A Study on Concentration, Identification, and Reduction of Airborne Microorganisms in the Military Working Dog Clinic.

BACKGROUND: The study was planned to show the status of indoor microorganisms and the status of the reduction device in the military dog clinic. METHODS: Airborne microbes were analyzed according to the number of daily patient canines. For identification of bacteria, sampled bacteria was identified using VITEK®2 and molecular method. The status of indoor microorganisms according to the operation of the ventilation system was analyzed. RESULTS: Airborne bacteria and fungi concentrations were 1000.6 ± 800.7 CFU/m3 and 324.7 ± 245.8 CFU/m3. In the analysis using automated identification system, based on fluorescence biochemical test, VITEK®2, mainly human pathogenic bacteria were identified. The three most frequently isolated genera were Kocuria (26.6%), Staphylococcus (24.48%), and Granulicatella (12.7%). The results analyzed by molecular method were detected in the order of Kocuria (22.6%), followed by Macrococcus (18.1%), Glutamicibacter (11.1%), and so on. When the ventilation system was operated appropriately, the airborne bacteria and fungi level were significantly decreased. CONCLUSION: Airborne bacteria in the clinic tend to increase with the number of canines. Human pathogenic bacteria were mainly detected in VITEK®2, and relatively various bacteria were detected in molecular analysis. A decrease in the level of bacteria and fungi was observed with proper operation of the ventilation system.

A point-of-care diagnostic system to influenza viruses using chip-based ultra-fast PCR.

In order to diagnose the infectious disease from clinical samples, the various protocols such as culturing microorganism, rapid diagnostic test using chromatographic method, ELISA, conventional PCR are developed. Since a novel strain of avian influenza can be cross-infected human as well as birds and livestock due to genetic reassortment, some strains of influenza such as H7N9 and H5N1 have emerged as a severe virus which can be threaten the health of poultry as well as human. Therefore, we explored the development of simultaneously and rapid diagnostic tool for seasonal influenza (A/H1N1, A/H3N2, B) and highly pathogenic avian influenza (A/H5N1, A/H7N9). We analyzed the unique nucleotide sequences of influenza types including three seasonal influenza, A/H7N9, and A/H5N1, and distinguished each type of influenza and diagnosed through One Step RT-PCR. In the results, Chip-based PCR technique can be diagnosed rapidly and directly from naked eye with EvaGreen the influenza also respiratory specimens within 23 min 15 s, including reverse transcription. The Chip-based PCR is a point-of-care system, and it is expected to reduce diagnosis time and to develop a diagnostic kit. Furthermore the Chip based PCR technique can be used for high risk pathogen in bioterror and/or biological warfare in the field.

Whole-genome sequence analysis of Zika virus, amplified from urine of traveler from the Philippines.

Zika virus (ZIKV) (genus Flavivirus, family Flaviviridae) is an emerging pathogen associated with microcephaly and Guillain-Barré syndrome. The rapid spread of ZIKV disease in over 60 countries and the large numbers of travel-associated cases have caused worldwide concern. Thus, intensified surveillance of cases among immigrants and tourists from ZIKV-endemic areas is important for disease control and prevention. In this study, using Next Generation Sequencing, we reported the first whole-genome sequence of ZIKV strain AFMC-U, amplified from the urine of a traveler returning to Korea from the Philippines. Phylogenetic analysis showed geographic-specific clustering. Our results underscore the importance of examining urine in the diagnosis of ZIKV infection.

Bacillus anthracis genomic DNA enhances lethal toxin-induced cytotoxicity through TNF-α production.

