Lactiplantibacillus plantarum K8-based paraprobiotics prevents obesity and obesity-induced inflammatory responses in high fat diet-fed mice.

This study was conducted to investigate the effect of the paraprobiotics, lactic acid bacteria lysates (LAB-P) prepared from Lactiplantibacillus plantarum K8, on obesity and obesity-induced inflammatory responses in high-fat diet (HFD)-fed mice. LAB-P (100 mg/kg) significantly decreased the HFD-induced increase in weight by approximately 20% compared to that in the HFD control. This result was accompanied by a decrease in adipose weight/size. The white adipose tissue weight of epididymis, subcutaneous inguinal region, and mesentery were decreased by 36%, 20%, and 40%, respectively, in LAB-P (100 mg/kg)-administered mice. The size of the epididymal white adipose tissue-derived adipocytes was reduced by 41%. The LAB-P-mediated reduction in adipose tissues was associated with downregulation of adipogenic factors, such as peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα). In addition, LAB-P administration reduced total cholesterol and low-density lipoprotein levels by 23% and 42%, respectively, with a 55% reduction in lactate dehydrogenase levels. Stromal vascular fraction-derived adipose tissue macrophages were favorably regulated by LAB-P administration; the expression of CD11c, an inflammatory marker, was reduced by 30%, and that of CD206, an anti-inflammatory marker, was increased by 9-fold. These results were shown to correlated with the inhibition of proinflammatory cytokines (IL-1ß and IL-6) and downregulation of NF-κB expression. Furthermore, LAB-P administration suppressed HFD-induced fatty liver by activating AMPKα, an energy metabolic sensor. This study indicates that LAB-P effectively prevents HFD-induced obesity and obesity-induced inflammatory responses and serves a valuable basic work for utilizing LAB-P as functional food ingredient to preventing obesity and treating obesity-associated inflammatory diseases.

Curcuma phaeocaulis Inhibits NLRP3 Inflammasome in Macrophages and Ameliorates Nanoparticle-Induced Airway Inflammation in Mice.

The activation of NLRP3 results in the assembly of inflammasome that regulates caspase-1 activation and the subsequent secretion of bioactive interleukin (IL)-1ß. Excessive activation of the NLRP3 inflammasome is mechanistically linked to diverse pathophysiological conditions, including airway inflammation. Here, we discovered that Curcuma phaeocaulis can suppress caspase-1 activation and processing of pro-IL-1ß into mature cytokine in macrophages stimulated with NLRP3 inflammasome activators, such as SiO2 or TiO2 nanoparticles. Furthermore, in the bronchoalveolar lavage fluids of animals administered the nanoparticles, the in vitro effects of C. phaeocaulis translated into a decrease in IL-1ß levels and cell infiltration. Demethoxycurcumin (DMC) and curcumin were found to be responsible for the inflammasome inhibitory activity of C. phaeocaulis. Interestingly, in contrast to the previously reported higher antioxidant- and NFκB-inhibitory activities of curcumin, DMC exhibited approximately two-fold stronger potency than curcumin against nanoparticle induced activation of NLRP3 inflammasome. In the light of these results, both compounds seem to act independently of their antioxidant- and NFκB-inhibitory properties. Although how C. phaeocaulis inhibits nanoparticle-activated NLRP3 inflammasome remains to be elucidated, our results provide a basis for further research on C. phaeocaulis extract as an anti-inflammatory agent for the treatment of disorders associated with excessive activation of NLRP3 inflammasome.

Characterization of culture supernatant proteins from Brucella abortus and its protection effects against murine brucellosis.

In this study, we characterized the secreted proteins of Brucella abortus into the enriched media under the bacterial laboratory growth condition and investigated the pathogenic importance of culture supernatant (CS) proteins to B. abortus infection. CS proteins from stationary phase were concentrated and analyzed using 2D electrophoresis. In MALDI TOF/TOF analysis, more than 27 proteins including CuZn SOD, Dps, Tat, OMPs, Adh, LivF, Tuf, SucC, GroEL and DnaK were identified. Cytotoxic effects of CS proteins were found to increase in a dose-dependent manner in RAW 264.7 cells. Upon B. abortus challenge into phagocytes, however, CS proteins pre-treated cells exhibited lower bacterial uptake and intracellular replication compared to untreated cells. Immunization with CS proteins induced a strong humoral and cell mediated immune responses and exhibited significant higher degree of protection against virulence of B. abortus infection compared to mice immunized with Brucella broth protein (BBP). Taken together, these results indicate that B. abortus secreted a number of soluble immunogenic proteins under laboratory culture condition, which can promote antibody production resulted in enhancing host defense against to subsequently bacterial infection. Moreover, further analysis of CS proteins may help to understand the pathogenic mechanism of B. abortus infection and host-pathogen interaction.

