Immunomagnetic separation of Vibrio vulnificus with antiflagellar monoclonal antibody.

Raw oysters are primary vectors for Vibrio vulnificus infections, and a rapid detection method for V. vulnificus in raw oysters before distribution would be an indispensable tool for the seafood industry. One approach to improving the recovery and detection of V. vulnificus without sacrificing assay time is through the use of immunomagnetic separation (IMS). The aim of this study was to develop and optimize an IMS protocol using anti-H (antiflagellar) antibody for determining the level of V. vulnificus in phosphate-buffered saline (PBS) suspensions and spiked oyster homogenate. Six monoclonal antibodies were produced by immunizing mice at 2-week intervals by injection of 50 microg of purified V. vulnificus ATCC 27562 flagellin. Antibodies that exhibited high anti-H titers were coated onto Cowan I Staphylococcus aureus cells and sheep anti-mouse immunoglobulin G immunomagnetic beads. The two reagents were used to determine the species specificity of the selected antibodies, which positively identified and coagglutinated 70 isolates identified genetically as V. vulnificus and did not react with 40 Vibrio parahaemolyticus isolates or nine other Vibrio species. The IMS protocol was optimized for PBS and oyster homogenate spiked with three different strains of V. vulnificus. IMS with V. vulnificus-spiked PBS yielded binding of 19 to 57%, and IMS with spiked oyster homogenate carried out at two V. vulnificus levels exhibited binding of 25 to 57%. The IMS protocol for V. vulnificus could be used to concentrate and detect V. vulnificus in seawater and shellfish homogenate.

Identification and characterization of two bacteriocin-producing bacteria isolated from garlic and ginger root.

Two bacteriocin-producing bacterial strains were isolated from garlic and ginger root by the agar overlay method. The bacteria were identified by 16S rRNA sequence analyses and fermentation patterns as Leuconostoc mesenteroides (garlic isolate) and Lactococcus lactis (ginger isolate). The bacteriocins were assigned the names leucocin BC2 and lactocin GI3, respectively. Physiochemical properties and antimicrobial spectra of the bacteriocins were determined by the spot-on-lawn method. Both bacteriocins were inhibited by proteolytic enzymes. Leucocin BC2 exhibited a narrow antimicrobial spectrum, inhibiting only Bacillus, Enterococcus, and Listeria species. Lactocin GI3 had a broader spectrum, inhibiting Bacillus, Clostridium, Listeria, Enterococcus, Leuconostoc, Pediococcus, and Staphylococcus species. Both bacteriocins remained active when heated at 90 degrees C for 15 min or 120 degrees C for 20 min. Leucocin BC2 assayed at 37 degrees C showed an inhibitory activity of 1,600 AU/ml, whereas at 8 degrees C the activity was 12,800 AU/ml. Conversely, lactocin GI3 activity was the same at both assay temperatures. Both bacteriocins remained active over a pH range of 2.0 to 9.0 and in various organic solvents. The activity of leucocin BC2 was increased when treated with 0.5% acetic acid and 0.5% lactic acid, whereas lactocin GI3 activity was decreased with either acid. The molecular mass values were 3.7 kDa for leucocin BC2 and 3.9 kDa for lactocin GI3. These results show that the inhibitory substances produced by the bacteria isolated from garlic and ginger are bacteriocins that appear to be different in some characteristics from previously reported bacteriocins.

Survival differences of Escherichia coli O157:H7 strains in apples of three varieties stored at various temperatures.

Differences in survival and growth among five different Escherichia coli O157:H7 strains in three apple varieties were determined at various temperatures. Jonathan, Golden Delicious, and Red Delicious apples were wounded and inoculated with E coli O157:H7 strains C7929 (apple cider isolate), 301C (chicken isolate), 204P (pork isolate), 933 (beef isolate), and 43890 (human isolate) at an initial level of 6 to 7 log CFU/g. The inoculated apples were stored at a constant temperature of 37, 25, 8, or 4 degrees C or at 37 degrees C for 24 h and then at 4 degrees C, and bacterial counts were determined every week for 28 days. By day 28, for Jonathan apples at 25 degrees C, the apple isolate counts were significantly higher than the chicken and human isolate counts. At 4 degrees C for 28 days, the human isolate inoculated into Jonathan, Golden Delicious, and Red Delicious apples was present in significantly smaller numbers than the other strains. The apple isolate survived significantly better at 4 degrees C, yielding the highest number of viable cells. By days 21 and 28, for apples stored at 37 degrees C for the first 24 h and then at 4 degrees C, the counts of viable E. coli O157:H7 apple and human isolates were 6.8 and 5.8 log CFU/g at the site of the wound, whereas for apples kept at 4 degrees C for the duration of storage, the respective counts were 5.6 and 1.5 log CFU/g. Our study shows that E. coli O157:H7 strains responded differentially to their ability to survive in these three apple varieties at 25 or 4 degrees C and produced higher viable counts when apples were temperature abused at 37 degrees C for 24 h and then stored at 4 degrees C for 27 days.

The leukocyte response of Japanese quail to Rous sarcoma virus-induced tumors.

