Lipopolysaccharides (LPSs) isolated from three Kanagawa-positive and three negative strains of Vibrio parahaemolyticus were characterized by using electrophoretic, immunochemical, and chemical methods. The results of this study indicated that the LPSs of all six strains of V. parahaemolyticus examined did not have an 0-specific side chain. These V. parahaemolyticus LPSs appeared to have molecular weights similar to that of the rough-type (Ra) LPS of Salmonella typhimurium TV-119 and might just contain lipid A and a core region. However, the microheterogeneity of V. parahaemolyticus LPS observed was greater than that of S. typhimurium LPS. The profile of V. parahaemolyticus LPS consisted of closely spaced triplet or quadruplet bands, but that of S. typhimurium consisted of doublet bands. Slower-moving bands appeared on sodium dodecyl sulfatepolyacrylamide gel electrophoresis gels only when large amounts of V. parahaemolyticus LPS were loaded. These bands were proven to be the aggregates of the fastest-moving low-molecular-weight bands by re-electrophoresis. The banding pattern of V. parahaemolyticus LPSs produced on nitrocellulose membranes by immunoblotting indicated that the V. parahaemolyticus LPSs did not have an 0-specific side chain. The low ratio of total carbohydrate to lipid A of V. parahaemolyticus LPSs also suggested that they were like rough-type LPS. The mobility and profile of V. parahaemolyticus LPS on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and its chemical composition were closely related to the serotype of a specific strain but not with the Kanagawa phenomenon.Vibrio parahaemolyticus is a gram-negative pathogen to humans and other creatures (47). Since it is an inhabitant of estuarine and coastal waters of the ocean (28), fresh seafood is often contaminated with this pathogen (41). Most gastroenteritis outbreaks resulting from seafood consumption are caused by V. parahaemolyticus (22). As seafood consumption is increasing in this country, it is becoming more and more important to understand this pathogen and its virulence factors. However, the virulence factors of pathogenic V. parahaemolyticus are still unknown and not all V. parahaemolyticus strains are pathogenic (23). Most (96.5%) of the pathogenic strains isolated from patients are able to lyse erythrocytes on blood agar medium containing 7% NaCl (44), i.e., they are Kanagawa positive (K+), but 99% of the strains isolated from natural environments do not have such hemolytic activity, i.e., they are Kanagawa negative (K-), and are usually nonpathogenic (23). Although the thermostable direct hemolysin which accounts for K+ activity has fluid-accumulating activity in the rabbit ileal loop (46), identification of thermostable direct hemolysin as the virulence factor is still unclear owing to its relatively low activity (21 Extraction of LPS. LPS was extracted from lyophilized cells by using the phenol-water method (45). This method had been demonstrated to be better than the phenol-chloroform-petroleum ether method (13) fo...

An improved spectrophotometric method for phosphorus determination in oils is proposed. The proposed new method has made significant improvements in safety, sensitivity, and efficiency in comparison with the current American Oil Chemists' Society (AOCS) Official Method (Ca 12-55, corrected 1992). The AOCS method employs hydrazine sulfate as the reducing agent to generate molybdenum blue. Hydrazine sulfate is a known carcinogen in laboratory animals and a suspected human carcinogen. The chemical also irritates skin and mucous membranes. [n the improved method ascorbic acid is used as the reducing agent. Ascorbic acid is equally effective as hydrazine sulfate for the color reaction. The improved method is approximately 75 times more sensitive than the AOCS method. The sensitivity is improved by measuring absorbance at the absorption peak, 825 nm, and reducing the final volume of reaction mixture for color reaction. The AOCS method determines phosphorus by measuring absorbance at 650 nm, which is about 40% of that at the absorption peak. The efficiency of this improved method is also significantly increased by reducing sample size and the volumes of sample preparation. Therefore, the improved method is more cost-effective than the AOCS method because less chemical reagents and smaller glassware are used, and the hazardous chemical waste disposal cost is eliminated. The improved method also avoids concentrated HCI and 50% KOH for sample preparation. ]AOCS 72, 881-885 (1995).

A total of 338 water samples were collected at 20 stations from three geographically shellfish-growing areas in Chesapeake Bay from May to September 1989. Samples were examined for standard plate count, total coliforms, fecal coliforms, Escherichia coli and coliphages. Salinity, dissolved oxygen and temperature varied slightly with the depth, season, and geographic area of water samples. The geometric means of standard plate count for the three areas were 135, 355 and 275/ml, respectively. The range of means of fecal coliform for these areas was from <3 to 93/100 mi. Escherichia coli counts were also low with a range of <3 to 93/100 mi and a mean of < 3/100 mi. The growing water area adjacent to cropland was found to have higher bacterial counts than those of the other two areas. Levels of male-specific phages were very low. Results indicate that shellfish-growing waters in all three areas were of satisfactory bacteriological quality.

