Because biochemical testing and 16S rRNA sequence analysis have proven inadequate for the differentiation of Vibrio parahaemolyticus from closely related species, we employed the gyrase B gene (gyrB) as a molecular diagnostic probe. ThegyrB genes of V. parahaemolyticus and closely related Vibrio alginolyticus were cloned and sequenced. Oligonucleotide PCR primers were designed for the amplification of a 285-bp fragment from within gyrB specific for V. parahaemolyticus. These primers recognized 117 of 117 reference and wild-type V. parahaemolyticus strains, whereas amplification did not occur when 90 strains of 37 otherVibrio species or 60 strains representing 34 different nonvibrio species were tested. In 100-μl PCR mixtures, the lower detection limits were 5 CFU for live cells and 4 pg for purified DNA. The possible application of gyrB primers for the routine identification of V. parahaemolyticus in food was examined. We developed and tested a procedure for the specific detection of the target organism in shrimp consisting of an 18-h preenrichment followed by PCR amplification of the 285-bp V. parahaemolyticus-specific fragment. This method enabled us to detect an initial inoculum of 1.5 CFU of V. parahaemolyticus cells per g of shrimp homogenate. By this approach, we were able to detect V. parahaemolyticus in all of 27 shrimp samples artificially inoculated with this bacterium. We present here a rapid, reliable, and sensitive protocol for the detection of V. parahaemolyticus in shrimp.
A new bacterial species belonging to the genus Pseudoalteromonas is described on the basis of phenotypic characterization, and sequence analysis of its 16S rRNA-coding and gyrase B (gyrB) genes. Ten strains, isolated from sea water of Yamato Island, Sea of Japan, were Gram-negative, yellow, motile, polarly flagellated, aerobic, rod-shaped eubacteria and had a GMC content of 42 mol %. Analysis of the 16S rDNA sequence revealed a clear affiliation between these strains and members of the γ-Proteobacteria. High similarity values were found with members of the genus Pseudoalteromonas and this was supported by fatty acid profiles. The 16S rDNA sequence similarity between strain F12-50-A1 T and Pseudoalteromonas piscicida was very high (99 1 %). However, molecular characterizations employing small subunit 16S rDNA sequences were at the limits of resolution for the differentiation of species in this genus. As a result, DNA-DNA hybridization and sequence analyses of a more rapidly evolving gyrB gene were performed. Our assertion that this strain represents a distinct bacterial species within the genus Pseudoalteromonas was supported by both of these molecular analyses. Species-specific PCR probes were designed for the gyrB gene and used for the rapid screening of F12-50-A1 T -like strains, thereby confirming the species. As these strains cleave complex protein compounds of the Mytilus edulis foot by secreting proteases, the name Pseudoalteromonas peptidolytica sp. nov. is proposed, with strain F12-50-A1 T (l MBICC F1250A1 T ) as the type strain.
SummaryIn the first study (Study 1), 4-wk-old Sprague-Dawley (SD) rats were fed high fat diets containing casein, Alaska pollack, yellowfin tuna, or chicken as the protein source for 28 d. The purpose of this study was to compare the effect of Alaska pollack protein with other animal proteins (casein, yellowfin tuna, and chicken) on the prevention of visceral fat accumulation. We found that Alaska pollack protein was a more potent inhibitor of visceral fat accumulation than the other proteins ( p Ͻ 0.05). In the second study (Study 2), we determined the quantity of Alaska pollack protein needed to have an effect. To test this, 4-wk-old SD rats were fed diets containing different percentages of Alaska pollack proteins (0, 3, 10, 30 or 100%) to replace casein as the protein source for 28 d. The diets with 30 or 100% Alaska pollack protein as the protein source prevented visceral fat accumulation and elevated plasma adiponectin levels. Based on these findings, an inhibitory effect on the accumulation of visceral fats can be achieved by consuming a diet in which 30% or more of the total protein content comes from Alaska pollack.
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