The role of a Brucella abortus lipoprotein in intracellular replication and pathogenicity in experimentally infected mice.

Brucella abortus, the causative agent of brucellosis, can survive and replicate within host cells. Understanding bacterial virulence factors and bacteria-host cell interactions is critical for controlling brucellosis. However, little is known regarding the pathogenic mechanisms of brucellosis. A lipoprotein mutant (Gene Bank ID: 3339351) of B. abortus showed a lower rate of intracellular replication than did the wild-type strain in HeLa cells and RAW 264.7 macrophages. The adherent activity of the lipoprotein mutant was slightly increased compared to that of the wild-type strain in HeLa cells. After infection into macrophages, the lipoprotein mutant co-localized with either late endosomes or lysosomes. In mice infected with the lipoprotein mutant, fewer lipoprotein mutants were recovered from the spleen at 8 weeks post-infection compared to the wild-type strain. The ability to protect the lipoprotein mutant against infection by the virulent B. abortus strain 544 was similar to that of strain RB51. Our results indicate that the B. abortus lipoprotein is an important factor for survival within phagocytes and mice, and the B. abortus lipoprotein mutant may help improve live vaccines used to control brucellosis.

Redundant effects of ketamine on the pathogenesis and severity of Brucella abortus infection.

Brucella abortus is an intracellular bacterium and leading to a serious debilitating disease known as brucellosis. Ketamine is an anesthetic and a sedative that affects the immunomodulatory activities of various immune cells. The current study was to elucidate the role of ketamine in B. abortus infection, focusing on the phagocytic activity and immune response of macrophages. Following incubation of murine macrophages with ketamine, the phagocytosis of B. abortus was markedly reduced compared with the unincubated control. Interestingly, ketamine-incubated cells displayed a decreased intensity of F-actin fluorescence compared with the B. abortus-induced amplification of intensity. Conversely, the intracellular replication of B. abortus within macrophages was notably enhanced by ketamine. Furthermore, the in vivo assessment using a mouse model revealed that continual injections with ketamine led to augmented bacterial burdens in the spleen, which was accompanied by decreased levels of mRNA expression of cytokines in the spleen. The elevations of serum cytokines such as IFN-γ, IL-12 and IL-6, as well as the chemokine MCP-1, were also reduced by ketamine. These findings verify that ketamine suppresses the phagocytic activity and immune response during B. abortus infection. Therefore, the current study might provide novel insights into the potential influences of ketamine on infectious diseases caused by B. abortus, considering the host-pathogen interaction.

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

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.

Anticoccidial effect of supplemental dietary Galla Rhois against infection with Eimeria tenella in chickens.

The anticoccidial effects of Galla Rhois (GR) powder, which contains a major tannin-derived component of 52.7%, were evaluated in chickens following oral infection with Eimeria tenella. One-day-old chickens were assigned to five groups (control, unsupplemented, GR 0.5% supplemented [GRS 0.5%], GRS 1.0% [GRS 1.0%] and salinomycin supplemented [SS]). The chickens were fed a standard diet supplemented or not supplemented with GR or salinomycin for 10 days prior to infection. The birds received the supplemented diets continuously until 10 days post infection. The effects of GR on a E. tenella infection were evaluated by several parameters, including body weight gain, feed intake, oocyst excretion, bloody diarrhoea, and lesion scores. Infected chickens on the GRS and SS diets had a relatively moderate body weight loss (reduction ratio < 15%) and improved feed conversion. GRS and SS chickens produced significantly fewer faecal oocysts (P<0.05) and showed milder bloody diarrhoea compared with the E. tenella-infected control group. Furthermore, the lesion scores of both the GRS 0.5% and GRS 1.0% groups were significantly lower than the scores of the unsupplemented group on day 5 post infection. The lesion scores for the GR groups were similar to the scores for the SS group. In conclusion, this study suggests that GR appears to be as efficacious as salinomycin against E. tenella infection. GR supplementation leads to a reduction in infected chickens, although infected chickens are still affected compared with the uninfected control group. GR-based diets may be beneficial in preventing or treating coccidial infections in poultry.

