St. Louis, MO) and 100 μL of 3N NaOH was added to stop the reaction. The optical density was measured at 405 nm with a microplate reader (Bio-Rad, USA).

Analysis of T-cells populations by FACS
To analyze T cell populations, blood samples were collected from non-vaccinated and vaccinated rats at one week after the final vaccination. The peripheral blood mononuclear cells were prepared using Histopaque-1077 (Sigma-Aldrich, St. Louis, MO) according to the manufacture's protocol. The cells (1.0 × 10 6 ) were washed three times in PBS and incubated either fluorescein isothiocyanate (FITC) labeled anti-CD4 or CD8 monoclonal antibodies at 4˚C for 30 min in the dark. After the incubation, all the samples were washed twice with PBS, and analyzed on a BD FACSCanto II flow cytometer (BD Biosciences, NJ, USA). Data analysis was done on BD FACSDiva software (BD Biosciences).

Determination of serum bactericidal activity
The bactericidal activity of the sera was tested as described previously [30]. Twenty-five μL of serum were mixed with 100 μL of S. typhimurium (1 × 10 6 CFU/mL) or S. enteritidis (1 × 10 6 CFU/mL) and incubated for 1 h at room temperature. After incubation, samples were plated on selective media and incubated for 24-48 h at 37˚C. Instead of serum, PBS was served as a control. The percentage of serum bactericidal activity was determined by using the following formula: SBA = {1 -(the number of viable bacteria after serum treatment/the number of viable bacteria after PBS treatment)} × 100%.

Bacterial clearance analysis
To evaluate the STG vaccine-induced protective immunity against homologous and heterologous challenges, the rats were sacrificed on 2 weeks post-challenge. The liver, lung, spleen, and kidney of each was harvested aseptically and homogenized in PBS using a tissue homogenizer (Sun MI Technology, Korea). Serial dilution of homogenates was then plated on selective media and incubated at 37˚C for 24-48 h. After incubation, CFUs were determined.

Statistical analysis
All statistical analyses were performed with one-way ANOVA using SPSS software (version 14.0) and data were expressed as mean ± standard error of the mean. Differences were considered statistically significant at P values of < 0.001, < 0.01 and < 0.05.

Production and characterization of S. typhimurium ghosts
In order to produce STG, we first determined the MIC of NaOH (3.13 mg/mL) against S. typhimurium by using the two-fold broth dilution method. STG was successfully produced by using the specific concentration of NaOH. At the end of lysis process, no viable colonies were detected in the MIC of NaOH-induced STG. This indicates that the efficiency of chemically-induced STG was 100%. Formation of the transmembrane tunnels in STG was examined by SEM (Fig 1A and 1B). Electron microscopic analysis revealed the presence of transmembrane lysis tunnel structures in STG (Fig 1B) as compared to the unlysed wild-type S. typhimurium cells (Fig 1A). Although the cellular morphology doesn't seem to be affected by the MIC of NaOH, some of the cell surface structures seem to be slightly affected.
Cytotoxicity tests and induction of cytokine mRNA expression in murine macrophages exposed to STG The cytotoxicity was compared using the viability of RAW 264.7 murine macrophages exposed to the FKC, wild-type bacterial cells and NaOH-induced STG, respectively (Fig 2). The given concentration of S. typhimurium LPS included in this analysis showed cell viability of 93.9%. The macrophages exposed to STG showed 84.9% cell viability (PBS: P < 0.01 and LPS: P < 0.05), which was similar to those exposed to FKC (85.6%) but significantly higher than those exposed to wild-type bacterial cells (66.5%) under the same concentration.
To determine the activation of macrophages exposed to STG to produce inflammatory cytokines, mRNA expression of pro-inflammatory cytokines and factor (TNF-α, IL-1β and iNOS), anti-inflammatory cytokine (IL-10) and dual properties of cytokine (IL-6) was investigated using RT-qPCR (Fig 2). In TNF-α mRNA, the highest mRNA level was found at 6 h in the macrophages exposed to wild-type bacterial cells (PBS and LPS: P < 0.001), which was much higher than those exposed to FKC and STG, respectively. In STG-exposed macrophages, the maximum accumulation of TNF-α mRNA was found at 2 h (PBS: P < 0.001 and LPS: P < 0.05), which was a similar quantity found in macrophages exposed to wild-type bacterial cells, and then declined in a time-dependent manner. Similarly, the induction of IL-1β mRNA was induced in a time-dependent manner in the macrophages exposed to STG. Its maximum level was found at 6 h, which was less than those exposed to FKC and wild-type bacterial cells. In iNOS, IL-6 and IL-10 mRNA, the respective maximum levels were found at 6 h in the macrophages exposed to wild-type bacterial cells (PBS and LPS: P < 0.001), which was slightly higher than those exposed to STG. Taken together, our data indicated that STG can activate macrophages to secrete both pro-inflammatory and anti-inflammatory cytokines.

