An aerolysin-like enterotoxin from Vibrio splendidus may be involved in intestinal tract damage and mortalities in turbot, Scophthalmus maximus (L.), and cod, Gadus morhua L., larvae.

Vibrio splendidus is a pathogen that can cause major losses during the early stages of larval turbot rearing when live feed (rotifers or Artemia) is used. As haemolytic bacteria have often been associated with larval rearing losses, we studied the role of the V. splendidus haemolysin in infection of larvae. From a bank of over 10,000 transposon mutants of V. splendidus, two different types of haemolysin-negative mutants were obtained. Both had lost virulence for larval fish, and immunohistochemistry showed that the transposon mutant studied colonized the turbot larval intestinal tract at a similar level to the wild-type organism but did not cause damage or signs of enteritis found with the wild-type organism. One transposon insertion site was located within a gene with high homology to aerolysin, the cytolytic toxin produced by several Aeromonas spp. The haemolysin, which we have termed vibrioaerolysin, had properties similar to aerolysin and osmotic protection studies showed that it formed pores in the membranes of erythrocytes of similar diameter to those of aerolysin. The Tn10 insertion site of the second transposon mutant was in an adjacent ToxR-like gene, suggesting that this might control expression of the vibrioaerolysin. The gastroenteritis caused by Aeromonas spp. in humans is considered to be due to production of aerolysin causing cyclic AMP-dependent chloride secretion in cells of the gastrointestinal tract. Damage to the intestinal tract of marine fish larvae could occur in a similar way, and it is possible that several Vibrio spp. found in the developing bacterial flora of the larval fish gut can secrete aerolysin-like toxins leading to death of larvae in the early rearing stages. Routine bacteriological screening on blood agar plates of live feed is recommended with measures to reduce the concentrations of haemolytic bacteria in rearing systems.

Francisella infections in fish and shellfish.

A series of recent reports have implicated bacteria from the family Francisellaceae as the cause of disease in farmed and wild fish and shellfish species such as Atlantic cod, Gadus morhua L., tilapia, Oreochromis spp., Atlantic salmon, Salmo salar L., three-line grunt, Parapristipoma trilineatum (Thunberg), ornamental cichlid species, hybrid striped bass Morone chrysops x M. saxatilis and, recently, a shellfish species, the giant abalone, Haliotisgigantea Gmelin. The range of taxa affected will very probably rise as it is likely that there has been considerable under-reporting to date of these disease agents. In common with other Francisella species, their isolation and culture require specialized solid and liquid media containing cysteine and a source of iron. This likely restricted earlier efforts to identify them correctly as the cause of disease in aquatic animals. The most information to date relates to disease in cod, caused by F. noatunensis and tilapia, caused by F. noatunensis subsp. orientalis (also termed F. asiatica), both causing granulomatous inflammatory reactions. Mortalities in both species can be high and, as the disease can likely be transferred via live fish movements, they pose a significant threat to tilapia and cod aquaculture operations. Although the fish-pathogenic Francisella species are classified in the same genus as the human pathogens F. tularensis, causative agent of tularemia, and F. philomiragia, the risk to humans from the fish and shellfish pathogenic Francisella species is considered very low.

Idiopathic muscle necrosis in the Norway lobster, Nephrops norvegicus (L.): aetiology, pathology and progression to bacteraemia.

The pathology and progression of idiopathic muscle necrosis (IMN) in Nephrops norvegicus and possible aetiologies have been investigated. Trawl capture, aerial exposure and handling initiate IMN, and the condition can be induced through periods of aerial exposure alone, in the absence of trawling. Within 24-48 h after trawl capture IMN progresses to a multi-species bacterial septicaemia, with moribund animals exhibiting clinical signs. The aetiology of this condition has been examined using molecular (16S rRNA gene sequencing) and biochemical (standard taxonomic assays, Biolog) criteria to characterize bacterial isolates from moribund and healthy animals. Histopathology of the IMN phase reveals a loss of sarcomeric structure with necrotic lesions containing pyknotic nuclei, fragments of myofibrils and connective tissue elements. In the bacterial phase there is extensive loss of abdominal muscle structure, and the presence of rod-shaped Gram-negative bacteria in the degrading tissues. The results demonstrate that the IMN condition is connected to stressful conditions imposed on N. norvegicus, but involves no pathogenic agents. This is followed by an opportunistic bacterial infection that causes further tissue spoilage. It is believed that the primary cause of both IMN and bacteraemia is imposed stress, but they are expressed in different time courses.

Vaccine-associated systemic Rhodococcus erythropolis infection in farmed atlantic salmon Salmo salar.