In male Japanese quail, different circulating leukocyte responses were observed for progressors (birds developing a massive tumor that persisted throughout the experiment) and regressors (birds developing a tumor that gradually disappeared) after initial challenge with Rous sarcoma virus (RSV). Blood was sampled before and at weekly intervals postinoculation. Blood smears were prepared and stained with Diff Quik, and a light microscope (1000 x) was used in a direct count of 50 fields. Leukocytes were classified as heterophils, lymphocytes, monocytes, or eosinophils. The significant increase (P < 0.05) in total leukocytes at 14 days in regressors and progressors was consistent with the increase in tumor growth. The regressors' individual percentage of leukocytes did not deviate from control values, whereas the progressors' percentages of heterophils and monocytes were significantly higher (P < 0.05) and of lymphocytes significantly lower (P < 0.05) than those of controls by 14 days postinoculation. Indicative of this was the progressors' heterophil to lymphocyte ratio, which was significantly higher (P < 0.05) than that of controls 14 days post RSV challenge and remained elevated throughout the experiment. These findings suggest that the progressors' immune response is suppressed by proliferation of malignant cells. Therefore, the heterophil to lymphocyte ratio may be used in addition to tumor size to identify those birds that will regress RSV-induced tumors.

A previously unrecognized promoter of LMO2 forms part of a transcriptional regulatory circuit mediating LMO2 expression in a subset of T-acute lymphoblastic leukaemia patients.

The T-cell oncogene Lim-only 2 (LMO2) critically influences both normal and malignant haematopoiesis. LMO2 is not normally expressed in T cells, yet ectopic expression is seen in the majority of T-acute lymphoblastic leukaemia (T-ALL) patients with specific translocations involving LMO2 in only a subset of these patients. Ectopic lmo2 expression in thymocytes of transgenic mice causes T-ALL, and retroviral vector integration into the LMO2 locus was implicated in the development of clonal T-cell disease in patients undergoing gene therapy. Using array-based chromatin immunoprecipitation, we now demonstrate that in contrast to B-acute lymphoblastic leukaemia, human T-ALL samples largely use promoter elements with little influence from distal enhancers. Active LMO2 promoter elements in T-ALL included a previously unrecognized third promoter, which we demonstrate to be active in cell lines, primary T-ALL patients and transgenic mice. The ETS factors ERG and FLI1 previously implicated in lmo2-dependent mouse models of T-ALL bind to the novel LMO2 promoter in human T-ALL samples, while in return LMO2 binds to blood stem/progenitor enhancers in the FLI1 and ERG gene loci. Moreover, LMO2, ERG and FLI1 all regulate the +1 enhancer of HHEX/PRH, which was recently implicated as a key mediator of early progenitor expansion in LMO2-driven T-ALL. Our data therefore suggest that a self-sustaining triad of LMO2/ERG/FLI1 stabilizes the expression of important mediators of the leukaemic phenotype such as HHEX/PRH.

Detection and enumeration of Vibrio vulnificus by direct colony immunoblot.

A direct colony immunoblot method (DCI) for the enumeration of Vibrio vulnificus was developed. Bacterial colonies were transferred from agar plates to membranes, which were then dried and blocked with bovine serum albumin. Subsequently, the membranes were treated with anti-V. vulnificus H antibodies, washed and incubated with peroxidase-conjugated goat anti-rabbit IgG. After a final wash, the membranes were exposed to a substrate mixture containing H(2)O(2) which resulted in the development of a purple color by V. vulnificus colonies. The DCI detected all clinical and environmental V. vulnificus strains tested and did not cross-react with other Vibrio species including V. cholerae, V. parahaemolyticus, or V. fluvialis. The DCI was then compared to the DNA hybridization procedure (DNAH) using V. vulnificus agar plates inoculated with mixed cultures of V. vulnificus and V. parahaemolyticus and V. vulnificus-seeded oyster homogenates. Both DCI and DNAH detected 1 to 2 log colony forming units (CFU)/mL V. vulnificus mixed with 4 log CFU/mL V. parahaemolyticus. Both methods were comparable and demonstrated no significant statistical differences when enumerating V. vulnificus in mixed cultures or in oyster homogenates seeded with levels of V. vulnificus from 2 to 6 log CFU/mL. The DCI demonstrated clearer color development and was less time consuming than the DNAH.

Growth and survival differences of Vibrio vulnificus and Vibrio parahaemolyticus strains during cold storage.