Identification of genes contributing to the intracellular replication of Brucella abortus within HeLa and RAW 264.7 cells.

Brucella abortus, the causative agent of brucellosis, can survive and replicate within host cells. Understanding bacterial virulence factors and bacteria-host cell interactions is critical for controlling brucellosis, yet very little is known about the virulence strategies and signaling pathways activated in phagocytes during infection to ensure their growth and survival. B. abortus was mutagenized by mini-Tn5Km2 transposon mutagenesis to identify virulence genes related to the internalization and intracellular replication of the bacteria. Of the total 2300 mutants used to infect HeLa cells, 23 mutants defective for intercellular growth and the mutated genes were identified. Sequence analysis of DNA flanking the transposon showed various insertion sites in bacterial genes that might be associated with virulence, including genes associated with lipoproteins, amino acid metabolism, translation, transcription, carbohydrate transport, coenzyme transport, inorganic ion transport, energy metabolism, membrane transport, and cell wall/membrane biogenesis. Moreover, mutants were classified into class I, class II and class III as higher, similar, and lower internalization, respectively, into HeLa cells. Furthermore, defective mutants for intracellular growth in HeLa cells were found to be defective in RAW 264.7 cells. Taken together, we suggest that the identified virulence associated genes might contribute to the intracellular growth and survival of B. abortus in phagocytes.

Evaluation of recombinant 28 kDa outer membrane protein of Brucella abortus for the clinical diagnosis of bovine brucellosis in Korea.

Brucella spp. are Gram-negative, facultative, intracellular coccobacilli that are pathogenic to a variety of mammals, including ruminants and humans. The conventional serological test for diagnosing brucellosis in cattle in Korea is the standard tube agglutination test. However, agglutination tests sometimes give false-positive results due to cross-reactions with other pathogens. The outer membrane proteins of Brucella species have been extensively studied for their immunogenicity and serodiagnostic applications. However, an application of B. abortus OMPs for serodiagnosis has not been successfully established. In this study, cloning and expression of B. abortus Omp28, a group 3 antigen, were accomplished by PCR amplification cloning into a pMAL expression system, and purification of a recombinant Omp28 (rOmp28). The immunogenicity of rOmp28 was confirmed by Western blot analysis with Brucella-positive bovine serum. To determine whether rOmp2 has a potential benefit for use in the serodiagnosis of bovine brucellosis, rOmp28-based ELISA and latex bead agglutination test were performed. B. abortus positive (n=122) or negative (n=88) from TAT were positive (118/122, 96.7%) or negative (84/88, 95.4%) in ELISA and were positive (94/122, 77%) or negative (71/88, 81.7%) in that the latex bead agglutination test, respectively. The sensitivity, specificity and accuracy were 96.7, 95.4, 96.2% in ELISA and 77, 80.6, 78.5% in latex bead agglutination test, respectively. These findings suggest that the rOmp28 of B. abortus might be a good candidate for developing serological diagnostic tools for bovine brucellosis.

Prevalence of Lawsonia intracellularis, Salmonella spp. and Eimeria spp. in healthy and diarrheic pet rabbits.

A total of 170 fresh fecal samples (healthy; n=137, diarrheic; n=33) were collected from pet rabbits. By using PCR and formol-ether concentration method, a total 13/137 healthy rabbit feces were positive for L. intracellularis, 6/137 for Salmonella, and 13/137 for Eimeria. On the other hand, a total 17/33 diarrheic rabbit fecal samples were positive for L. intracellularis, 10/33 for Salmonella, and 21/33 for Eimeria. From these results, more than 20% of clinically normal and 97% of diarrheic rabbits were positive for single or concurrent infection of three pathogens. To the best of our knowledge, this is the first report to describe the prevalence of the microorganisms L. intracellularis, Salmonella and Eimeria in pet rabbits.

