In Vitro Antibacterial Effect of the Combination of Galla rhois ethanol extracts and Sodium chlorate against Intramacrophage Brucella abortus

Chun-Nam Cha,Il-Hwa Hong,Eun-Ah Yu,Eun-Kee Park,Chang-Yeol Yoo,Suk Kim,Hu Jang Lee 한국식품위생안전성학회 2014 한국식품위생안전성학회지 Vol.29 No.3

본 연구는 오배자 에탄올 추출물 (GRE), 염소산나트륨 (SC) 그리고 오배자 에탄올 추출물과 염소산나트륨 합제(GS)의 B. abortus에 대한 항균효과를 확인하기 위해 수행 되었다. GRE, SC 그리고 GS를 B. abortus에 처리하여 배양한 후, B. abortus의 생존수를 확인하였으며, 마우스 탐식세포 내 감염된 B. abortus의 증식 억제효과를 경시별 (2, 24, 48시간)로 조사하였다. GRE, SC 그리고 GS는 각각 400 μg/mL 이하, 15 mM 그리고 0.6GS (GS 1, GRE 1,000 μg/mL + SC 30 mM) 이하의 농도에서 세포독성을 나타나지 않았다. 모든 처리구에서 B. abortus의 생존율은 용량- 의존적으로 현저하게 감소하는 결과를 나타내었다. 또한, GRE (400 μg/mL), SC (15 mM) 그리고 0.5GS (GRE 500 μg/mL + SC 15 mM)를 처리한 세포에서 배양 48시간 후에, B. abortus의 증식이 통계적으로 유의성 있게 감소하였으며 (GRE, p < 0.01; SC and 0.5GS, p < 0.001), 특히, GS를 처리한 경우, B. abortus의 세포내 증식이 GRE와 SC의 상승작용에 의한 강력한 항균효과를 나타내었다. 결론적으로, GS는 B. abortus에 대한 항균물질로서 유용할 뿐만 아니라, 식육과 우유 위생 분야에 적용할 수 있을 것으로 생각된다. This study investigated the antibacterial effects of GR ethanol extracts (GRE), sodium chlorate (SC) and a combination of GRE and SC (GS) on Brucella abortus (B. abortus). The antibacterial activities of GRE, SC and GS towards B. abortus were evaluated by incubating B. abortus with GRE, SC and GS. Following treatment with GRE, SC and GS, B. abortus survival and intracellular proliferation in macrophages were monitored. In the cellular cytotoxicity assay, GRE, SC and GS are not cytotoxic at concentrations less than 400 μg/ml, 15 mM and 0.6GS (1 of GS, GRE 1,000 μg/ml + SC 30 mM), respectively. The viability of B. abortus was markedly decreased in a dosedependent manner in all treatment groups. In addition, B. abortus intracellular proliferation within macrophages was significantly reduced in cells treated with GRE (400 μg/mL), SC (15 mM) and 0.5GS (GRE 500 μg/mL + SC 15 mM) after 48 hr-incubation (GRE, p < 0.01; SC and 0.5GS, p < 0.001). Especially, in the treatment of GS, the synergistic effect of GRE and SC treatment on B. abortus in macrophage was observed. In conclusion, GS is useful as an antibacterial candidate against B. abortus, and can be applied in the field of meat and milk hygiene.

In Vitro Antibacterial Effect of the Combination of Galla rhois ethanol extracts and Sodium chlorate against Intramacrophage Brucella abortus

Cha, Chun-Nam,Hong, Il-Hwa,Yu, Eun-Ah,Park, Eun-Kee,Yoo, Chang-Yeol,Kim, Suk,Lee, Hu Jang The Korean Society of Food Hygiene and Safety 2014 한국식품위생안전성학회지 Vol.29 No.1

