Subunit vaccines are being developed as a potential therapy for preventing microbial pathogen infection. In this study, the immunogenicity of recombinant Brucella (B.) abortus Fe/Mn superoxide dismutase (rFe/Mn SOD) protein as a subunit vaccine against B. abortus was investigated in BALB/c mice model. Brucella Fe/Mn SOD gene was cloned into a pcold-TF DNA vector. The bacterial recombinant protein was expressed using the Escherichia coli DH5α strain with a size of 82.50 kDa. The western blotting assay showed that rFe/Mn SOD reacted with Brucella-positive serum, indicating the potential immunoreactivity of this recombinant protein. After the second and third vaccinations, the peripheral CD4+ T cell population was increased significantly in the rFe/Mn SOD-immunized mice group compared to the PBS control group. Moreover, immunization of this recombinant protein increased the CD4+ T cell population from the first vaccination to the third vaccination. Meanwhile, the CD8+ T cells were slightly enhanced after the second vaccination compared to the first vaccination and compared to control groups. Fourteen days after the bacterial infection, the splenomegaly and the number of bacteria in the spleen were evaluated. The result showed that both rFe/Mn SOD and positive control RB51 decreased the bacterial replication in the spleen and the splenomegaly compared to control groups. Altogether, these results suggested that rFe/Mn SOD could induce host immunity against B. abortus infection.
Chlorine dioxide (ClO2) has recently emerged as an ideal disinfectant and has shown a wide range of antimicrobial activities in various pathogenic microorganisms. In this study, the virucidal effect of ClO2 at low concentration (0.02 ppm) and higher concentration (0.06 – 0.09 ppm) against Adenovirus and Herpesvirus was evaluated based on the NF T 72-281 and ASTM 1053-11 standard methods at different exposure times. The virus suspension was dried onto the carrier and then exposed to gaseous ClO2 (gClO2) at 22 ± 2∘C. For Adenovirus, exposure at a low concentration of ClO2 at the middle height resulted in the average log10 reduction of 0.95, 2.65, and 5.30 after 1, 3, and 6 h post-exposure (pe), respectively. Moreover, more than 4-log10 reduction was achieved at 4 and 6 h pe with higher concentrations of ClO2. On the other hand, the antiviral activity of gClO2 at the middle height was also effective against Herpesvirus. In particular, at 1 h pe, a less than 4-log10 reduction was observed at all examined concentrations of ClO2, whereas exposure for 3 and 6 h (with low concentration) or 2 h (with higher concentration) inactivated completely viruses attached to the carrier. These results suggested that ClO2 fumigation is a potential alternative method for disinfecting healthcare facilities, high-containment laboratories, and households with a safe concentration for human health.
We investigated the effect of a synthetic complement peptide C3a on the outcome of Brucella abortus 544 infection in a murine macrophage cell line RAW264.7 cell. First, we determined the highest non-cytotoxic concentration of the peptide in the cell line. We also found that the peptide significantly increased the growth of the bacteria at 8 and 24 h. Although the number of bacterial CFU was also elevated at 48 and 72 h, the increases were not significant as compared to controls. We further investigated the effect of C3a peptide on the growth of Brucella by pre-incubating the peptide at various temperatures and found that the effect was reversed at 24 h post-incubation suggesting that incubation of peptide at high temperatures including 65°C or 95°C could inactivate its action. This also could indicate the beneficial effect of high temperature during infection. Although several studies reported the inhibitory effect of different antimicrobial peptides including C3a, the present study preliminarily revealed that it had no positive contribution on the control of B. abortus 544 infection in vitro and indirectly to its receptor, CD88, which belongs to GPCR. Moreover, the encouraged further exploration of the effect of other similar peptides would be performed for the purpose of finding Brucella-host cell interaction for the control of disease progression.
