The (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) colorimetric assay for the quantitation of Actinobacillus pleuropneumoniae cytotoxin.

Using swine neutrophils as target cells, two MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) colorimetric assay systems, one with and one without phorbol 12-myristate 13-acetate (PMA) stimulation were established for the quantitation of Actinobacillus pleuropneumoniae cytotoxin. The MTT assays were optimized for the number of neutrophils, incubation time, and PMA concentration by a series of experiments. The optimal conditions were 25 x 10(4) cells/well incubated for four hours for the assay system without PMA stimulation, and 12.5 x 10(4) cells/well incubated for two hours for the assay system with PMA stimulation. One culture supernatant of a toxigenic Pasteurella multocida strain and five A. pleuropneumoniae cytotoxin preparations produced from three A. pleuropneumoniae strains were used to test assay reproducibility. Results showed both assays were reproducible with a coefficient of variation ranging from 7.8 to 18% for the assay system without PMA stimulation and from 10.7 to 18.2% for the assay system with PMA stimulation. The PMA-stimulated assay had 40 to 60-fold higher sensitivity than the nonstimulated MTT assay. The MTT assay also was applied to the measurement of neutralizing antibody titers against A. pleuropneumoniae cytotoxin.     

Effect of acibenzolar-S-methyl, strobilurins and other fungicide treatments on phylloplane fungi of crops

Strobilurins, broad-spectrum fungicides used to control various diseases, may also inhibit the growth of nontarget fungi in the natural environment. Acibenzolar-S-methyl (ASM) is a plant activator; however, the effect of ASM on the growth of microflora on the leaf surface is not clear yet. ASM, strobilurins and other fungicides were sprayed on Japanese pear and cucumber plants in the field. Variations in the morphological characteristics and rDNA-ITS nucleotide sequence of isolated phylloplane fungi were evaluated. The isolation frequency of Alternaria spp., Aureobasidium spp., Cladosporium spp. and Myrothecium spp. on Japanese pear leaves treated with kresoxim-methyl and polycarbamate was lower than on nontreated pear leaves. Moreover, the frequency of Alternaria spp., Cladosporium spp., Epicoccum spp. and Fusarium spp. on leaves of two cucumber cultivars treated with azoxystrobin, triflumizole and mancozeb was lower than the frequency of fungal isolation on nontreated cucumber leaves. The effect of ASM on fungal isolation frequency was not significantly different from that on nontreated pear or cucumber leaves. The fungal genera observed and isolation frequency on the leaves treated with polycarbamate, azoxystrobin, triflumizole and mancozeb tended to be reduced compared to the no-spray treatment.     

Identification of lactoferrin and glutamate receptor‐interacting protein 1 in bovine cervical mucus: A putative marker for oestrous detection

Accurate detection of oestrus is important for artificial insemination. The aim of this study was to identify oestrous‐specific bovine cervical mucus proteins that could be used to determine the optimal time for artificial insemination. Non‐oestrous and controlled internal drug release (CIDR)‐induced oestrous‐stage mucus proteins were purified and subjected to surface‐enhanced laser desorption/ionization time‐of‐flight mass spectrometry, sodium dodecyl sulphate polyacrylamide gel electrophoresis and MALDI‐TOF/TOF. Among differentially expressed proteins, lactoferrin (LF) and glutamate receptor‐interacting protein 1 (GRIP1) showed a twofold increase during the CIDR‐induced oestrous stage compared to the levels in non‐oestrous stage in bovine cervical mucus. The RT‐PCR, Western blotting and immunohistochemistry results showed that LF and GRIP1 expression was significantly increased during the oestrous stage in the uterus. This study demonstrated that bovine LF and GRIP1 exist during the oestrous stage, but not during the non‐oestrous stage, suggesting that cervical mucus LF and GRIP1 are useful oestrous detection markers in cattle.     

Application of a solid-phase fluorescence immunoassay to determine oxytetracycline and tetracycline residues in tissue of olive flounder (Paralichthys olivaceus)

Parallux, a solid-phase fluorescence immunoassay (SPFIA) developed for antibotics residue detection in milk, was applied for analysis of fish tissue. The recommended therapeutic doses of oxytetracycline (OTC, 100 g/ton water, withdrawal period 30 days) and tetracycline (TC, 150 g/ton water, withdrawal period 30 days) were treated to a group of 35 olive flounders (Paralichthys olivaceus) using dipping administration. Muscle was sampled before and after drug treatment 1st, 2nd, 3rd, 5th, 7th, and 14th day. The concentration of oxytetracycline in muscle, determined by SPFIA, was compared with that of internal standard (100 ppb as oxytetracycline). The S/C ratio of sample inhibition value to cutoff inhibition value was employed as an index to determine the muscle residue in olive flounder. To investigate the recovery rate, and standard solutions were added to muscle samples to give final concentrations in muscle of 0.1 and 0.5 microg/ml. The recovery rates of all spiked samples were >89% of the spiked value. OTC and TC were detected in muscle of fishes treated until the 3rd day of withdrawal period. The present study showed that the SPFIA can be easily adopted in predicting tissue residues for OTC and TC in farmed fishes.     

Study of a Small‐Scale Laboratory Wet‐Milling Procedure for Wheat

The objectives of this research were to study the effects of slurry specific gravity, starch table slope, slurry pumping rate, and their interactions on starch recovery and purity; and to propose a small‐scale laboratory wet‐milling procedure for wheat. First‐order and second‐order response surface regression models were developed to study the effects and interactions of slurry specific gravity, starch table slope, and slurry pumping rate on starch and gluten separation for a 100‐g wheat wet‐milling procedure. The starch and starch protein content data fit the first‐order models (R² = 0.99 and 0.96) better than the second‐order models (R² = 0.98 and 0.93). Regression results from the first‐order models indicated that specific gravity, table slope, pumping rate, and their interactions all had a significant effect on starch yield and purity. However, these effects could be simplified as the effect of the resident time of starch and gluten slurry on the starch table and the specific gravity. Starch yield increased as resident time increased and specific gravity decreased. Protein content in starch decreased as the resident time decreased and the specific gravity increased. The separation condition with specific gravity of 3 Bé, table slope of 1.04 cm/m, and pumping rate of 50 mL/min was recommended. Under this condition, starch recovery was 85.6% and protein content of starch was 0.42%, which was similar to the 1.5‐kg laboratory methods in starch recovery. Total solids recovery was 98.1%, which is similar to that from 1.5‐kg laboratory methods. These results indicated that precision of the 100‐g wheat wet‐milling procedure was similar to that of the 1.5‐kg laboratory methods.     

Physicochemical Properties of Sonicated Mung Bean,Potato, and Rice Starches

Mung bean, potato, and rice starch solutions (5%, w/w) were sonicated for up to 5 min after heating, and their physicochemical properties were investigated. Alkaline viscosities, including the apparent and inherent viscosities of starches, decreased. The residues of the swollen starch granules after pasting and centrifugation were also reduced prominently by sonication. Average degree of polymerization did not change with sonication. The starch paste became more transparent, and the hot paste viscosity measured at 70°C decreased remarkably. Results indicate that changes in the physicochemical properties of starch were induced by the disruption of swollen granules rather than the breakage of glucosidic linkages with sonication.