Seong Bin Park

Development of an immunochromatography assay kit for rapid detection of ranavirus

Ranaviruses are large, double-stranded DNA viruses of the family Iridoviridae and are known to be primary pathogens in frogs, fish and other amphibians. These viruses have been shown to be highly adaptable and have the ability to cross species barriers, making them a potent threat to global biodiversity. There is therefore, a need for rapid and efficient diagnostic methods to control the spread of these viruses. To address this, monoclonal antibodies (MAbs) were developed against ranavirus strain FV-3 (standard frog virus 3) to detect the major capsid protein and FV-3gorf19R related hypothetical protein in both the FV-3 and KRV-1 (Korean ranavirus) strains. The antibodies were then applied on a colloidal gold-immunochromatographic assay (GICA) as a kit for the detection of ranaviruses. The kit was able to detect low concentrations of the virus (10(1)TCID50/ml) and showed analytical specificity when tested against other viral pathogens, including those belonging to the same family. It was possible to detect ranavirus in experimentally infected frogs within 30 min using the kit. The kit described here is expected to be a valuable and informative tool for on-site detection of ranavirus in frog.

Outer membrane vesicles from β-lactam-resistant Escherichia coli enable the survival of β-lactam-susceptible E . coli in the presence of β-lactam antibiotics

Outer membrane vesicles (OMVs) containing various bacterial compounds are released from mainly gram-negative bacteria. Secreted OMVs play important roles in the ability of a bacterium to defend itself, and thus contribute to the survival of bacteria in a community. In this study, we collected OMVs from β-lactam antibiotic-resistant Escherichia coli established by conjugation assay and the parental β-lactam antibiotic-susceptible strain, and performed comparative proteomic analysis to examine whether these OMVs carried β-lactam-resistant compounds. We also investigated whether both types of OMVs could protect susceptible cells from β-lactam-induced death and/or directly degrade β-lactam antibiotics. Several proteins that can be involved in degrading β-lactam antibiotics were more abundant in OMVs from β-lactam-resistant E. coli, and thus OMVs from β-lactam resistant E. coli could directly and dose-dependently degrade β-lactam antibiotics and fully rescue β-lactam-susceptible E. coli and other bacterial species from β-lactam antibiotic-induced growth inhibition. Taken together, present study demonstrate that OMVs from β-lactam-resistant E. coli play important roles in survival of antibiotic susceptible bacteria against β-lactam antibiotics. This finding may pave the way for new efforts to combat the current global spread of antibiotic resistances, which is considered to be a significant public health threat.

116 INCORPORATION AND DEVELOPMENTAL TOXICITY OF QUANTUM DOT NANOPARTICLES IN AMPHIBIAN LARVAE

The use of nanoparticles both commercially and pharmaceutically has increased over the past decade, including fluorescent quantum dot nanoparticles (QD) in biochemical research for in vivo imaging. Previous studies have reported the toxic effects of nanoparticles, but their effects on larval metamorphosis and animal development and growth have not been thoroughly examined. Additionally, the method of uptake of nanoparticles by larval systems is unknown. Amphibian larvae are an ideal model for assessing toxicity because of their sensitivity to environmental contaminants and rapid and easily observable developmental stages. We used Anaxyrus fowleri tadpoles to investigate QD (≤ 25nm diameter) integration into larvae and possible deleterious effects on their growth and development. Tadpoles (A. fowleri; n=5/group) were placed in 24-well plates containing 1mL of distilled water and increasing concentrations of QD (0, 1, and 2 nM) 72h post-hatch. The fluorescence emission of QD in wells was detected at various time points (1, 2, 24, 48, and 72h) using the in vivo imaging system (IVIS). A subset of tadpoles was killed (MS-222) and sectioned for histopathology. Remaining tadpoles were monitored throughout development. Fluorescence emission of QD in sectioned tadpoles was visualised using an EVOS Cell Imaging System. Developmental metrics of living tadpoles were recorded until metamorphosis. Fluorescence intensity between controls and dosage groups were analysed by ANOVA-1, followed by Students l.s.d. test to evaluate the effects of QD concentration and exposure time. The threshold of significance was P<0.05. The rate of incorporation of QD into tadpoles was determined using the equation y=C+Ao*2(-x/t1/2), where t1/2 is the half-life of QD remaining in solution. The IVIS imaging revealed a rapid decrease of QD fluorescence (total flux) signals from the aqueous tadpole environment. Decreases in fluorescence occurred within 1h post-exposure and appeared dose and time dependent, with signal nearly gone within 48h. Half-life of total flux (time necessary for tadpoles to absorb half of the QD in solution) is 20.75h (R2=0.92) and 2.54h (R2=0.96) for 1nm and 2nm QD in solution, respectively. The EVOS imaging revealed integration of QD and localization into tadpole tissues. Fluorescence was exclusively found within the mouth, gills, and sections of the intestinal lumen of exposed tadpoles within the first hour. Dose-dependent increases in fluorescence within tissue were observed at each time-point. No signal was observed in controls. In remaining live tadpoles, QD treated tadpoles were smaller in size [t(34)=2.35, P=0.024] than controls. Findings reveal that (1) A. fowleri tadpoles integrate and accumulate nanoparticles, without detectable excretion within 72h post-exposure, and (2) nanoparticles impede normal tadpole development. Ongoing studies are determining the effects of QD exposure on complete tadpole metamorphosis.