RGS2-mediated intracellular Ca2+ level plays a key role in the intracellular replication of Brucella abortus within phagocytes.

BACKGROUND: Brucella abortus can proliferate within professional and nonprofessional phagocytic host cells and thereby successfully bypass the bacteriocidal effects of phagocytes. However, the intracellular survival mechanism and factors of virulence are not fully understood. METHODS: We have investigated the role of the regulator of G protein signaling 2 (RGS2), an intracellular calcium ([Ca(2+)](i)) regulator of the host cell, in the intracellular survival of B. abortus within phagocytes. RESULTS: B. abortus infection markedly induced RGS2 messenger RNA expression in early phase and increased the [Ca(2+)](i) level up to 24 hours postinfection within macrophages from wild-type mice. The [Ca(2+)](i) level, however, was not influenced by B. abortus infection within macrophages from RGS2-deficient mice. Furthermore, B. abortus survival was reduced within RGS2-deficient macrophages, and hence bacterial proliferation was inhibited in RGS2-deficient mice. Moreover, treatment with the Ca(2+) chelator ethylenediaminetetraacetic acid (EDTA) or 1,2-bis-(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and the L-type Ca(2+) channel-blocking agent nifedipine or genistein also showed a reduced intracellular replication of B. abortus within macrophages. CONCLUSION: These results indicate that B. abortus infection induces host RGS2 expression and that up-regulation of [Ca(2+)](i) levels is an essential factor for the intracellular survival of B. abortus within phagocytes.

Intracellular replication inhibitory effects of Galla Rhois ethanol extract for Brucella abortus infection.

ETHNOPHARMACOLOGICAL RELEVANCE: Galla Rhois (GR) has long been applied in traditional Korean and Oriental medicine. Although GR has an anti-bacterial effect, the anti-bacterial mechanism and therapeutic efficiency of GR for intracellular parasitic Brucella infection are still unclear. AIM OF THE STUDY: The objective of this study was to investigate the antibacterial and therapeutic effects of GR ethanol extract (GRE), which is a natural antibacterial component for the treatment of Brucella abortus infection. MATERIALS AND METHODS: The antibacterial activity of GRE towards Brucella abortus was evaluated by incubating Brucella abortus with GRE. Following treatment with GRE, Brucella abortus adherence, uptake, intracellular growth, and intracellular trafficking in macrophages were monitored. Mice were infected intraperitoneally with Brucella abortus and treated orally with GRE for 14 days, and then the weight and CFUs from each spleen were monitored. RESULTS: The viability of Brucella abortus was markedly decreased in a dose-dependent manner. Moreover, Brucella abortus internalization and intracellular growth within macrophages were reduced in GRE-treated cells. The number of bacteria that adhered to GRE-pretreated cells was significantly lower than that of untreated cells. With regards to intracellular trafficking, treatment with GRE augmented the colocalization of Brucella abortus-containing phagosomes with LAMP-1. GRE-treated mice showed considerably decreased weight and bacterial burdens in the spleen compared to untreated mice. CONCLUSION: GRE exhibits antibacterial and protective effects on Brucella abortus in vitro and in vivo. These results highlight the beneficial effects of GRE in the prevention and treatment of brucellosis.

Characterization and identification of Erysipelothrix rhusiopathiae isolated from an unnatural host, a cat, with a clinical manifestation of depression.

Erysipelothrix rhusiopathiae is pathogenic for humans, many domestic animals and wild birds, but infectious cases with clinical symptoms in cats have not been reported. E. rhusiopathiae was recovered from a 4-month Russian blue breed cat with a very poor body condition score of 1 (BCS: 1/5). The isolate was typed as serotype 2b. Mice experimentally infected with the clinical isolate of E. rhusiopathiae through subcutaneous or intraperitoneal routes survived, and the organism was recovered from the spleen and synovial and pericardial fluids. Cats experimentally inoculated with the isolate either orally or subcutaneously survived but commonly exhibited depression and emaciation together with localized erythemal lesion of the skin accompanied by purulent ocular discharge. On hematological analysis, the number of total white blood cells was high compared with that in normal cats. Histological examination revealed congestion and moderate inflammation with focal necrosis. This observation may provide insight on E. rhusiopathiae infection in cats with the possible epidemiological significance and implications as a potential source of infection to other animals and humans.