Vibrio vulnificus and Vibrio parahaemolyticus are the most common Vibrio species associated with seafood illness in the United States. Our study was conducted to determine if strain-to-strain differences exist in the growth and survival of 8 different V. vulnificus and V. parahaemolyticus strains at low temperatures. By day 10, V. vulnificus strain 515-4C2 had significantly higher counts (P < 0.05) (1.97 log CFU/g) compared with strains 3315, 1007, 29306 at 5 degrees C, which reached nondetectable levels. At 8 degrees C, strain 515-4C2 had significantly higher counts (P < 0.05) (2.23 log CFU/mL) compared with 1007, 33815, 541(O) 49C, which reached nondetectable levels. At 10 degrees C, only V. vulnificus strain 33815 reached nondetectable levels. At 5 degrees C, V. parahaemolyticus strain 541(O) 57C had the highest counts (5.28 log CFU/g) by day 10 while strain 33847 had significantly lower counts (3.46 log CFU/g). After 10 d at 8 degrees C, V. parahaemolyticus strain M350A had the highest counts (7.97 log CFU/mL) while strain 541(O) 57C had the lowest counts (4.80 log CFU/mL). At 10 degrees C, V. parahaemolyticus strain NY477 had significantly higher counts (P < 0.05) with 8.31 log CFU/mL compared with strain 33847, which had the lowest counts (6.77 log CFU/mL). Our research has shown that various V. vulnificus and V. parahaemolyticus strains vary in their ability to survive and grow at refrigeration temperatures.

Immunomagnetic separation and coagglutination of Vibrio parahaemolyticus with anti-flagellar protein monoclonal antibody.

Mice were immunized by injection of Vibrio parahaemolyticus ATCC 17802 polar flagellin in order to produce monoclonal antibodies (mAbs). mAbs were analyzed by anti-H enzyme-linked immunosorbent assay using V. parahaemolyticus polar flagellar cores. The mAb exhibiting the highest anti-H titer was coated onto Cowan I Staphylococcus aureus cells at a concentration of 75 microg/ml cell suspension and used for slide coagglutination. Of 41 isolates identified genetically as V. parahaemolyticus, 100% coagglutinated with the anti-H mAb within 30 s, and the mAb did not react with 30 isolates identified as Vibrio vulnificus. A strong coagglutination reaction with V. parahaemolyticus ATCC 17802 was still observed when the S. aureus cells were armed with as little as 15 microg of mAb/ml S. aureus cell suspension. At this concentration, the mAb cross-reacted with three other Vibrio species, suggesting that they share an identical H antigen or antigens. The anti-H mAb was then used to optimize an immunomagnetic separation protocol which exhibited from 35% to about 45% binding of 10(2) to 10(3) V. parahaemolyticus cells in phosphate-buffered saline. The mAb would be useful for the rapid and selective isolation, concentration, and detection of V. parahaemolyticus cells from environmental sources.

Control of Listeria monocytogenes and Salmonella anatum on the surface of smoked salmon coated with calcium alginate coating containing oyster lysozyme and nisin.

This study investigated the antimicrobial effect of oyster lysozyme with or without nisin added to calcium alginate (CaAlg) coated on the surface of smoked salmon against Listeria monocytogenes and Salmonella anatum. L. monocytogenes or S. anatum inoculated smoked salmon samples (1 g) were dipped into CaAlg with either oyster lysozyme (OysL) or hen egg white lysozyme (HEWL), with or without added nisin (N), then stored at 4 degrees C for 35 d. Our results indicated that the effectiveness of oyster lysozyme or hen egg white lysozyme was enhanced when added to calcium alginate coatings. After 35 d at 4 degrees C the growth of L. monocytogenes and S. anatum was suppressed in the range of 2.2 to 2.8 log CFU/g with CaAlgNOysL or CaAlgNHEWL coatings compared to the control nontreated samples. There was no significant difference between oyster lysozyme and hen egg white lysozyme treatments against L. monocytogenes or S. anatum inoculated on the surface of salmon. Calcium alginate coatings containing lysozyme with nisin or without could be used to reduce the growth of L. monocytogenes and S. anatum on the surface of ready-to-eat smoked salmon at refrigerated temperatures.

Selected quality characteristics of fresh-cut sweet potatoes coated with chitosan during 17-day refrigerated storage.

Selected quality characteristics of fresh-cut sweet potatoes (FCSP) coated with chitosan were evaluated during 17-d refrigerated storage. The FCSP cubes were coated with a solution (1%, w/v) of chitosan having 470 or 1110 kDa. Color (L*, a*, b*) values of uncoated and chitosan-coated FCSP during storage were generally affected by storage time as well as coating treatments (P < 0.05). No significant changes in color lightness (L*) of 470 kDa-coated FCSP were observed during the 17-d storage. During days 3 to 17, 470 kDa-coated FCSP had significantly higher redness (a*) and yellowness (b*) values than did uncoated and 1110 kDa-coated FCSP. Texture firmness of uncoated and chitosan-coated FCSP exhibited minimal changes during the 17-d storage. Although actual weight loss values (%) of uncoated and chitosan-coated FCSP were not significantly different at day 17, the weight loss difference (%) between day 3 and day 17 for uncoated FCSP (3.02%) was slightly higher compared to those (2.24% to 2.26%) of chitosan-coated FCSP. The initial total aerobic count was 4.7 log(10) CFU/g which then gradually increased to 8.54 and 9.67 log(10) CFU/g after 17 d of storage for 470 kDa-coated and uncoated FCSP, respectively. After day 6, the total aerobic counts of uncoated FCSP were higher than those of 470 kDa-coated FCSP. The yeast and mold count of chitosan-coated FCSP was about 2.5 log(10) CFU/g at day 17. Overall, consumers could not differentiate between 470 kDa-coated FCSP at day 17 and uncoated FCSP at day 0.