본 연구는 오배자 에탄올 추출물 (GRE), 염소산나트륨 (SC) 그리고 오배자 에탄올 추출물과 염소산나트륨 합제 (GS)의 B. abortus에 대한 항균효과를 확인하기 위해 수행되었다. GRE, SC 그리고 GS를 B. abortus에 처리하여 배양한 후, B. abortus의 생존수를 확인하였으며, 마우스 탐식세포 내 감염된 B. abortus의 증식 억제효과를 경시별 (2, 24, 48시간)로 조사하였다. GRE, SC 그리고 GS는 각각 $400{\mu}g/mL$ 이하, 15 mM 그리고 0.6GS (GS 1, GRE $1,000{\mu}g/mL$ + SC 30 mM) 이하의 농도에서 세포독성을 나타나지 않았다. 모든 처리구에서 B. abortus의 생존율은 용량-의존적으로 현저하게 감소하는 결과를 나타내었다. 또한, GRE ($400{\mu}g/mL$), SC (15 mM) 그리고 0.5GS (GRE $500{\mu}g/mL$ + SC 15 mM)를 처리한 세포에서 배양 48시간 후에, B. abortus의 증식이 통계적으로 유의성 있게 감소하였으며 (GRE, p < 0.01; SC and 0.5GS, p < 0.001), 특히, GS를 처리한 경우, B. abortus의 세포내 증식이 GRE와 SC의 상승작용에 의한 강력한 항균효과를 나타내었다. 결론적으로, GS는 B. abortus에 대한 항균물질로서 유용할 뿐만 아니라, 식육과 우유 위생 분야에 적용할 수 있을 것으로 생각된다. This study investigated the antibacterial effects of GR ethanol extracts (GRE), sodium chlorate (SC) and a combination of GRE and SC (GS) on Brucella abortus (B. abortus). The antibacterial activities of GRE, SC and GS towards B. abortus were evaluated by incubating B. abortus with GRE, SC and GS. Following treatment with GRE, SC and GS, B. abortus survival and intracellular proliferation in macrophages were monitored. In the cellular cytotoxicity assay, GRE, SC and GS are not cytotoxic at concentrations less than $400{\mu}g/ml$, 15 mM and 0.6GS (1 of GS, GRE $1,000{\mu}g/ml$ + SC 30 mM), respectively. The viability of B. abortus was markedly decreased in a dose-dependent manner in all treatment groups. In addition, B. abortus intracellular proliferation within macrophages was significantly reduced in cells treated with GRE ($400{\mu}g/mL$), SC (15 mM) and 0.5GS (GRE $500{\mu}g/mL$ + SC 15 mM) after 48 hr-incubation (GRE, p < 0.01; SC and 0.5GS, p < 0.001). Especially, in the treatment of GS, the synergistic effect of GRE and SC treatment on B. abortus in macrophage was observed. In conclusion, GS is useful as an antibacterial candidate against B. abortus, and can be applied in the field of meat and milk hygiene.

Nucleomodulin BspJ as an effector promotes the colonization of Brucella abortus in the host

Zhongchen Ma,Shuifa Yu,Kejian Cheng,Yuhe Miao,Yimei Xu,Ruirui Hu,Wei Zheng,Jihai Yi,Huan Zhang,Ruirui Li,Zhiqiang Li,Yong Wang,Chuangfu Chen 대한수의학회 2022 Journal of Veterinary Science Vol.23 No.1

Background: Brucella infection induces brucellosis, a zoonotic disease. The intracellular circulation process and virulence of Brucella mainly depend on its type IV secretion system (T4SS) expressing secretory effectors. Secreted protein BspJ is a nucleomodulin of Brucella that invades the host cell nucleus. BspJ mediates host energy synthesis and apoptosis through interaction with proteins. However, the mechanism of BspJ as it affects the intracellular survival of Brucella remains to be clarified. Objectives: To verify the functions of nucleomodulin BspJ in Brucella's intracellular infection cycles. Methods: Constructed Brucella abortus BspJ gene deletion strain (B. abortus ΔBspJ) and complement strain (B. abortus pBspJ) and studied their roles in the proliferation of Brucella both in vivo and in vitro. Results: BspJ gene deletion reduced the survival and intracellular proliferation of Brucella at the replicating Brucella-containing vacuoles (rBCV) stage. Compared with the parent strain, the colonization ability of the bacteria in mice was significantly reduced, causing less inflammatory infiltration and pathological damage. We also found that the knockout of BspJ altered the secretion of cytokines (interleukin [IL]-6, IL-1β, IL-10, tumor necrosis factor-α, interferon-γ) in host cells and in mice to affect the intracellular survival of Brucella. Conclusions: BspJ is extremely important for the circulatory proliferation of Brucella in the host, and it may be involved in a previously unknown mechanism of Brucella's intracellular survival.