Proteomic analysis of antimicrobial effects of pegylated silver coated carbon nanotubes in Salmonella enterica serovar Typhimurium

BackgroundSynthesis of silver nano-compounds with enhanced antimicrobial effects is of great interest for the development of new antibacterial agents. Previous studies have reported the antibacterial properties of pegylated silver-coated carbon nanotubes (pSWCNT-Ag) showing less toxicity in human cell lines. However, the mechanism underlining the pSWCNT-Ag as a bactericidal agent remained unfolded. Here we assessed the pSWCNT-Ag effects against foodborne pathogenic bacteria growth and proteome profile changes.ResultsMeasurements of bioluminescent imaging, optical density, and bacteria colony forming units revealed dose-dependent and stronger bactericidal activity of pSWCNT-Ag than their non-pegylated counterparts (SWCNT-Ag). In ovo administration of pSWCNT-Ag or phosphate-buffered saline resulted in comparable chicken embryo development and growth. The proteomic analysis, using two-dimensional electrophoresis combined with matrix assisted laser desorption/ionization time of flight/time of flight mass spectrometry, was performed on control and surviving Salmonella enterica serovar Typhimurium to pSWCNT-Ag. A total of 15 proteins (ten up-regulated and five down-regulated) differentially expressed proteins were identified. Functional analyses showed significant reduction of proteins associated with biofilm formation, nutrient and energy metabolism, quorum sensing and maintenance of cell structure and cell motility in surviving S. Typhimurium. In contrast, proteins associated with oxygen stress, DNA protection, starvation, membrane rebuilding, and alternative nutrient formation were induced as the compensatory reaction.ConclusionsThis study provides further evidence of the antibacterial effects of pSWCNT-Ag nanocomposites and knowledge of their mechanism of action through various protein changes. The findings may lead to the development of more effective and safe antimicrobial agents.

Real-Time Bioluminescence Analysis of Escherichia coli O157:H7 Survival on Livestock Meats Stored Fresh, Cold, or Frozen

Foodborne bacteria such as Escherichia coli O157:H7 can cause severe hemorrhagic colitis in humans following consumption of contaminated meat products. Contamination with pathogenic bacteria is frequently found in the food production environment, and adequate household storage conditions of purchased foods are vital for illness avoidance. Real-time monitoring was used to evaluate bacterial growth in ground horse, beef, and pork meats maintained under various storage conditions. Various levels of E. coli O157:H7 carrying the luxCDABE operon, which allows the cells to emit bioluminescence, were used to inoculate meat samples that were then stored at room temperature for 0.5 day, at 4°C (cold) for 7 or 9 days, or -20°C (frozen) for 9 days. Real-time bioluminescence imaging (BLI) of bacterial growth was used to assess bacterial survival or load. Ground horse meat BLI signals and E. coli levels were dose and time dependent, increasing during room temperature and -20°C storage, but stayed at low levels during 4°C storage. No bacteria survived in the lower level inoculum groups (101 and 103 CFU/g). With an inoculum of 107 CFU/g, pork meats had higher BLI signals than did their beef counterparts, displaying decreased BLI signals during 7 days storage at 4°C. Both meat types had higher BLI signals in the fat area, which was confirmed with isolated fat tissues in the beef meat. Beef lean and fat tissues contrasted with both pork fat and lean tissues, which had significantly higher BLI signals and bacterial levels. BLI appears to be a useful research tool for real-time monitoring of bacterial growth and survival in various stored livestock meats. The dependence of E. coli O157:H7 growth on meat substrate (fat or lean) and storage conditions may be used as part of an effective antibacterial approach for the production of safe ground horse, beef, and pork meats.

