Optimization of a Staphylococcus aureus adhesion assay for equine corneocytes

Meticillin-resistant Staphylococcus aureus (MRSA) causes serious skin and soft-tissue infections of humans and animals. Multiple strains of MRSA have been characterized, and one in particular, designated as strain USA 500, causes infections predominantly of horses and the people who work with them. The purpose of this study was to optimize an assay which could subsequently be used to compare the relative avidity of different S. aureus strains for equine corneocytes. Corneocytes were collected from the perineal skin of 10 healthy horses onto adhesive discs. The discs were then incubated at 37°C with an S. aureus field strain at each of three concentrations [10(7), 10(8) and 10(9) colony forming units (CFU)/mL] and for each of three incubation periods (45, 90 and 180 min). After standardized rinsing and staining procedures, discs were examined at ×1,000 magnification and areas containing confluent corneocytes photographed. The percentage of surface area occupied by adherent bacteria was analysed using image processing and analysis software. Significant colour space image processing was required to distinguish bacteria from the ubiquitous melanin granules present within equine corneocytes. Objective and subjective methods were used to determine optimal conditions for specific adherence without introducing confounding factors. A bacterial concentration of 10(8) CFU/mL incubated with corneocytes for 45 min produced maximal bacterial adhesion with the least amount of interbacterial clumping. Future studies should utilize these conditions for optimal assay performance.     

Pulmonary thromboembolism associated with Blastomyces dermatitidis in a dog

An 8-year-old, male castrated golden retriever presented for cough and increased respiratory effort. Radiographs revealed an alveolar pattern in the right caudal lung lobe and an opacity at the carina suspected to be enlarged tracheobronchial lymph nodes. The disease progressed to involve the right middle lung lobe. Cytopathology of a fine-needle aspirate and bronchoalveolar lavage fluid were nondiagnostic. Surgical removal of the right caudal lung lobe and biopsy of the perihilar lymph nodes revealed pulmonary thromboembolism and reactive lymph nodes. The dog died several days postoperatively, and necropsy revealed diffuse pulmonary thromboembolism. Additionally, Blastomyces dermatitis organisms were identified in a pyogranulomatous mass surrounding the trachea near the carina. In an extensive literature search, no reports of pulmonary thromboembolism associated with blastomycosis were identified. It is suspected that the inflammation secondary to blastomycosis caused the thromboembolism.     

Optimization of lycopene extraction from tomato cell suspension culture by response surface methodology

Radioisotope-labeled lycopene is an important tool for biomedical research but currently is not commercially available. A tomato cell suspension culture system for the production of radioisotope-labeled lycopene was previously developed in our laboratory. In the current study, the goal was to optimize the lycopene extraction efficiency from tomato cell cultures for preparatory high-performance liquid chromatography (HPLC) separation. We employed response surface methodology (RSM), which combines fractional factorial design and a second-degree polynomial model. Tomato cells were homogenized with ethanol, saponified by KOH, and extracted with hexane, and the lycopene content was analyzed by HPLC-PDA. We varied five factors at five levels: ethanol volume (1.33-4 mL/g); homogenization period (0-40 s/g); saturated KOH solution volume (0-0.67 mL/g); hexane volume (1.67-3 mL/g); and vortex period (5-25 s/g). Ridge analysis by SAS suggested that the optimal extraction procedure to extract 1 g of tomato cells was at 1.56 mL of ethanol, 28 s homogenization, 0.29 mL of KOH, 2.49 mL of hexane, and 17.5 s vortex. These optimal conditions predicted by RSM were confirmed to enhance lycopene yield from standardized tomato cell cultures by more than 3-fold.     

Spatial Variability of Agricultural Soil Fertility Parameters in a Gleyic Podzol of Belgium

Precise information about the spatial variability of soil nutrients is essential in developing site-specific management, e.g., variable rate application of nutrients. In this study 5 fields (4 grassland, 1 arable land) on a Gleyic Podzol, each of about 1 ha, were sampled according to a 20 m×20 m grid. On these samples pH, carbon content and nutrient status (P, K, Ca, Mg, and Na) were measured. The variogram analysis of the different soil fertility parameters revealed quite different ranges for these parameters. Carbon content, pH and calcium have high ranges, between 110 and 170 m. On the other hand, ranges for potassium and sodium are much smaller, 60 m and 30 m respectively. For the elements potassium and sodium, high coefficients of variation are found, especially on the grassland fields. These differences in ranges of spatial relationship indicate that the sampling strategy for estimating variability of soil fertility, should be adopted to the different selected soil parameters and the soil use.     

The effects of dietary HUFA level on flame angelfish (Centropyge loriculus) spawning,egg quality and early larval characteristics

This study was conducted to determine the effects of dietary highly unsaturated fatty acids (HUFA) on flame angelfish (Centropyge loriculus) reproduction, and egg and larval quality. In the experiment, formulated diets containing 1.8, 2.9 or 3.6% n‐3 HUFA were fed to flame angelfish broodstock (n = 4) for 5 months. Mean fecundity (daily egg production), egg fertilization rates and embryo viability were used as indicators of egg quality. In addition, mean egg diameter, oil globule diameter, per cent hatch, larval size at hatch, per cent survival to yolk exhaustion and larval size at yolk exhaustion were recorded for each treatment. Flame angelfish fed the diet containing 3.6% n‐3 HUFA exhibited significantly increased fecundity, fertilization rates and embryo viability than fish that were fed the other two formulated diets. Egg diameter, egg oil globule diameter, larval size at hatch, larval survival to yolk exhaustion and larval size at yolk exhaustion from the 3.6% n‐3 HUFA broodstock treatment group were not significantly different from those derived from the Control broodstock treatment. These data revealed that flame angelfish egg quality could respond rapidly (within weeks) to maternal dietary changes. Results from this study further support that dietary HUFA composition can significantly affect broodstock reproductive performance as well as subsequent performance of eggs and larvae.     

Phosphorus Fractionation in Sediment Cores Collected In 2005 Before and After Onset of an Aphanizomenon flos-aquae Bloom in Upper Klamath Lake, OR, USA

We tested the hypothesis that there would be measurable losses of phosphorus (P) from surficial sediments of Upper Klamath Lake (UKL), Oregon, if sediments were a source of P during an algal bloom. We compared concentrations of total and forms of P at various depths in cores collected before and after the onset of a large Aphanizomenon flos-aquae bloom. Concentrations of inorganic P were determined in extraction solutions of MgCl₂ (1 M, pH 8), citrate-dithionite-bicarbonate, and 1 M HCl. Sediments below 2 cm were dominated by residual P which is defined as total P minus inorganic P. During the study period, data from the top 2-cm of sediment indicated (a) significant decrease in total P concentration, primarily associated with iron oxyhydroxides at one site, and (b) significant increase in total P concentration associated with residual P at a second site. Data from two other sites indicated no net changes in concentrations of total P.