Humoral immune response
To evaluate humoral immune responses of the STG vaccine against S. typhimurium and S. enteritidis antigens, the titers of total IgG in the sera of vaccinated and non-vaccinated animals

Fig 2. Cell viability and pro-and anti-inflammatory cytokine production of STG.
To determine cytotoxicity, murine macrophages (RAW 264.7) were exposed to PBS buffer, LPS purified from S. typhimurium, FKC, wild-type bacterial cells (ST) and NaOH-induced STG, respectively (bars). At 24 h postexposure, macrophages were collected for analysis of cell viability using Cell counting Kit-8. Absorbance was measured at 450 nm and all experiments were performed in triplicate. Cytotoxic activity is expressed as the percentage of cell viability by the formula described in Materials and Methods. At given time post-exposure with PBS buffer, LPS purified from S. typhimurium, FKC, ST and STG, respectively (bars), macrophages were collected for analysis of mRNA expression for pro-and anti-inflammatory cytokines and factor (TNF-α, IL-1β, iNOS, IL-6 and IL-10) using RT-qPCR. Data are representative of triplicate experiments with each sample run in triplicate. All statistical analyses were performed with one-way ANOVA using SPSS software (version 14.0) and data were expressed as mean ± standard error of the mean. *** = P < 0.001; ** = P < 0.01; * = P < 0.05 (significant difference from PBS) and ### = P < 0.001; ## = P < 0.01; # = P < 0.05 (significant difference from LPS).
https://doi.org/10.1371/journal.pone.0185488.g002 were determined by indirect ELISA (Fig 3). Rats from FKC and STG groups elicited higher serum IgG antibody responses as compared to the PBS control group (Fig 3A). However, there were significant differences in the levels of serum IgG antibodies between FKC and STG at weeks 2 and 6. At week 9 post-challenge, rats vaccinated with either STG or FKC showed significantly higher IgG antibody responses than PBS control rats (P < 0.01). Most importantly, serum IgG antibodies in STG group rats increased robustly as compared to PBS group (P < 0.01) and FKC group (P < 0.05) throughout the vaccination and S. enteritidis challenge period (Fig 3B).

Cellular immune response
The CD4 + and CD8 + T-cell populations in vaccinated and non-vaccinated animals were observed by FACS (Fig 4A and 4B). As shown in Fig 4C, CD4 + T-cell populations significantly increased in all vaccinated animals (FKC: P < 0.05 and STG: P < 0.01) than the non-vaccinated control PBS group, but no significant difference detected between FKC and STG. Similarly, animals from FKC (P < 0.05) and STG (P < 0.01) had statistically higher percentage of CD8 + T-cells compared to the control PBS group (Fig 4D). Meanwhile, the statistical difference between FKC and STG was not significant.