Simultaneous RNA-seq based transcriptional profiling of intracellular <i>Brucella abortus</i> and <i>B. abortus</i>-infected murine macrophages

Hop, Huynh Tan,Arayan, Lauren Togonon,Reyes, Alisha Wehdnesday Bernardo,Huy, Tran Xuan Ngoc,Min, WonGi,Lee, Hu Jang,Son, Jee Soo,Kim, Suk Elsevier 2017 Microbial pathogenesis Vol.113 No.-

<P><B>Abstract</B></P> <P> <I>Brucella</I> is a zoonotic pathogen that survives within macrophages; however the replicative mechanisms involved are not fully understood. We describe the isolation of sufficient <I>Brucella abortus</I> RNA from primary host cell environment using modified reported methods for RNA-seq analysis, and simultaneously characterize the transcriptional profiles of intracellular <I>B. abortus</I> and bone marrow-derived macrophages (BMM) from BALB/c mice at 24 h (replicative phase) post-infection. Our results revealed that 25.12% (801/3190) and 16.16% (515/3190) of the total <I>B. abortus</I> genes were up-regulated and down-regulated at >2-fold, respectively as compared to the free-living <I>B. abortus</I>. Among >5-fold differentially expressed genes, the up-regulated genes are mostly involved in DNA, RNA manipulations as well as protein biosynthesis and secretion while the down-regulated genes are mainly involved in energy production and metabolism. On the other hand, the host responses during <I>B. abortus</I> infection revealed that 14.01% (6071/43,346) of BMM genes were reproducibly transcribed at >5-fold during infection. Transcription of cytokines, chemokines and transcriptional factors, such as tumor necrosis factor (<I>Tnf</I>), interleukin-1α (<I>Il1α</I>), interleukin-1β (<I>Il1β</I>), interleukin-6 (<I>Il6</I>), interleukin-12 (<I>Il12</I>), chemokine C-X-C motif (<I>CXCL</I>) family, nuclear factor kappa B (<I>Nf-κb</I>), signal transducer and activator of transcription 1 (<I>Stat1</I>), that may contribute to host defense were markedly induced while transcription of various genes involved in cell proliferation and metabolism were suppressed upon <I>B. abortus</I> infection. In conclusion, these data suggest that <I>Brucella</I> modulates gene expression in hostile intracellular environment while simultaneously alters the host pathways that may lead to the pathogen's intracellular survival and infection.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The transcriptional profiles of <I>B. abortus</I> and BMM were analyzed by RNA-seq approach. </LI> <LI> The up-regulated <I>B. abortus</I> genes are involved in DNA, RNA manipulations and protein biosynthesis. </LI> <LI> The down-regulated <I>B. abortus</I> genes are involved in energy production and metabolism. </LI> <LI> The host defense genes are induced but cell proliferation and metabolism genes are suppressed in <I>B. abortus</I> infected BMM. </LI> </UL> </P>

Efficacy of Brucella abortus strain RB51 vaccine in Korean mongrel dogs against virulent strains of B. abortus biotype 1 and B. canis

( Jin Hur ),( Byeong Kirl Baek ) 한국가축위생학회 2010 韓國家畜衛生學會誌 Vol.33 No.1

This study was performed to test the hypothesis that Brucella abortus strain RB51 (SRB51) might protect Korean indigenous mongrel dog against challenge with either virulent B. abortus biotype 1 or B. canis. A total of 12 Korean mongrel dogs were divided into four groups (Group A, B, C and D). Dogs belonging to Group A and C were inoculated subcutaneously with 1×109 CFU of SRB51 in 1ml of sterile phosphate buffered saline (PBS). Dogs of Group B and D were inoculated subcutaneously with 1ml of sterile PBS as control. At 12 weeks post vaccination, dogs of Group A and B were challenged by oral inoculation of virulent strain of B. canis (5.0×109 CFU) and dogs of Group C and D were challenged by oral inoculation of virulent strain of B. abortus biotype 1 (4.4×1010 CFU). The serum antibodies titers in all dogs were monitored at regular interval for eight weeks after challenge (AC) by standard tube agglutination test, plate agglutination test, rose bengal test, 2-mercaptoethanol rapid slide agglutination test and 2-mercaptoethanol tube agglutination test. No antibody titers in Group A and C was detected. Also, the challenge strains were not found from blood of all dogs of Group A and C from 1 week AC till the end of the experiment by culture and modified AMOS-PCR, whereas B. canis and B. abortus challenge strains were detected from blood of Group B and D, respectively. In addition, neither of two challenge bacteria was recovered from liver, spleen, kidneys, lymph nodes and reproductive tracts of Group A and C dogs after postmortem. However, B. canis and B. abortus challenge strains were isolated from these tissues of Group B and D, respectively. These data suggest that SRB51 could be a promising vaccine candidate for immunizing dogs to control canine brucellosis caused by B. canis or B. abortus.

