Temperature dependent characteristics of a recombinant infectious hematopoietic necrosis virus glycoprotein produced in insect cells.
(9/793)
A recombinant infectious hematopoietic necrosis virus (IHNV) glycoprotein (G protein) was produced in insect cells using a baculovirus vector (Autographa californica nuclear polyhedrosis virus). Characteristics of this protein were evaluated in relation to native viral G protein. A full-length (1.6 kb) cDNA copy of the glycoprotein gene of IHNV was inserted into the baculovirus vector under control of the polyhedrin promoter. High levels of G protein (approximately 0.5 microgram/1 x 10(5) cells) were produced in Spodoptera frugiperda (Sf9) cells following recombinant baculovirus infection. Analysis of cell lysates by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot revealed a recombinant IHNV G of slightly higher mobility on the gel than the viral G protein. Differences in mobility were abrogated by endoglycosidase treatment. When the recombinant G protein was produced in insect cells at 20 degrees C (RecGlow), immunostaining and cell fusion activity demonstrated surface localization of the protein. In contrast, when recombinant protein was produced at 27 degrees C (RecGhigh), G protein was sequestered within the cell, suggesting that at the 2 different temperatures processing differences may exist. Eleven monoclonal antibodies (MAbs) were tested by immunoblotting for reactivity to the recombinant G protein. All 11 MAbs reacted to the reduced proteins. Four MAbs recognized both RecGhigh and RecGlow under non-reducing conditions; however, 1 neutralizing MAb (92A) recognized RecGlow but failed to react to RecGhigh under non-reducing conditions. This suggests that differences exist between RecGlow and RecGhigh which may have implications in the development of a properly folded recombinant G protein with the ability to elicit protective immunity in fish. (+info)
Elevated temperature treatment as a novel method for decreasing p57 on the cell surface of Renibacterium salmoninarum.
(10/793)
Renibacterium salmoninarum is a Gram-positive diplo-bacillus and the causative agent of bacterial kidney disease, a prevalent disease of salmonid fish. Virulent isolates of R. salmoninarum have a hydrophobic cell surface and express the 57-58 kDa protein (p57). Here we have investigated parameters which effect cell hydrophobicity and p57 degradation. Incubation of R. salmoninarum cells at 37 degrees C for > 4 h decreased cell surface hydrophobicity as measured by the salt aggregation assay, and decreased the amount of cell associated p57. Incubation of cells at lower temperatures (22, 17, 4 or -20 degrees C) for up to 16 h did not reduce hydrophobicity or the amount of cell associated p57. Both the loss of cell surface hydrophobicity and the degradation of p57 were inhibited by pre-incubation with the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Cell surface hydrophobicity was specifically reconstituted by incubation with extracellular protein (ECP) concentrated from culture supernatant and was correlated with the reassociation of p57 onto the bacterial cell surface as determined by western blot and total protein stain analyses. The ability of p57 to reassociate suggests that the bacterial cell surface is not irreversibly modified by the 37 degrees C treatment and that p57 contributes to the hydrophobic nature of R. salmoninarum. In summary, we describe parameters effecting the removal of the p57 virulence factor and suggest the utility of this modification for generating a whole cell vaccine against bacterial kidney disease. (+info)
Unexpected BII conformer substate population in unoriented hydrated films of the d(CGCGAATTCGCG)2 dodecamer and of native B-DNA from salmon testes.
(11/793)
Conformational substates of B-DNA had been observed so far in synthetic oligonucleotides but not in naturally occurring highly polymeric B-DNA. Our low-temperature experiments show that native B-DNA from salmon testes and the d(CGCGAATTCGCG)2 dodecamer have the same BI and BII substates. Nonequilibrium distribution of conformer population was generated by quenching hydrated unoriented films to 200 K, and isothermal structural relaxation toward equilibrium by interconversion of substates was followed by Fourier transform infrared spectroscopy. BI interconverts into BII on isothermal relaxation at 200 K, whereas on slow cooling from ambient temperature, BII interconverts into BI. Our estimation of the dodecamer's BI-to-BII conformer substate population by curve resolution of the symmetrical stretching vibration of the ionic phosphate is 2.4 +/- 0.5 to 1 at 200 K, and it is 1.3 +/- 0.5 to 1 between 270 and 290 K. Pronounced spectral changes upon BI-to-BII interconversion are consistent with base destacking coupled with migration of water from ionic phosphate toward the phosphodiester and sugar moieties. Nonspecific interaction of proteins with the DNA backbone could become specific by induced-fit-type interactions with either BI or BII backbone conformations. This suggests that the BI-to-BII substate interconversion could be a major contributor to the protein recognition process. (+info)
Time course tissue distribution of infectious salmon anaemia virus in experimentally infected Atlantic salmon Salmo salar.
(12/793)
Atlantic salmon Salmo salar L. were injected intraperitoneally with infectious salmon anaemia virus (ISAV)-infective tissue homogenate to clarify the tissue distribution of ISAV in a time course study. Fish were sampled at 11 different intervals between 1 and 40 d post-infection (p.i.) and mid-kidney, head kidney, liver, spleen, intestine, gills, muscle and heart were tested for the presence of ISAV by reverse transcriptase polymerase chain reaction (RT-PCR). The results showed that during a disease outbreak, ISAV is present in most organs. It was possible to detect ISAV at all sampling times in at least 1 of the fish examined. However, for the first 8 d p.i. positive RT-PCR results were predominantly found in samples from the head kidney and mid-kidney. Fish giving positive samples after Day 13 p.i. were RT-PCR positive in most organs. These results indicated that between Days 8 to 13 p.i. considerable replication of the virus occurred, combined with wide tissue dissemination. (+info)
Aspects of the epizootiology of pancreas disease in farmed Atlantic salmon Salmo salar in Ireland.