Determination of serum bactericidal activity
Serum bactericidal activity from rats vaccinated with the STG vaccine was tested against S. typhimurium (Fig 5A) and S. enteritidis (Fig 5B). As can be seen in Fig 5, the group vaccinated with the STG vaccine showed significantly higher levels of bactericidal antibodies compared to FKC and PBS control groups (P < 0.01). However, at weeks 4 and 6, bactericidal activity was not significantly different between STG and FKC against homologous strain. Interestingly, FKC vaccinated rats showed significant bactericidal antibodies against heterologous strain at week 6 (P < 0.05). Overall, the results suggest that STG vaccinated group increased bactericidal activities throughout the vaccination period (weeks 2, 4, and 6).

Homologous and heterologous protections of S. typhimurium ghost vaccine
To examine the ability of the STG vaccine against S. typhimurium challenge, rats were challenged orally with S. typhimurium two weeks after the final booster vaccination. The bacterial loads in the liver, lung, spleen, and kidney were significantly lower in FKC (P < 0.05) and STG (P < 0.01) than in PBS (Fig 7A-7D). The protection against S. typhimurium afforded by the STG vaccine was significantly greater than that afforded by the FKC vaccine (P < 0.05). In order to determine the cross-protective efficacy of the STG vaccine, rats were vaccinated with STG, FKC or PBS and challenged with a virulent S. enteritidis strain. The results of the bacterial loads in liver, lung, spleen, and kidney are shown in Fig 7E-7H. The bacterial count in liver, lung, spleen, and kidney were significantly lower in STG than those in PBS and FKC (P < 0.05). These results suggest that the vaccination with STG vaccine provided protection against S. enteritidis strain, but FKC vaccinated rats failed to protect against S. enteritidis strain.