This study aims to investigate the effects of exogenous succinic acid (SCA) on Brucella (B.) abortus infection in macrophage RAW 264.7 cells and ICR mice. Firstly, the in vitro experiment was conducted by MTT cytotoxicity and bacterial internalization assay to evaluate the uptake of B. abortus into macrophage cells. Two non-cytotoxic concentrations of SCA demonstrated attenuated invasion of Brucella into macrophages at 30 and 45 min post- infection (pi). Secondly, ICR mice were treated with SCA and infected with B. abortus. On day-14 pi, spleen and blood serum were collected to evaluate the bacterial burden and total spleen weight as well as the production of cytokine/chemokine, respectively. The results showed that SCA treatment promoted bacterial growth and reduced the total spleen weight in mice. Furthermore, SCA treatment increased the level of IL-10 cytokine in the sera, while dampening the production of MCP-1 chemokine compared to the control. The results of bacterial load in spleen and spleen weight together with cytokine/chemokine production profile in the sera indicated that SCA induced the host anti-inflammatory response which is beneficial for the survival of Brucella. Therefore, these findings suggest that SCA contributed to host immunity against Brucella infection and the emerging potential topic-immunometabolism should be invested for further investigations.
We investigated the cytotoxic potential of three different commercially available absorbent feminine hygiene products and one transdermal patch using direct contact and extract exposure methods. Two different cell lines were used – mouse fibroblast L929 and normal human skin fibroblast CCD-986sk cell lines. The test samples were extracted using three different methods in accordance to International Organization for Standardization (ISO). Viability of cells was analyzed using MTT assay and morphology of the cells were also observed using phase contrast microscopy. Overall, the direct contact method using L929 cells showed that all the test samples had no toxic effect when exposed to extracts for 1 h. For the exposure method, no toxic effect was observed in both L929 and CCD986sk cells incubated with all the test samples regardless of the extraction methods used.
This study investigated the efficacy of four Brucella (B.) abortus recombinant proteins, namely adenylate kinase (Adk), nucleoside diphosphate kinase (Ndk), 50S ribosomal protein (L7/L12) and preprotein translocase subunit (SecB), as a combined subunit vaccine (CSV) against B. abortus infection in BALB/c mice. Immunoblotting assay showed that these four recombinant proteins as well as pcold-TF vector reacted individually with Brucella-positive serum, but not with Brucella-negative serum. The peripheral blood CD4+ T cell population was increased in CSV-immunized mice compared to PBS and pcold-TF vector groups. In addition, CSV and pcold-TF groups displayed induced IgG1 and IgG2a antibodies production compared to PBS and RB51 group, whereas IgG2a titer was higher than IgG1 titer in CSV group. The secretion profiles of IgG1 and IgG2a production together with an enhancement of CD4+ T cell population suggested that CSV did not only induce T helper 1 (Th1) T cell immunity but also humoral immunity. Therein, Th1 T cell immunity is more predominant in eliminating intracellular bacteria B. abortus. Furthermore, CSV immunization significantly reduced the bacterial burden in the spleen as well as the spleen weight in comparison to PBS and pcold-TF groups. Altogether, combination of these antigens could be potential to induce protective immunity against B. abortus infection in animals.
To attenuate and control the spread of infectious disease, a body of research has been conducted to generate safe vaccines and to continue national-level surveillance. However, understanding on viability and persistence of avian influenza virus (AIV) in infected carcasses, and effective disposal approaches are still limited up to date. Here, using HA test and RT-PCR, we assessed active status of AIV and degradation of viral RNA in collected specimens at different sites and time points. First, AIV infectivity was recovered until day 2, and viral nucleic acids persisted to day 14 and 21 in inorganic and organic samples, respectively, in sealed vials incubated at room temperature. Second, AIV was totally inactivated in all examined specimens, and viral RNA was not detectable at all time points tested at least one month post-infection in AIV-inoculated carcasses buried directly in soil or fiber reinforced plastic (FRP) bin. Lastly, among different burial sites in South Korea, 6 out of 17 sampling sites in Jeonbuk province showed the presence of viral genetic materials, while the rest of the field samples displayed neither the presence of infective AIV nor detectable viral RNA. This study showed a linear relation between time and degradation degree of viral RNA in buried samples suggesting that burial disposal method is effective for the control or at least attenuation of spread of AI infection in infected animals although consistent monitoring is required to verify safety of disposal.