Maternal nutrient restriction alters uterine artery hemodynamics and placentome vascular density in Bos indicus and Bos taurus

The objective was to examine uterine artery blood flow (UBF) as well as macroscopic and microscopic placentome vascular density in nutrient-restricted Angus and Brahman heifers. Angus (n = 6) and Brahman (n = 6) heifers were bred to a single sire and pregnancy confirmed at 30-d postbreeding. Heifers were randomly assigned to 1 of 2 dietary treatments consisting of 100% (control-fed; CON; n = 6) or 60% (total nutrient-restricted; RES; n = 6) based from net energy requirements for gestating heifers. Nutritional treatments were imposed from days 50 to 180 of gestation. On day 175 of gestation, UBF was collected ipsilateral and contralateral to the conceptus via Doppler ultrasonography. Heifers underwent Cesarean sections for collection of 2 adjacent placentomes on day 180 of gestation. The primary cotyledonary artery of 1 placentome was perfused with Alexa Fluor 647 Con A conjugate to examine macroscopic cotyledonary vascular density via an in vivo imaging system. The second placentome was fixed for microscopic immunofluorescence labeling of capillaries and separated into maternal (caruncle) and fetal (cotyledon) components for determination of angiogenic factor mRNA expression. Main effects of nutritional treatment and breed are reported in the absence of a significant nutritional treatment by breed interaction. Ipsilateral UBF was decreased (P < 0.05) by 48% in RES vs. CON, whereas breed did not influence ipsilateral UBF. Contralateral UBF was not different between nutritional treatments; however, contralateral UBF was decreased (P < 0.05) by 63% in Brahman vs. Angus cattle. Macroscopic cotyledonary vascular density was increased (P < 0.05) by 36% in RES vs. CON and 82% in Brahman vs. Angus heifers. Percent capillary area and capillary perimeter were increased (P < 0.05) in RES vs. CON and increased (P < 0.05) in Brahman vs. Angus heifers. Dietary treatments did not alter angiogenic factor expression; however, transcript abundance of caruncle and cotyledon ANGP1, FLT1, and KDR was increased (P < 0.05) in Brahman vs. Angus heifers. In summary, these data indicate compensatory responses in macroscopic and microscopic placentome blood vessel density during maternal nutrient restriction-induced reductions in UBF. Moreover, a greater macroscopic density of cotyledonary blood vessels was observed in Brahman vs. Angus heifers.

106 ASSESSMENT OF ANTI-BACTERIAL EFFECTS OF PEGYLATED SILVER-COATED CARBON NANOTUBES ON CAUSATIVE BACTERIA OF BOVINE INFERTILITY USING BIOLUMINESCENCE IMAGING SYSTEM

Pathogenic bacteria including Escherichia coli and Salmonella sp. are the major causative agents of endometritis and can cause infertility in livestock animals. Antibiotics are commonly used to terminate bacterial infections, but the development of bacterial antibiotic resistance is often encountered. Nanotechnology associated with silver nanoparticles has been highlighted as an alternative anti-bacterial agent, and pegylated silver-coated single-walled carbon nanotubes have high anti-bacterial effects and are non-toxic to human and murine cells in vitro. Here we verified whether a real-time bioluminescence monitoring system could be an alternative tool to assess anti-bacterial effects of nanotubes in a noninvasive approach. Escherichia coli and Salmonella sp. were transfected with plasmids containing constructs for luciferase enzyme (LuxCDABE) and substrate (luciferin) to create self-illuminating bioluminescent bacteria. Pathogens were grown in LB broth at 37°C, adjusted to 107 cfumL-1, and placed in 96-well plates for treatments. Pegylated (pSWCNTs-Ag) and non-pegylated (SWCNTs-Ag) nanotubes were prepared and added to culture wells at various concentrations (31.25-125µgmL-1). The control group corresponded to bacteria without nanotubes (0µgmL-1). Anti-bacterial effects of nanotubes were determined every 10min until 1h, then every 30min up to 6h incubation through optical density (600nm) measurements and bioluminescence imaging (BLI) and quantification using an IVIS system. Optical density and BLI data were compared at each time-point using 2-way ANOVA, with P<0.05 set for significance. Bioluminescence signals emitted by both bacteria stains appeared within 10min of incubation. Thereafter, control bacteria showed exponential growth that was detected as early as 25min post-incubation. Bioluminescence imaging revealed dose-dependent anti-bacterial activities of both pSWCNTs-Ag and SWCNTs-Ag on each E. coli and Salmonella sp. (P<0.05). Contrary to BLI, the OD values did not always reflect bacteria concentrations, and varied according to nanotube concentrations. No significant differences in anti-bacterial activities were revealed between pSWCNTs-Ag and SWCNTs-Ag based on OD values during 6h of incubation (P>0.05); meanwhile, pSWCNTs-Ag nanotubes exhibited stronger anti-bacterial effects than SWCNTs-Ag during the same period using BLI (P<0.05). In summary, we confirmed previous reports showing dose-dependent eliminations of pathogenic bacteria by silver nanotubes. Pegylated nanotubes exhibited high anti-bacterial activity compared to non-pegylated nanotubes. Bioluminescence imaging system revealed superior resolution to enable precise investigation of anti-bacterial kinetics of silver nanotubes. This feature could be useful for the study of bacterial infections that impair livestock fertility.