In 7 instances between 2000 and 2003, clinical investigation of populations of fresh- and seawater-reared, vaccinated, Atlantic salmon Salmo salar suffering total losses of between 0.1 and 35 % revealed infection with a Gram-positive rod-shaped bacterium. The isolations were geographically widespread, occurring in both Norway and Scotland. In all cases, a Gram-positive bacterium, subsequently identified as Rhodococcus erythropolis, was isolated in pure culture. Infections, although systemic, were focused within the peritoneal cavity. While initial attempts to reproduce the disease by intraperitoneal injection of unvaccinated Atlantic salmon failed, Koch's postulates were subsequently fulfilled in fish vaccinated with a commercially available oil-adjuvanted vaccine.

Vibrio splendidus biotype 1 as a cause of mortalities in hatchery-reared larval turbot, Scophthalmus maximus (L.).

AIMS: To characterize bacteria associated with turbot larvae feeding on Artemia and identify pathogens causing mortalities in larvae. METHODS AND RESULTS: To identify bacteria associated with mortalities in larval turbot rearing, bacteria were isolated from homogenates of Artemia or from several batches of well-performing or poorly performing turbot larvae. Samples were plated onto marine agar and were characterized using biochemical tests and BIOLOG GN plates. Total culturable aerobic bacteria ranged from 1.9 x 10(5) to 1.8 x 10(6) CFU per larva and >96% of bacteria identified were vibrios. Almost all bacteria were haemolytic and clustered into two phenons represented by Vibrio alginolyticus and Vibrio splendidus. The bacterial flora of Artemia was almost entirely V. alginolyticus, whereas V. splendidus biotype 1 dominated the larval turbot gut flora (69/115 isolates in seven experiments) and formed four different groups based on BIOLOG GN reactions. Of 16 isolates tested for virulence towards turbot larvae, four of the 11 V. splendidus biotype 1 isolates were lethal and all belonged to the same group of V. splendidus biotype 1 isolates. CONCLUSIONS: In a commercial turbot hatchery, the microbial flora of the larval gut was dominated by V. splendidus biotype 1. Four of the 11 V. splendidus biotype 1 isolates caused mortalities in larval turbot and all belonged to one group of the biotype 1 strains identified. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of four isolates of V. splendidus that are pathogenic for turbot larvae from three separate batches of larval turbot will allow these to be compared with avirulent isolates to define how V. splendidus causes mortalities in larval turbot.

Isolates of Piscirickettsia salmonis from Scotland and Ireland show evidence of clonal diversity.

Salmonid rickettsial septicemia, caused by Piscirickettsia salmonis, causes major mortalities in Chilean salmonid aquaculture and is an increasing problem in Atlantic salmon in Ireland and Scotland. Analysis of 16S-to-23S internal transcribed sequences and 16S ribosomal DNA (rDNA) shows that Irish isolates of P. salmonis form two new groups of the organism while Scottish isolates cluster together with Norwegian and Canadian isolates from Atlantic salmon.

Salinity effects on immune parameters of Ruditapes philippinarum challenged with Vibrio tapetis.

The occurrence of brown ring disease (BRD) in farmed Manila clams Ruditapes philippinarum is seasonal. Development of the disease is believed to require the presence of the infective agent Vibrio tapetis and particular environmental conditions. This paper studies the effect of salinity (20 to 40 per thousand) on measurable immune parameters of Manila clams, and the progression of BRD in experimentally infected individuals. At 20 per thousand salinity, the total haemocyte count was reduced and disease prevalence was highest. At 40 per thousand salinity significantly fewer clams presented signs of BRD, and this was correlated with increases in the total haemocyte count, hyalinocyte count, phenoloxidase levels and phagocytic activity of haemocytes. Inoculation of clams with V. tapetis did not have a significant effect on the immune parameters measured. Thus, this laboratory-based study relates environmental stress to disease development.

Pasteurella skyensis sp. nov., isolated from Atlantic salmon (Salmo salar L.).

From four separate incidents of disease in farmed Atlantic salmon over a four-year period, gram-negative rod-shaped bacteria were consistently isolated by culture on sea-water blood agar. Biochemical and physiological tests indicated that the organism was related to the family Pasteurellaceae, and this was confirmed from the 16S rRNA gene sequence. Comparison of the 16S rRNA gene sequence with those of 45 members of the Pasteurellaceae showed that the closest phylogenetic relationship was with an organism termed 'Pasteurella phocoenarum', isolated from a porpoise, for which the 16S rRNA gene sequence has been recorded but for which the properties have yet to be published. It is proposed that this bacterium isolated from salmon should be classified as a new species, namely Pasteurella skyensis sp. nov. The type strain of Pasteurella skyensis sp. nov. is strain 95A1T (= NCTC 13204T = NCIMB 13593T).

