Infectivity of a Scottish isolate of Piscirickettsia salmonis for Atlantic salmon Salmo salar and immune response of salmon to this agent.

A Scottish isolate of Piscirickettsia salmonis (SCO-95A), previously shown by intraperitoneal injection to have a lethal dose (LD50) of < 2 x 10(3) infectious rickettsial units, was tested for virulence by bath challenge, surface application to the skin, or dorsal median sinus injection. Atlantic salmon Salmo salar post-smolts were used in all experiments, and exposure to 1 x 10(5) tissue culture infective doses (TCID) of P. salmonis ml(-1) for 1 h in a bath challenge resulted in only 1 mortality, 18 d later, in 10 exposed fish. Application of 2.5 x 10(6) TCID of P. salmonis SCO-95A to paper discs on the skin failed to induce any mortalities within 42 d. Intraperitoneally, fish were administered vaccines containing 10(9) heat-inactivated (100 degrees C, 30 min) or 10(9) formalin-inactivated P. salmonis SCO-95A in adjuvant, with a control group receiving phosphate-buffered saline (PBS) in adjuvant. After an induction period of over 6 mo fish were challenged by injection of P. salmonis into the dorsal median sinus. Mortalities in the control group reached 81.8% and the heat-inactivated and formalin-inactivated vaccines gave significant protection from P. salmonis, with relative percentage survivals of 70.7 and 49.6%, respectively. The nature of the protective antigen is unknown, but could be lipopolysaccharide or a heat-stable outer membrane protein. Fish that survived a dorsal median sinus challenge of P. salmonis or were cohabitants showed a strong immune response to P. salmonis.

Growth of Piscirickettsia salmonis to high titers in insect tissue culture cells.

Piscirickettsia salmonis was grown in established insect, frog, and fish tissue culture cells. The yield of P. salmonis in Sf21 cells was up to 100 times that obtained in CHSE-214 cells, and virulence for Atlantic salmon was retained. The ceiling temperature for growth of P. salmonis in Sf21 cells was 24 degrees C.

Bacterial influences on Atlantic halibut Hippoglossus hippoglossus yolk-sac larval survival and start-feed response.

A bacteria-free halibut larval rearing system was used to test 20 bacterial isolates, from British halibut hatcheries, for their toxicity towards halibut yolk-sac larvae under microbially controlled conditions. The isolates tested spanned a range of genera and species (Pseudoalteromonas, Halomonas marina, Vibrio salmonicida-like, Photobacterium phosphoreum and V. splendidus species). A pathogen of turbot, Scophthalmus maximus, V. anguillarum 91079, and 2 isolates from adult halibut were also included. Isolates were inoculated, at a concentration of 5 x 10(2) cfu ml(-1), into flasks containing 25 recently hatched axenic halibut larvae, using a minimum of 3 flasks for each treatment. Control survivals to 38 d post-hatch for the 3 experiments averaged 84, 51.5 and 49%, respectively. With the exception of V. anguillarum 91079, which was highly pathogenic towards halibut yolk-sac larvae, there was no statistically significant difference in survival between the controls and the different treatments. This suggests that most of the bacteria routinely isolated from halibut hatcheries are not harmful to yolk-sac larvae, even though most flasks contained in excess of 5 x 10(6) cfu m(-1) of the inoculated organism when the experiments were terminated. Three organisms previously shown to inhibit growth of bacteria in vitro were tested for their ability to protect halibut yolk-sac larvae against invasion by V. anguillarum. In 4 separate challenge experiments none of the test isolates, a Pseudoalteromonas strain and 2 Carnobacterium-like organisms, showed any protective effect. To investigate how particular bacteria influence their start-feed response, larvae were fed axenic and gnotobiotic Artemia colonized with a range of different Vibrio spp., and examined after 8 d. There were no statistically significant between-treatment differences in the proportion of Artemia-containing larvae, indicating that bacterial contamination of the live food does not appear to influence initiation of the feeding response.

Mechanism and kinetics of delta-lysin interaction with phospholipid vesicles.

Delta-lysin is a 26 amino acid, hemolytic peptide toxin secreted by Staphylococcus aureus. It has been reported to form an amphipathic helix upon binding to lipid bilayers and is often cited as a typical example of the barrel-stave model for pore formation in lipid bilayer membranes. However, the exact mechanism by which it lyses cells and the physical basis of its target specificity are still unknown. Moreover, the evidence for delta-lysin insertion and pore formation in the membrane stems largely from theoretical modeling of the toxin and lacks experimental confirmation. We investigated binding and insertion of delta-lysin into phospholipid bilayer vesicles. The kinetics of these processes were studied by stopped-flow fluorescence with two types of experiments: (a) carboxyfluorescein release from the vesicles upon peptide-vesicle interaction, with concomitant relief of dye self-quenching; (b) fluorescence energy transfer from the intrinsic tryptophan of the peptide to a membrane-bound lipid probe. We formulated a detailed kinetic mechanism with explicit molecular rate constants for peptide binding, association, and insertion, obtaining a quantitative description of the experimental results. delta-Lysin insertion is strongly dependent on the peptide-to-lipid ratio, suggesting that association of a critical number of monomers on the membrane is required for activity. However, we found no evidence for a stable membrane-inserted pore. Rather, the peptide appears to cross the membrane rapidly and reversibly and cause release of the lipid vesicle contents in this process.