Immunogenic proteins of Brucella abortus to minimize cross reactions in brucellosis diagnosis

Ko, K.Y.,Kim, J.W.,Her, M.,Kang, S.I.,Jung, S.C.,Cho, D.H.,Kim, J.Y. Elsevier Scientific Pub. Co 2012 Veterinary microbiology Vol.156 No.3

To overcome the limitations of serological diagnosis, including false positive reactions caused by other pathogens, specific antigens for diagnosis of brucellosis other than LPS have been required. The present study was conducted to separate and identify immuno-dominant insoluble proteins of Brucella abortus against the antisera of cattle infected with B. abortus, or/and Yersinia enterocolitica, or the sera of non-infected cattle. After separating insoluble proteins of B. abortus by two dimensional electrophoresis (2-DE), their immuno-reactivity was determined by western blotting. A portion of the immunogenic spots against the positive antisera of B. abortus that have the potential for use as specific antigens were identified by MS/MS analysis. Overall, 18 immunogenic insoluble proteins of B. abortus 1119-3 showed immuno-reactivity against only the positive antisera of B. abortus, but failed to have immunogenicity toward both the positive sera of Y. enterocolitica and the negative sera of B. abortus. Identification of these proteins revealed the following: F0F1 ATP synthase subunit β, solute-binding family 5 protein, 28kDa OMP, Leu/Ile/Val-binding family protein, Histidinol dehyddrogenase, Hypothetical protein, Twin-arginine translocation pathway signal sequence domain-containing protein, Dihydroorotase, Serine protease family protein, β-hydroxyacyl-(acyl-carrier-protein) dehydratase FabA, Short-chain dehydrogenase-/reductase carbonic anhydrase, Orinithine carbamoyltransferase, Leucyl aminopeptidase, Cold shock DNA-binding domain-containing protein, Cu/Zn superoxide dismutase, and Methionine aminopeptidase. The 18 immunogenic proteins separated in the present study can be considered candidate antigens to minimize cross reaction in the diagnosis of brucellosis and useful sources for Brucella vaccine development.

DNA fingerprinting of Brucella abortus isolated from bovine brucellosis outbreaks by repetitive element sequence (rep)-PCR

Suh, Dong Kyun The Korean Society of Veterinary Science 2005 大韓獸醫學會誌 Vol.45 No.2

DNA fingerprint patterns of 8 Brucella reference strains and 15 B. abortus field isolates were characterized by repetitive element sequence-based PCR (rep-PCR) using BOX- and ERIC-primers in this study. AMOS PCR differentiated all Brucella field isolates from B. abortus RB51, a vaccine strain by producing a B. abortus-specific 498 bp band. Rep-PCR using BOX-primer produced 13 to 18 bands with sizes of between 230 and 3,300 bp, and discriminated Brucella strains to the species level except B. canis and B. suis. PCR products amplified with ERIC primers were, however, not appropriate for differentiating the Brucella isolates. DNA fingerprint patterns for all B. abortus field isolates were identical among them and were put on one cluster with B. abortus biovar 1 reference strain in the dendrogram, indicating they were highly clonal. These results suggested that rep-PCR using BOX primer might to be a useful tool for calculating genetic relatedness among the Brucella species and for the study of brucellosis epidemiology.