BACKGROUND: Bacillus anthracis is the etiological agent of anthrax. Lethal toxin (LT) produced by B. anthracis is a well-known key virulence factor for anthrax because of its strong cytotoxic activity. However, little is known about the role of B. anthracis genomic DNA (BAG) in anthrax pathogenesis. RESULTS: We examined the effect of BAG on TNF-α production and LT-mediated cytotoxicity during B. anthracis spore infection in mouse macrophage cell lines (RAW264.7 cells and J774A.1) and BALB/c mice. Infection of RAW264.7 cells with B. anthracis spores induced TNF-α expression in a multiplicity of infection (MOI)-dependent manner, and this enhancement was attenuated by the toll-like receptor (TLR) 9 inhibitor oligodeoxynucleotide (ODN)2088. BAG led to TNF-α expression in a dose- and time-dependent manner when applied to RAW264.7 cells. TNF-α expression induced by BAG was reduced by either pretreatment with TLR9 inhibitors (ODN2088 and chloroquine (CQ)) or transfection with TLR9 siRNA. Furthermore, BAG-induced TNF-α production in TLR9(+/+) macrophages was completely abrogated in TLR9(-/-) macrophages. BAG enhanced the phosphorylation of mitogen-activated protein kinases (MAPK), and BAG-induced TNF-α expression was attenuated by pretreatment with MAPK inhibitors. A reporter gene assay and confocal microscopy demonstrated that BAG increased NF-κB activation, which is responsible for TNF-α expression. Treatment with BAG alone showed no cytotoxic activity on the macrophage cell line J774A.1, whereas LT-mediated cytotoxicity was enhanced by treatment with BAG or TNF-α. Enhanced LT-induced lethality was also confirmed by BAG administration in mice. Furthermore, LT plus BAG-mediated lethality was significantly recovered by administration of Infliximab, an anti-TNF-α monoclonal antibody. CONCLUSIONS: Our results suggest that B. anthracis DNA may contribute to anthrax pathogenesis by enhancing LT activity via TLR9-mediated TNF-α production.

Monoclonal antibody against the poly-gamma-D-glutamic acid capsule of Bacillus anthracis protects mice from enhanced lethal toxin activity due to capsule and anthrax spore challenge.

BACKGROUND: The poly-gamma-D-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, protects bacilli from immune surveillance and allows its unimpeded growth in the host. Recently, the importance of the PGA in the pathogenesis of anthrax infection has been reported. The PGA capsule is associated with lethal toxin (LT) in the blood of experimentally infected animals and enhances the cytotoxicity of LT. METHODS: To investigate the role of anti-PGA Abs on progression of anthrax infection, two mouse anti-PGA mAbs with K(d) values of 0.8 microM and 2.6 microM respectively were produced and in silico three dimensional (3D) models of mAbs with their cognitive PGA antigen complex were analyzed. RESULTS: Anti-PGA mAbs specifically bound encapsulated B. anthracis H9401 and showed opsonophagocytosis activity against the bacteria with complement. The enhancement effect of PGA on LT-mediated cytotoxicity was confirmed ex vivo using mouse bone marrow-derived macrophages and was effectively inhibited by anti-PGA mAb. Passive immunization of mAb completely protected mice from PGA-enhanced LT toxicity and partially rescued mice from anthrax spore challenges. 3D structure models of these mAbs and PGA complex support specific interactions between CDR and cognitive PGA. These results indicate that mouse mAb against PGA capsule prevents the progress of anthrax disease not only by eliminating the vegetative form of encapsulated B. anthracis but also by inhibiting the enhanced cytotoxic activity of LT by PGA through specific binding with PGA capsule antigen. GENERAL SIGNIFICANCE: Our results suggest a potential role for PGA antibodies in preventing and treating anthrax infection.

The poly-γ-D-glutamic acid capsule of Bacillus anthracis enhances lethal toxin activity.

The poly-γ-D-glutamic acid (PGA) capsule is one of the major virulence factors of Bacillus anthracis, which causes a highly lethal infectious disease. The PGA capsule disguises B. anthracis from immune surveillance and allows its unimpeded growth in the host. The PGA capsule recently was reported to be associated with lethal toxin (LT) in the blood of experimentally infected animals (M. H. Cho, et al., Infect. Immun. 78:387-392, 2010). The effect of PGA, either alone or in combination with LT, on macrophages, which play an important role in the progression of anthrax disease, has not been thoroughly investigated. In this study, we investigated the effect of PGA on LT cytotoxicity using the mouse macrophage cell line J774A.1. PGA produced a concentration-dependent enhancement of the cytotoxicity of LT on J774A.1 cells through an enhancement in the binding and accumulation of protective antigen to its receptors. The increase of LT activity was confirmed using Western blot analysis, which showed that the combination of PGA and LT produced a greater degree of degradation of mitogen-activated protein kinase kinases and an increased level of the activation of the proform of caspase-1 to its processed form compared to the effects of LT alone. In addition, mice that received a tail vein injection of both PGA and LT had a significantly increased rate of death compared to that of mice injected with LT alone. PGA had no effect when added to cultures or administered to mice in the absence of LT. These results emphasize the importance of PGA in the pathogenesis of anthrax infection.