Discussion
The development of safe and effective vaccines against different Salmonella serotypes is an urgent need for the control and prevention of Salmonella infections. It is known that macrophages and dendritic cells are the key cells to be infected by S. typhimurium, and these antigenpresenting cells transport the bacteria to the liver and spleen, the major organs of bacterial replication [33,34]. Both cells are critical components to the immune response to S. typhimurium infection, with macrophages controlling bacterial growth in the early phase of infection and dendritic cells initiating the T cell response [35][36][37].
In this study, the higher cell viability in the macrophages exposed STG than that of wildtype bacterial cells, suggesting that the MIC of NaOH reduces the cytotoxic effect of wild-type bacterial cells. Previously, alkaline treatment to a Gram-negative bacterial LPS resulted in reduced toxicity and deacylated LPS. The resultant LPS showed intact amide-linked fatty acids and removal of ester-linked fatty acids [38], but it was antigenically poor [38,39] or deficient [40]. However, our previous studies demonstrated that immunization with the NaOH-induced BG from a Gram-negative bacterium or a Gram-positive bacterium induces effective immune responses and provides a good protection against virulent challenge [17,18]. The present study also supports previous studies and suggests that the NaOH treatment does not affect the immunogenicity of BG or its potential as a vaccine candidate. Furthermore, our previous results showed that LPS of BG is modified or party lost by NaOH treatment and suggested that immunogenicity of the NaOH-induced BG could be derived from other cell envelope components rather than LPS [19].
Furthermore, we demonstrate that the NaOH-induced STG vaccine has the ability to induce both humoral and cellular immune responses and protects rats from S. typhimurium and S. enteritidis challenges. The cellular morphology and cell surface structures were unaffected by the MIC of NaOH during lysis process. This result indicates that the MIC of NaOH successfully induced non-living STG vaccine. Previously, we produced the chemically induced BG from a Gram-positive bacterium Staphlococcus aureus and a Gram-negative bacteria S. enteritidis and Vibrio parahaemolyticus with 100% lysis efficiency [17][18][19][20].
In our previous study with chemically-induced S. enteritidis ghosts (SEG), the SEG vaccine has shown the usefulness of detecting serum IgG antibody response after challenge and provided the protective immunity that might be mediated by induction of humoral immune responses [17]. Peng et al [46] also demonstrated that humoral immune responses play an important role in preventing Salmonella infections. In this regard, serum IgG antibodies in STG vaccinated rats after challenge with S. typhimurium and S. enteritidis strains were evaluated. Compared with the levels of antibody response in PBS and FKC control groups, STG vaccinated animals had higher serum IgG antibody response throughout the vaccination as well as the challenge period. Similar results were shown in S. enteritidis challenge studies, suggesting STG vaccine has the ability to induce protective immune responses against different Salmonella serotypes. In contrast, the FKC vaccine induced significant protective immunity against the S. typhimurium strain, but failed to induce significant antibody responses and protective immunity against the S. enteritidis strain.
Western blot analysis supported the difference between immune response induced by the STG vaccine and FKC vaccine. Compared with the FKC vaccine, the STG vaccinated sera showed stronger protein bands against the S. typhimurium and S. enteritidis antigen. This could be due to the loss of major surface antigenic components in the FKC vaccine. Hofstra et al [47] described that antigenic cross-reactivity of the major outer membrane proteins (30-42 kDa) was a general phenomenon in Enterobacteriaceae. The lack of heterologous antibodies revealed in the western blot analysis could be a reason for the lesser protection against the S. enteritidis challenge. In both the S. typhimurium and S. enteritidis strains, the serum bactericidal antibodies were significantly induced in STG vaccinated animals as compared to FKC and PBS vaccinated animals. Particularly, serum bactericidal analysis revealed that rats vaccinated with FKC vaccine produced higher levels of antibodies to the S. typhimurium strain. These antibodies could not induce cross-protective antibodies to the S. enteritidis strain. The data from the present study indicate that vaccination with the STG vaccine induces a strong protective immunity against S. typhimurium and S. enteritidis challenges.
It has been reported that BG from pathogenic bacteria has the ability to induce strong humoral and cellular immune responses and protect experimental animals from virulent challenge [48,49]. CD4 + MHC II-and CD8 + MHC I-restricted T cells play an important role in destroying intracellular bacteria, such as Salmonella [50][51][52]. Our study showed that STG vaccinated rats significantly induced CD4 + and CD8 + T cell populations compared to FKC and PBS control groups. Jawale et al [25] demonstrated that BG vaccine-induced CD4 + and CD8 + T-cells were involved in protection against Salmonella infections. However, recent studies have suggested that B-cell and T-cell immune responses are equally important for protective immunity against S. typhimurium [53,54].
Recently, genetically inactivated BG vaccines decreased bacterial load in tissue homogenates and provided protection against Salmonella infections [25,46]. Similarly, immunization with the chemically-induced SEG vaccine reduced bacterial clearance in liver, lung, spleen, and kidney and protected rats from S. enteritidis challenge [17]. In a previous study with Salmonella live vaccines, aromatic amino acid-dependent, streptomycin dependent and galE mutants [55][56][57][58] effectively induced protective immunity against a homologous challenge. However, these vaccines have limited potential to induce protective immunity against heterologous challenge. Effective Salmonella vaccination in livestock needs the induction of homologous and heterologous protective immunity. Based on these results, vaccination with the STG vaccine strongly induced protective immune responses and protected experimental animals from S. typhimurium and S. enteritidis infections.

Conclusion
In conclusion, vaccination with the STG vaccine induced higher levels of humoral and cellmediated immunity than the FKC vaccine and exhibited a significant reduction in bacterial colonization against S. typhimurium and S. enteritidis strains. The potential of chemicallyinduced BG to heterologous protection is now being evaluated. Moreover, the results obtained from this study suggest that the envelope antigen expression could be an important component of inducing cross-protective immune responses to the S. enteritidis strain. Therefore, the STG vaccine could be a promising candidate for the development of inactivated vaccines against Salmonella infections.
Supporting information S1 Table. Primers sequences of the targeted genes in real-time PCR. (DOCX)