BALB/c mice were vaccinated with Brucella (B.) abortus recombinant protein L27 (50S ribosomal protein L27) cloned into a pMal vector system. L27 was induced, purified and injected intraperitoneally (IP). Mice were vaccinated on 0-, 15- and 35-day. Serum cytokines were evaluated on 36- and 49-day from first vaccination. Mice were intraperitoneally infected with 5×104 CFU of virulent B. abortus 544 on day-50 and sacrificed after two weeks from infection. Bacterial burden from the spleen was quantified and showed a 0.7- and 0.9-log reduction in vaccinated mice in comparison to PBS and MBP (maltose binding protein) groups respectively. Cytokines in the serum demonstrated increased interferon-gamma (IFN-γ) and other pro-inflammatory cytokines such as macrophage chemoattractant protein-1 (MCP-1) and interleukin 6 (IL-6). On the other hand, interleukin 10 (IL-10) was attenuated in the sera of vaccinated mice. This cytokine profile is indicative of a cell-mediated type of immune response which is favorable for the eradication of intracellular infections. The current study showed the potential of another B. abortus ribosomal protein in inducing protective immunity against B. abortus infection.
In this study, we examined the protective immunity of a combination of seven Brucella abortus recombinant proteins; superoxide dismutase (rSodC), riboflavin synthase subunit beta (rRibH), 50S ribosomal protein (50s rL7/L12), nucleoside diphosphate kinase (rNdk), malate dehydrogenase (rMDH), arginase (rRocF), and elongation factor (rTsf) cloned in a pMal vector system and expressed in DH5α. Mice groups were immunized thrice with a combined subunit vaccine (CSV-7) at 0, 2, and 5 weeks and subsequently challenged with B. abortus at 5 × 104 CFU at 6 weeks. At four weeks post-infection, the mice were sacrificed and the bacterial burden in their spleens was quantified. Results revealed bacterial log reductions of 0.63 and 0.34 in comparison to PBS and maltose-binding protein (MBP), respectively. Cytokine profiling revealed a marked increase in IFN-γ (interferon-gamma), MCP-1 (macrophage chemoattractant protein-1) and IL-6 (interleukin 6) cytokines at 5-weeks post-immunization. On the other hand, only TNF was heightened at 7-weeks post-immunization. In general, this cytokine profile is consistently reflective of a Th1 immune response, which is beneficial for host immunoresistance.
This study evaluated the protective effects of a combination of eight B. abortus recombinant proteins that were cloned and expressed into a pMal vector system and DH5α: nucleoside diphosphate kinase (rNdk), 50S ribosomal protein (rL7/L12), malate dehydrogenase (rMDH), DNA starvation/stationary phase protection protein (rDps), elongation factor (rTsf), arginase (rRocF), superoxide dismutase (rSodC), and riboflavin synthase subunit beta (rRibH). The proteins were induced, purified, and administered intraperitoneally into BALB/c mice. The mice were immunized three times at weeks 0, 2, and 5 and then infected intraperitoneally (IP) with 5×104 CFU of virulent B. abortus 544 one week after the last immunization. The spleens were collected and the bacterial burden was evaluated at four weeks post-infection. The results showed that this combination produced a significant reduction of the bacterial burden in the spleen with a log reduction of 1.01 compared to the PBS group. Cytokine analysis revealed induction of the cell-mediated immune response in that TNF (tumor necrosis factor) and proinflammatory cytokines IL-6 (Interleukin 6) and MCP-1 (macrophage chemoattractant protein-1) were elevated significantly. In summary, vaccination with a combination of eight different proteins induced a significant protective effect indicative of a cell mediated immune response.