The Vibrio cholerae VarS/VarA two-component system controls the expression of virulence proteins through ToxT regulation.

Although the conditions for inducing virulence protein expression in vitro are different, both classical and El Tor biotypes of Vibrio cholerae have been reported to regulate the expression of virulence proteins such as cholera toxin (CT) and toxin-coregulated pili (Tcp) through the ToxR/S/T system. The transcription activator ToxR responds to environmental stimuli such as pH and temperature and activates the second transcriptional regulator ToxT, which upregulates expression of virulence proteins. In addition to the ToxR/S/T signalling system, V. cholerae has been proposed to utilize another two-component system VarS/VarA to modulate expression of virulence genes. Previous study has shown that VarA of the VarS/VarA system is involved in the regulation of virulence proteins in the classical V. cholerae O395 strain; however, no further analysis was performed concerning VarS. In this study, we constructed varS mutants derived from the classical O395 and El Tor C6706 strains and demonstrated that VarS is also involved in the expression of the virulence proteins CT and Tcp from the V. cholerae classical and El Tor strains. This expression is through regulation of ToxT expression in response to environmental changes due to different toxin-inducing conditions.

Regulation of hemagglutinin/protease expression by the VarS/VarA-CsrA/B/C/D system in Vibrio cholerae.

In this study, through the analysis of Vibrio cholerae 2740-80 mutant strains produced by the cholera toxin subunit B gene containing Mariner-based transposon, we found that disruption of the varS gene, a member of the recently reported sensory system VarS/VarA-CsrA/B/C/D, resulted in altered expression of hemagglutinin/protease A. To further investigate the connection between VarS and HapA, we generated an additional varS mutant, V. cholerae 2740-80-VS, and examined the effect of this mutation on expression of HapA and of genes in the VarS/VarA-CsrA/B/C/D system. 2740-80-VS showed decreased expression of varS, csrB/C, hapR, and hapA along with increased biofilm production. Interestingly, expression of the alternative sigma factor sigma(s), which is important for adaptation to environmental stress, was also decreased in this mutant. These results indicate that the VarS/VarA-CsrA/B/C/D system is involved in the control of HapA expression and biofilm production in V. cholerae 2740-80 through HapR regulation, and also that VarS/VarA controls expression of sigma(s) for HapA regulation.

Effects of a mutation in the Drosophila porin gene encoding mitochondrial voltage-dependent anion channel protein on phototransduction.

Mitochondrial porins, also know as VDACs (voltage-dependent anion channels), play an important role in regulating energy metabolism, apoptosis, and the transport of metabolites across the mitochondrial outer membrane. So far three distinct isoforms of VDAC (VDAC1-3) have been reported in vertebrates, but their functions remain unknown. The annotation database of the Drosophila melanogaster genome sequence has identified four genes (porin, CG17137, CG17139, and CG17140) encoding different isoforms of VDACs. We identified post-translational modifications of PORIN that are specific to D. melanogaster eyes. We also identified the P-element insertion in the porin gene, porin(G2294), that is homozygous viable whereas all the porin mutants previously reported are homozygous lethal at the pupal stage. The mutant does not show any defects in fly morphology, survival, and photoreceptor structure. The mutant, however, produces <10% of the normal level of wild-type (WT) porin transcripts and 16.5% of WT level of the PORIN protein. The P-element insertion affects only the expression of Class I transcript but not Class II transcript of the porin gene. Unlike in WT, the mutant displays an ERG (electroretinogram) that is not maintained during a prolonged light stimulus. The revertant obtained from remobilization of the P-element in the mutant produces the WT level of porin transcripts and PORIN protein, and shows a normal ERG response. Our data suggest that the PORIN protein is important in maintaining a photoreceptor response during prolonged stimulation.