Isolation of Vibrio vulnificus serovar E from aquatic habitats in Taiwan.
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The existence of strains of Vibrio vulnificus serovar E that are avirulent for eels is reported in this work. These isolates were recovered from water and oysters and differed from eel virulent strains in (i) fermentation and utilization of mannitol, (ii) ribotyping after HindIII digestion, and (iii) susceptibility to eel serum. Lipopolysaccharide of these strains lacked the highest molecular weight immunoreactive bands, which are probably involved in serum resistance. (+info)
Acidification of the phagosome in Crassostrea virginica hemocytes following engulfment of zymosan.
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Phagocytic hemocytes are responsible for engulfing and internally degrading foreign organisms within the hemolymph and tissue of the eastern oyster, Crassostrea virginica. Since rapid acidification of the phagosome lumen is typically essential for activation of hydrolytic and reactive oxygen intermediate (ROI) producing enzymes in vertebrate cells, we measured phagosomal pH in oyster hemocytes by using the emission fluorescence of two fluorescent probes, rhodamine and Oregon Green 488 (OG 488), conjugated to zymosan to determine whether oyster hemocyte phagosomes become acidified after phagocytosis of zymosan. The average pH of 1079 phagosomes within 277 hemocytes 1 h after phagocytosis of zymosan was 3.9 +/- 0.03. Observations of 141 hemocytes with internalized zymosan by light microscopy revealed that, over a 60-min time period, 51% of highly granular hemocytes became partially granular, and 29% became agranular. In addition, 83% of partially granular hemocytes containing zymosan at time = 0 became agranular within 60 min. A comparison revealed that the phagosomes of agranular hemocytes were much more acidic (pH 3.1 +/- 0.02) than those of highly granular hemocytes (4.9 +/- 0.02; P < 0.05). These values are significantly lower than most reported in the literature for blood cells from metazoan organisms. (+info)
Use of antibacterial agents To elucidate the etiology of juvenile oyster disease (JOD) in Crassostrea virginica and numerical dominance of an alpha-proteobacterium in JOD-affected animals.
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Since 1988, juvenile oyster disease (JOD) has resulted in high seasonal losses of cultured Eastern oysters (Crassostrea virginica) in the Northeast. Although the cause of JOD remains unknown, most evidence is consistent with either a bacterial or a protistan etiology. For the purpose of discerning between these hypotheses, the antibacterial antibiotics norfloxacin and sulfadimethoxine-ormetoprim (Romet-B) were tested for the ability to delay the onset of JOD mortality and/or reduce the JOD mortality of cultured juvenile C. virginica. Hatchery-produced C. virginica seed were exposed in triplicate groups of 3,000 animals each to either norfloxacin, sulfadimethoxine-ormetoprim, or filter-sterilized seawater (FSSW) and deployed in floating trays on the Damariscotta River of Maine on 17 July 1997. Each week thereafter, a subset of animals from each group was reexposed to the assigned treatment. Repeated immersion in either a sulfadimethoxine-ormetoprim or a norfloxacin solution resulted in a delay in the onset of JOD mortality in treated animals and reduced weekly mortality rates. Weekly treatments with either norfloxacin or sulfadimethoxine-ormetoprim also resulted in a statistically significant reduction in cumulative mortality (55 and 67% respectively) compared to animals treated weekly with FSSW (81%) or those that had received only a single treatment with either norfloxacin, sulfadimethoxine-ormetoprim, or FSSW (77, 84, and 82%, respectively). Bacteriological analyses revealed a numerically dominant bacterium in those animals with obvious signs of JOD. Sequence analysis of the 16S rRNA gene from these bacteria indicates that they are a previously undescribed species of marine alpha-proteobacteria. (+info)
Purification and partial genome characterization of a herpes-like virus infecting the Japanese oyster, Crassostrea gigas.
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First observed in 1972 in Crassostrea virginica, herpes-like viruses of bivalves were more recently found to be associated with high mortality rates in other cultured oyster species, such as Crassostrea gigas and Ostrea edulis. The diagnosis of herpes-like virus infections is performed currently by laborious histological and transmission electron microscope examinations. Preparation of specific reagents for use in more amenable diagnostic techniques prompted purification of virus particles and investigation of the viral genome. This paper is the first description of the purification of a virus pathogen from a bivalve mollusc. A procedure was developed which facilitated purification of large amounts of virus particles on the 40-50% interface of sucrose gradients. Transmission electron microscopy showed that a purified virus suspension contained capsids and enveloped virus particles. High molecular mass viral DNA was extracted, and the genome size was estimated by the summation of the sizes of restriction endonuclease fragments to be approximately 180 kbp. Partial cloning of the virus genome was achieved and the specificity of certain cloned fragments was established by dot blot hybridization. (+info)
Factors influencing in vitro killing of bacteria by hemocytes of the eastern oyster (Crassostrea virginica).