We investigated the effects of two Brucella proteins expressed in a pMAL expression system, RocF and EF-Ts, as subunit vaccines on immune modulation and protective efficacy using a mouse model. Mice vaccinated with MBP-RocF and MBP-EF-Ts displayed increased production of TNF, IFN-, MCP-1, IL-10 and IL-6, and TNF and MCP-1, respectively. Furthermore, mice vaccinated with MBP-EF-Ts showed decreased induction of IFN- and Th2-related cytokines, IL-10 and IL-6. Higher proportions of CD4+ and CD8+ T cells were observed in the blood of mice vaccinated with MBP-RocF than in the PBS-vaccinated group, although the increases were not significant. Furthermore, significantly reduced Brucella proliferation in the spleens of the MBP-RocF and MBP-EF-Ts groups were observed, but inflammation of these organs was not attenuated. Overall, these results indicate that RocF and EF-Ts could be potential subunit vaccine candidates against animal brucellosis.
This study evaluated the antimicrobial efficacy of different concentrations of ozonated water with organic matter, fetal bovine serum, at different concentrations and incubation times with bacteria. In the absence of organic matter, total eradication of up to 5 log of Escherichia (E.) coli was achieved, however, interference by organic matter led to inefficiency of ozonated water as a disinfecting agent. In addition, diminishing antimicrobial effects at higher temperatures, even in the absence of organic matter, were also demonstrated. These findings indicate that ozonated water will be a safe and effective disinfectant agent that could be useful in meat processing, especially an intestine processing, in Korean slaughter houses.
Treatment of dextran sodium sulfate(DSS) on HeLa cells results to an enhanced susceptibility to Brucella(B.) abortus infection. An increase in the adherence, invasion and intracellular replication of B. abortus was observed in DSS-treated cells. Furthermore, a marked elevation in the intensity of F-actin fluorescence was also observed in DSS-treated cells compared with untreated B. abortus-infected cells. An upregulation of phagocytic signaling proteins by Western blot analysis demonstrated an apparent activation of ERK, p38α and JNK phosphorylation levels in B. abortus-infected DSS-treated cells compared with the control. Colocalization with LAMP-1 proteins was attenuated in DSS-treated cells upon intracellular trafficking of the pathogen compared with control cells. The results of this study demonstrated consistency with other pathogens. The uptake and intracellular replication of B. abortus is hypothesized to be stimulated by various dextran receptors such as C-type lectins that are involved in phagocytosis which can either be direct phagocytic receptors, modulators of the expression of other receptors or as opsonins leading to an enhanced internalization of B. abortus. The complexity of these interactions thus would warrant further investigation into the role of DSS in the pathogenesis of brucellosis. In summary, we conclude that DSS enhanced adhesion, phagocytosis and intracellular replication of B. abortus in epithelial cells which could lead to suppression of the innate immune system in chronic Brucella infection.
The objective of this study was to determine the efficacy of ozone in sanitizing water experimentally inoculated with the gram-positive food-poisoning bacterium Staphylococcus aureus. The bactericidal effect was measured after experimentally inoculated solutions were exposed to 0, 0.5, and 1.0 ppm ozone at several time points and different temperatures, in the presence of varying concentrations of different organic matter, namely, fetal bovine serum (FBS) or cattle liver. Results revealed inhibition of the bactericidal effect in the presence of the lowest percentage of FBS, but a lower extent of the inhibition occurred when liver was used as the organic matter. It was also apparent that a higher temperature and shorter ozone exposure time had led to a more reduced bactericidal efficacy than that under a lower temperature and longer ozone exposure. This study provides insight into the potential use of ozonated water as an effective and safe disinfectant in an abattoir setting.
In Korea, a serious amphibian disease caused by the fungus Batrachochytrium dendrobatidis (Bd) has been reported from historical samples collected in the 1900s. In this study, we continue to evaluate the current prevalence of chytridiomycosis in the Korean Peninsula and we include imported frogs from America to our analysis. Non-invasive skin swabs were taken from 275 apparently healthy frogs, and Bd was detected in five free living frogs by the nested PCR protocol consisting of two species: Bombina orientalis and Rana catesbeiana, from Gyeongnam and Cheonbuk provinces. These frogs comprised about 2% of the total number of free living samples. This study might be useful for understanding amphibian chytridiomycosis in Korea.