Biological and antibacterial activities of the natural herb Houttuynia cordata water extract against the intracellular bacterial pathogen salmonella within the RAW 264.7 macrophage.

Salmonellosis is a major bacterial zoonosis that causes a variety of disease syndromes, from self-limiting enteritis to fatal infection in animals and food-borne infection and typhoid fever in humans. Recently, the emergence of multidrug-resistant strains of Salmonella sp. has caused more serious problems in public health. The present study investigated the antibacterial effects of Houttuynia cordata water extract (HCWE) against murine salmonellosis. In RAW 264.7 cells, there was no detectable cytotoxic effect of HCWE at any concentration between 25 and 100 microg/ml after 8-h incubation. The antibacterial activity of HCWE was then examined in a Salmonella enterica serovar (Salmonella typhimurium), and was found to increase in a dose-dependent manner at concentrations from 25 to 100 microg/ml during 8-h incubation. HCWE also affected RAW 264.7 cells including morphologic change and bacterial uptake, but there was no significant difference in bacterial replication in RAW 264.7 cells. With HCWE alone, nitric oxide (NO) production by RAW 264.7 cells did not increase, but when RAW 264.7 cells were infected by S. typhimurium, with or without HCWE, NO production with HCWE was 2-fold higher than that without HCWE. Treatment with HCWE did not affect inducible NO synthase (iNOS) mRNA expression by RAW 264.7 cells, but when RAW 264.7 cells with HCWE were infected by S. typhimurium, iNOS mRNA expression was increased during 8-h incubation. Furthermore, HCWE showed virulence reduction effects in S. typhimurium-infected BALB/c mice. After a lethal dose of S. typhimurium, the mortality rate in the HCWE untreated group was 100% at 7 d, but the HCWE 25, 50, and 100 microg/ml groups survived until 11, 17, and 23 d, respectively. These data suggest that HCWE is stable and beneficial in the treatment of bacterial infection including intracellularly replicating pathogens and may solve antimicrobial misuse and overuse.

Ceftiofur distribution in plasma and tissues following subcutaneously administration in ducks.

A study was performed to determine the residues in blood and edible tissues of healthy ducks (25 days old, mean body weight 1.0+/-0.13 kg) after subcutaneous administration of ceftiofur sodium at a dose rate of 2 mg/kg body weight (Group I) and 4 mg/kg body weight (Group II). Blood, muscle, liver, kidney, and fat samples were collected from all of ducks on the 1st, 2nd, 3rd, 4th, and 5th day after treatment of drug, and ceftiofur was analyzed with a high-performance liquid chromatography (HPLC) assay with results reported as ceftiofur-free acid equivalent (CFAE). To study the spiked recovery, blank plasma and tissues were spiked with two different concentrations of ceftiofur sodium (0.1, 0.5 microg/g). Average recovery values for all samples ranged from 70.3 to 87.3%. In the group I, desfuroylceftiofur acetamide (DCA) was not detected in all of plasma, muscle, liver, and fat tissues on the 1st day after treatment. But, kidney samples on the 1st day were detected DCA (0.059+/-0.01 microg CFAE/g tissue). On the 2nd day of post-treatment, the concentrations of DCA in all tissues were lower than the detection limit, 0.05 microg CFAE /g tissue. In the group II on the 1st day after treatment, the concentration of DCA was 0.124+/-0.06 microg CFAE/g tissue, 0.103+/-0.03 microg CFAE/g tissue, and 0.071+/-0.010 microg CFAE/g tissue in plasma, kidney, and muscle samples, respectively. On the 2nd day after treatment of ceftiofur, the concentrations of DCA in all tissues were lower than 0.05 microg CFAE/g tissue. According to our results, the concentrations of DCA on the 1st day after treatment with 2 mg/kg body weight were below 0.05 microg CFAE/g tissue equivalent in all tissues except for kidney. On the 2nd day after administration at the dose of 4 mg/kg body weight, no DCA was also detected in all of the tissues although DCA was detected in all samples on the 1st day.