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A tetrazolium dye reduction assay was used to study factors governing the killing of bacteria by oyster hemocytes. In vitro tests were performed on bacterial strains by using hemocytes from oysters collected from the same location in winter and summer. Vibrio parahaemolyticus strains, altered in motility or colonial morphology (opaque and translucent), and Listeria monocytogenes mutants lacking catalase, superoxide dismutase, hemolysin, and phospholipase activities were examined in winter and summer. Vibrio vulnificus strains, opaque and translucent (with and without capsules), were examined only in summer. Among V. parahaemolyticus and L. monocytogenes, significantly (P < 0.05) higher levels of killing by hemocytes were observed in summer than in winter. L. monocytogenes was more resistant than V. parahaemolyticus or V. vulnificus to the bactericidal activity of hemocytes. In winter, both translucent strains of V. parahaemolyticus showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. In summer, only one of the V. parahaemolyticus translucent strains showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. No significant differences (P > 0.05) in killing by hemocytes were observed between opaque (encapsulated) and translucent (nonencapsulated) pairs of V. vulnificus. Activities of 19 hydrolytic enzymes were measured in oyster hemolymph collected in winter and summer. Only one enzyme, esterase (C4), showed a seasonal difference in activity (higher in winter than in summer). These results suggest that differences existed between bacterial genera in their ability to evade killing by oyster hemocytes, that a trait(s) associated with the opaque phenotype may have enabled V. parahaemolyticus to evade killing by the oyster's cellular defense, and that bactericidal activity of hemocytes was greater in summer than in winter. (+info)
Oyster IKK-like protein shares structural and functional properties with its mammalian homologues.
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In our search for genes involved in oyster immunity we isolated a cDNA encoding a polypeptide closely related to the mammalian IkappaB kinase (IKK) family. IKK proteins play a central role in cell signaling by regulating nuclear factor-kappaB (NF-kappaB) activation. We report here the cloning of an oyster IKK-like protein (oIKK) which possesses the characteristic organization of the mammalian IKK proteins, namely an amino-terminal kinase domain followed by a leucine zipper region and a carboxyl-terminal helix-loop-helix motif. When transfected into human cell lines, oIKK activated the expression of NF-kappaB-controlled reporter gene, whereas transfections with mutants of oIKK deleted within the kinase domain or within the helix-loop-helix motif respectively abolished and greatly reduced reporter gene activation. These results indicate that oIKK can replace the hIKK-alpha in catalyzing NF-kappaB nuclear translocation, and in triggering gene expression. Our results sustain the concept of an evolutionarily conserved signaling machinery in which IKK plays a major role. (+info)
Perkinsus marinus extracellular protease modulates survival of Vibrio vulnificus in Eastern oyster (Crassostrea virginica) hemocytes.
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The in vitro effects of the Perkinsus marinus serine protease on the intracellular survival of Vibrio vulnificus in oyster hemocytes were examined by using a time-course gentamicin internalization assay. Results showed that protease-treated hemocytes were initially slower to internalize V. vulnificus than untreated hemocytes. After 1 h, the elimination of V. vulnificus by treated hemocytes was significantly suppressed compared with hemocytes infected with invasive and noninvasive controls. Our data suggest that the serine protease produced by P. marinus suppresses the vibriocidal activity of oyster hemocytes to effectively eliminate V. vulnificus, potentially leading to conditions favoring higher numbers of vibrios in oyster tissues. (+info)
Cryptosporidium parvum in oysters from commercial harvesting sites in the Chesapeake Bay.
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Oocysts of Cryptosporidium parvum, a zoonotic waterborne pathogen, can be removed by bivalve molluscs from contaminated water and retained on gills and in hemolymph. We identified oocysts of C. parvum in oysters from seven sites in the Chesapeake Bay area. These findings document the presence of C. parvum infectious for humans in oysters intended for human consumption. (+info)