Fast, cheap and sufficient serodiagnostic tools needs to be developed for the early detectionof brucellosis. Currently the tools cannot differentiate an active infection from vaccinated, norcan it differentiate other bacterial infections with lipopolysaccharides, especially Yersiniainfections. In this study, we purified recombinant outer membrane protein 10 and 28(rOmp10,rOmp28), and a dipstick assay(indirect or sandwich) was constructed with single(rOmp10 orrOmp28) and combined rOmps(rOmp10 and rOmp28) from Brucella(B.) abortus 544 to evaluatebovine Brucella positive serum collected during the beginning of the Korean outbreak from2006 to 2015. In application with single rOmp, rOmp10(70%; indirect, 92.11%; sandwichdipstick) and rOmp28(72.5%; indirect, 86.84%; sandwich dipstick) had comparable results. Inaddition, results indicated that dipstick with combined rOmps(rOmps10 and rOmp28) weresuperior in detecting positive serum samples, at 85% indirect and 100% sandwich dipstick. Surprisingly, the results were the same in detecting negative results at 97.78% for both singleand combined indirect dipsticks. The dipstick tools with rOmp10 and rOmp28 would be usefulfor a rapid screen method for bovine brucellosis.
The bacterial lipopolysaccharide (LPS) mainly contributes to the structural integrity, survival and protection barrier against harsh environments. Therefore, the early stages in LPS or lipid A biosynthesis are attractive targets in the identification and development of inhibitors which would be effective against infections caused by Gram-negative bacteria. The bacterial outer membrane proteins (OMPs) meanwhile function as maintenance for structure, adhesion to other cells and substances, as well as development of resistance to antimicrobials. The LPS and LPS-related molecules, and OMPs are important immunogenic components of several important pathogens including Brucella, which have been extensively used in immunological studies and in the diagnosis of diseases. Here we review the importance, structure, functions and immunogenic aspects of LPS and OMPs particularly of Brucella which can be targeted for the prevention and diagnosis of brucellosis.
To date, most serodiagnostic methods for brucellosis screening are based on antibodies against lipopolysaccharides of Brucella spp. However, this approach has the drawback of yielding false-positive results due to cross-reactivity with lipopolysaccharides of other related pathogens, especially Yersinia enterocolitica O:9. In this study, Brucella abortus AspC was cloned and expressed by PCR amplification into a pCold TF expression system to obtain recombinant AspC (rAspC). The immunogenicity of rAspC was confirmed by western blotting of Brucella-positive bovine serum. rAspC-based ELISA was performed to determine whether rAspC could be used in the serodiagnosis of bovine brucellosis. rAspC reacted strongly with anti-Brucella antibodies in positive sera in the tube agglutination test (TAT), but did not show strong reaction with most negative samples. In particular, the average OD492 value at the highest TAT titer showed a 1.4-fold increase with respect to the cutoff value. The accuracy, specificity, and sensitivity of rAspC were 71.88%, 78.33%, and 68%, respectively. These findings suggest that rAspC might be valuable for the serological diagnosis of bovine brucellosis.
Brucellosis is an important and re-emerging zoonotic disease worldwide. The prevention of human infection is achieved predominantly through the control of brucellosis in agricultural animals, which in turn depends on accurate diagnosis and vaccination. However, conventional serological diagnosis of brucellosis has several limitations, and currently available vaccines for animals have several drawbacks, including the ability to cause infection in humans. Phosphoglycerate kinase (Pgk) is one of the specific proteins reactive with mouse sera in the early stage of Brucella infection, and deletion of the pgk gene in B. abortus strain 2308 resulted in extreme attenuation of this strain in vitro and in vivo. Furthermore, the B. abortus pgk mutant has been used as a live vaccine, and in challenge experiments, it induced protection that was superior to that conferred by commercial strains. In this study, the pgk gene from Brucella abortus 544 was successfully amplified and cloned into a maltose binding protein fusion protein expression vector (pMAL). The recombinant protein was expressed in Escherichia coli DH5α and purified. The immunogenicity of purified recombinant B. abortus 544 Pgk (rPgk) was evaluated by western blot analysis using Brucella-positive mouse sera. rPgk could be used as an antigenic component for future serological tests and potential vaccine development.