(13/793)
A computerised database containing information on over 17.8 million salmon contained within 49 separate marine populations was used to study the epidemiology of pancreas disease (PD) in Ireland. Of the 43 recorded PD outbreaks, 57% occurred in the 3 mo period August to October inclusive (17 to 32 wk post-transfer). Analysis of variance of mortality rates during PD outbreaks occurring on 6 marine sites over a 5 yr period showed that mortality rates vary significantly between sites (p < 0.001) but not between years over this time period. The mortality rate during PD outbreaks ranged from 0.1 to 63%. Mortality rates were significantly higher when PD outbreaks occurred earlier in the year (y = -1.28x + 59, SE of b 0.33). The mean length of a PD outbreak was 112 d (SE = 7.7, n = 37). There was no correlation between PD mortality rate and smolt input weight, initial stocking density and transfer mortality. (+info)
Production of acylated homoserine lactones by psychrotrophic members of the Enterobacteriaceae isolated from foods.
(14/793)
Bacteria are able to communicate and gene regulation can be mediated through the production of acylated homoserine lactone (AHL) signal molecules. These signals play important roles in several pathogenic and symbiotic bacteria. The following study was undertaken to investigate whether AHLs are produced by bacteria found in food at temperatures and NaCl conditions commercially used for food preservation and storage. A minimum of 116 of 154 psychrotrophic Enterobacteriaceae strains isolated from cold-smoked salmon or vacuum-packed chilled meat produced AHLs. Analysis by thin-layer chromatography indicated that N-3-oxo-hexanoyl homoserine lactone was the major AHL of several of the strains isolated from cold-smoked salmon and meat. AHL-positive strains cultured at 5 degrees C in medium supplemented with 4% NaCl produced detectable amounts of AHL(s) at cell densities of 10(6) CFU/ml. AHLs were detected in cold-smoked salmon inoculated with strains of Enterobacteriaceae stored at 5 degrees C under an N(2) atmosphere when mean cell densities increased to 10(6) CFU/g and above. Similarly, AHLs were detected in uninoculated samples of commercially produced cold-smoked salmon when the level of indigenous Enterobacteriaceae reached 10(6) CFU/g. This level of Enterobacteriaceae is often found in lightly preserved foods, and AHL-mediated gene regulation may play a role in bacteria associated with food spoilage or food toxicity. (+info)
Efficacy of orally administered oxolinic acid and Vetoquinol, an oxolinic acid ester, for the treatment of furunculosis in Atlantic salmon Salmo salar held in seawater.
(15/793)
This study was performed to determine the efficacy of orally administered oxolinic acid and Vetoquinol, an oxolinic acid ester, in the treatment of experimental induced furunculosis in Atlantic salmon Salmo salar held in seawater. Two strains of the causative bacterium Aeromonas salmonicida subsp. salmonicida, 1 sensitive (VI-88/09/03175) and 1 resistant (3475/90) to oxolinic acid, were used. In 2 trials, cohabitational challenges were performed by introducing 8 fish challenged in advance by an intraperitoneal injection of 2.2 x 10(4) colony forming units of strain 3475/90 (Trial 1) or strain VI-88/09/03175 (Trial 2) to 10 aquaria each containing 40 healthy fish. The treatment groups in both trials consisted of 4 groups receiving either oxolinic acid (2 groups) or Vetoquinol (2 groups) and 1 control group. An unchallenged, unmedicated group was used to determine the natural mortality in the population. The recommended therapeutic dose of 25 mg oxolinic acid kg-1 fish at Days 1, 2, 4, 6, 8 and 10 following initiation of treatment was used. Oral medication initiated at Day 10 (Trial 1) or Day 11 (Trial 2) following challenge significantly (p < 0.05) lowered the specific mortality in all drug-treated groups compared to the untreated control groups. Mortality in Vetoquinol-treated groups was significantly (p < 0.05) lower than in oxolinic acid-treated groups in Trial 1 whereas no significant (p < 0.05) difference in survival rate was found between the medicated groups in Trial 2. (+info)
Inhibitory effects of protamines on proteolytic and adhesive activities of Porphyromonas gingivalis.
(16/793)
Protamines (salmine prepared from sperm DNA of salmon and clupeine from herring sperm), which are basic peptides rich in arginine, were found to inhibit the proteolytic activity of arginine-specific cysteine protease (RC-protease) from Porphyromonas gingivalis. Lineweaver-Burk plot analysis revealed that the protamines competitively inhibited proteolytic activity with cleavage of benzoyl-L-arginine-p-nitroanilide, a synthetic substrate of RC-protease. Furthermore, the protamines were capable of binding strongly to P. gingivalis fimbriae and inhibited fimbrial interaction with immobilized fibronectin. These results clearly show that protamines are potent inhibitors of the proteolytic and adhesive activities of P. gingivalis. (+info)