국내 소에서 분리한 Brucella abortus의 병원성 분석

임정주,김정화,김동혁,이진주,김대근,전무형,김상훈,장홍희,이후장,민원기,김석,Lim, Jeong Ju,Kim, Jeong-Hwa,Kim, Dong Hyeok,Lee, Jin Ju,Kim, Dae Geun,Jun, Moo-Hyung,Kim, Sang Hun,Chang, Hong Hee,Lee, Hu Jang,Min, Won-Gi,Kim, Suk 대한수의학회 2011 大韓獸醫學會誌 Vol.51 No.1

In this study, we isolated 12 of Brucella (B.) spp. from cattle, which have been positive in Rose Bangal test and tube agglutination test in Gyeongbuk province in 2009. According to AMOS PCR analysis, isolated 12 strains were identified as B. abortus. Murine derived macrophage, RAW 264.7 cells, were infected with isolated 12 strains or reference strain (B. abortus 544), and bacterial internalization were characterized. According to these results, we divided the isolated strains into the following three groups: class I, lower internalization than that of B. abortus 544; class II, similar internalization to that of that of B. abortus 544; class III, higher internalization than that of B. abortus 544 within RAW 264.7 cells. Furthermore, intracellular growth, bacterial adherent assay, LAMP-1 colocalization, virulence in mice and surface protein pattern were characterized. From these results, representative strains of class III showed lower LAMP-1 colocalization, higher adherent efficiency, higher virulence in mice than those of B. abortus 544, and showed different pattern of surface proteins. These results suggest that B. abortus field strains, isolated from cattle in Korea, possess various virulence properties and higher internalization ability of field strain may have an important role for its virulence expression.

Immunization of Mice with Recombinant Brucella abortus Organic Hydroperoxide Resistance (Ohr) Protein Protects Against a Virulent Brucella abortus 544 Infection

( Huynh Tan Hop ),( Alisha Wehdnesday Bernardo Reyes ),( Hannah Leah Tadeja Simborio ),( Lauren Togonon Arayan ),( Won Gi Min ),( Hu Jang Lee ),( Jin Ju Lee ),( Hong Hee Chang ),( Suk Kim ) 한국미생물 · 생명공학회 2016 Journal of microbiology and biotechnology Vol.26 No.1

In this study, the Brucella abortus ohr gene coding for an organic hydroperoxide resistance protein (Ohr) was cloned into a maltose fusion protein expression system (pMAL), inserted into Escherichia coli, and purified, and its immunogenicity was evaluated by western blot analysis using Brucella-positive mouse sera. The purified recombinant Ohr (rOhr) was treated with adjuvant and injected intraperitoneally into BALB/c mice. A protective immune response analysis revealed that rOhr induced a significant increase in both the IgG1 and IgG2a titers, and IgG2a reached a higher level than IgG1 after the second and third immunizations. Additionally, immunization with rOhr induced high production of IFN-γ as well as proinflammatory cytokines such as TNF, MCP-1, IL-12p70, and IL-6, but a lesser amount of IL-10, suggesting that rOhr predominantly elicited a cell-mediated immune response. In addition, immunization with rOhr caused a significantly higher degree of protection against a virulent B. abortus infection compared with a positive control group consisting of mice immunized with maltose-binding protein. These findings showed that B. abortus rOhr was able to induce both humoral and cell-mediated immunity in mice, which suggested that this recombinant protein could be a potential vaccine candidate for animal brucellosis.

Protective effects of recombinant Brucella abortus Omp28 against infection with a virulent strain of Brucella abortus 544 in mice

Jeong Ju Lim,김동혁,Jin Ju Lee,Dae Geun Kim,민원기,이후장,이만휘,김석 대한수의학회 2012 Journal of Veterinary Science Vol.13 No.3

The outer membrane proteins (OMPs) of Brucella (B.) abortus have been extensively studied, but their immunogenicity and protective ability against B. abortus infection are still unclear. In the present study, B. abortus Omp28, a group 3 antigen, was amplified by PCR and cloned into a maltose fusion protein expression system. Recombinant Omp28 (rOmp28) was expressed in Escherichia coli and was then purified. Immunogenicity of rOmp28 was confirmed by Western blot analysis with Brucella-positive mouse serum. Furthermore, humoral- or cell-mediated immune responses measured by the production of IgG1 or IgG2a in rOmp28- immunized mice and the ability of rOmp28 immunization to protect against B. abortus infection were evaluated in a mouse model. In the immunogenicity analysis, the mean titers of IgG1 and IgG2a produced by rOmp28-immunized mice were 20-fold higher than those of PBS-treated mice throughout the entire experimental period. Furthermore, spleen proliferation and bacterial burden in the spleen of rOmp28-immunized mice were approximately 1.5-fold lower than those of PBS-treated mice when challenged with virulent B. abortus. These findings suggest that rOmp28 from B. abortus is a good candidate for manufacturing an effective subunit vaccine against B. abortus infection in animals.