A total of 472 samples of soft-shell clams (Mya arenaria), collected from three major clam harvest areas in the Chesapeake Bay and dockside check stations, was analyzed for standard plate count (SPC), total coliforms, fecal coliforms, Escherichia coli, and coliphages. SPC increased during the summer season. SPC geometric means of 2.6 × 104, 6.9 × 104, and 7.2 × 104/g, respectively, were found in three major harvest areas. Fecal coliforms remained relatively stable with geometric means of 30, 54, and 62/100 g. As seasonal temperatures increased, the total coliform geometric means declined slightly ranging from 1,500 to 6,300/100 g. E. coli means were low (< 27/100 g). The occurrence and levels of male-specific coliphages were also low and did not correlate with bacteriological quality. No significant microbiological quality difference was found between soft-shell clams sampled from harvest waters and check stations. Results indicate that the microbiological quality of soft-shell clams either at harvest or check stations was satisfactory.

The occurrence of 2-keto-3-deoxy-D-manno-octonic acid (KDO) in lipopolysaccharides (LPS) of Vibrio parahaemolyticus was demonstrated for the first time by gas chromatography-mass spectrometry after dephosphorylation, reduction, and methylation. KDO was virtually completely phosphorylated, since no KDO was detected by either gas chromatography or thiobarbituric acid assay before dephosphorylation. The level of KDO in all six strains of V. parahaemolyticus investigated ranged from 0.37 to 0.69%, which was considerably lower than that in enterobacterial LPS.2-Keto-3-deoxy-D-manno-octonic acid (KDO) is a unique eight-carbon sugar commonly found in the inner core region of gram-negative bacterial lipopolysaccharides (LPS) (14), but it was considered virtually missing in vibrios (9, 10). However, the observed KDO-specific antigenicity of Vibrio cholerae LPS led to the finding of phosphorylated KDO by gas chromatography-mass spectrometry (GC-MS) (1). By using a thiobarbituric acid (TBA) assay alone, KDO has been recognized in thiobarbiturate-negative LPS prepared from Pseudomonas spp., Bacteroides spp., and Aeromonas spp. after dephosphorylation (4). No conclusive evidence for the general existence of KDO in Vibrio parahaemolyticus LPS has been reported. The purpose of this study was to demonstrate the existence of KDO in V. parahaemolyticus LPS by GC-MS and chemical methods.Six strains of V. parahaemolyticus were studied. Their serotypes and Kanagawa phenomena were as follows: 38C1, 04:K11 and K+; P7, 04:K4 and K+; P26, 02:K3 and K+; 38C6, 03:K30 and K-; P68, 04:K34 and K-; and P6, 02:K30 and K-. Kanagawa-positive (K+) strains are able to lyse red blood cells on blood agar medium containing 7% NaCl (16). Virtually all pathogenic V. parahaemolyticus strains are Kanagawa positive (17). Three strains of either K+ or K-V. parahaemolyticus were used for this study to determine whether there is any correlation between the presence of KDO and Kanagawa phenomenon. Bacteria were grown in PPBE broth (1% proteose peptone, 0.2% beef extract, 2.5% NaCl) at 35°C. Cells were harvested at an optical density at 660 nm of 1.0 to 1.2 and then lyophilized. LPS was extracted by the phenol-water method (18) and purified by repeated high-speed centrifugation (10,000 x g, 30 min) and ultracentrifugation (105,000 x g, 3 h) (7). LPS was dephosphorylated in 48% HF at 4°C for 72 h in a polyethylene centricone. HF was removed either by evaporation under a vacuum (13) or by dialysis (12). Mild acid hydrolysis with 5% acetic acid at 100°C for 5 h was used to break the glycosidic linkage between polysaccharide and lipid A in control (untreated) and dephosphorylated LPS. KDO in degraded polysaccharide was derivatized by the procedure described by Waeghe et al. (15) tions. Degraded polysaccharide underwent the following derivatization steps: (i) prereduction of the keto group by NaBD4; (ii) methylation of the hydroxyl groups of sugars by methylsulfinyl carbanion (CH3 * SO -CH2-, Na+) and CH3I by the Hakomori method (6) (methylsulfinyl carbanion was pr...