Comparison of paralytic shellfish toxin (PST) production by the dinoflagellates Alexandrium lusitanicum NEPCC 253 and Alexandrium tamarense NEPCC 407 in the presence and absence of bacteria.

The ability of two Alexandrium species to produce paralytic shellfish toxins (PST) in laboratory culture following the generation of bacteria-free cultures was investigated. The dinoflagellates Alexandrium lusitanicum NEPCC 253 and Alexandrium tamarense NEPCC 407 were cultured in the presence of antibiotics and tested for residual bacteria. After treatment with a cocktail of streptomycin, ciprofloxacin, gentamicin and penicillin G, bacteria could not be detected in either of the treated Alexandrium cultures using 17 different solid and broth bacterial growth media, by epifluorescence microscopy with the dye Sybr green 1, or polymerase chain reaction amplification using universal eubacterial primers designed to target the 16S rRNA gene. Subsequent analysis of A. lusitanicum for PST using high performance liquid chromatography demonstrated that the growth rate and toxin profile remained similar in both bacteria-free and control cultures, although the quantity of toxins produced differed with the bacteria-free culture producing generally more of each compound and also having a greater toxin content in terms of saxitoxin equivalents. A. tamarense also retained similarities between the bacteria-free and control cultures in terms of growth rates and toxin profile, although in this instance, depending on the growth stage and the toxin, the control culture produced more of some toxins than the bacteria-free culture. The control culture was also more toxic in terms of saxitoxin equivalents than the axenic culture. These results suggest that bacteria can influence toxin production in laboratory cultures of Alexandrium species although the mechanisms remain unknown.

Suppression of the humoral immune response of Atlantic salmon, Salmo salar L. by the 64 kDa serine protease of Aeromonas salmonicida.

Bacteria-free supernatants of broth cultures of Aeromonas salmonicida inhibited the humoral immune response, but not the cell-mediated immune response, of Atlantic salmon to bacteriophage MS2. The immunosuppressive factor was the 64 kDa serine protease secreted by A. salmonicida. The suppressive activity was not due to degradation of epitopes of MS2, and although serine protease degraded the heavy chain of salmon IgM in vitro there was no evidence for significant degradation in vivo. The principal lethal toxin of A. salmonicida, the glycerophospholipid: cholesterol acyltransferase did not inhibit the immune response of salmon.

Comparison of the Growth and Survival of Larval Turbot in the Absence of Culturable Bacteria with Those in the Presence of Vibrio anguillarum, Vibrio alginolyticus, or a Marine Aeromonas sp.

Larval turbot (Scophthalmus maximus) were reared on rotifers (Brachionus plicatilis) in the absence of culturable bacteria for up to 14 days and exhibited growth and high rates of survival (>55% in five experiments). Low numbers of known bacteria were introduced into similar cultures by exposure of the rotifers to a suspension of bacteria prior to addition of rotifers to the larval cultures; Vibrio anguillarum 91079 caused a highly significant decrease (P <0.01) in the proportion of survivors in two separate trials. With an Aeromonas sp. previously isolated from a healthy batch of copepod-fed larvae, there was no significant difference in survival compared with control larvae, even though the density of bacteria in the water of larval cultures reached 10(sup7) ml(sup-1). Bacteria colonized the gut of larvae exposed to Aeromonas-treated rotifers to levels similar to those in conventionally reared fish (>4 x 10(sup4) CFU per larva). Rearing of larvae in the presence of known bacteria provides a means of investigating the interaction of specific bacteria with turbot larvae and could provide a method for the selection of bacteria which may restrict the growth of opportunistic pathogens which would be harmful to turbot larvae.

Short communication: characterization of streptolysin s (sls): storage and stability.

The stability of streptolysin S during storage under different conditions has been studied. It is completely stable in 0.1% (w/v) bovine serum albumin supplemented with 20% (v/v) glycerol at -20 degrees C for more than 6 months. The in vitro study suggests that removal of SLS from cellular environment subjects it to variety of conditions and processes that can lead to loss of activity.

Effect of phosphate concentration on production of tetrodotoxin by Alteromonas tetraodonis.

Tetrodotoxin production by Alteromonas tetraodonis occurred during the stationary phase of growth and was regulated by phosphate concentration; toxin production was repressed if phosphate was added at the onset of stationary phase and was over 100-fold greater in phosphate-limited cultures than in cultures in which phosphate was not limiting.

Characterization of staphylococcal gamma-lysin.

gamma-Lysin was purified from Staphylococcus aureus strains Smith 5R and PG23 (a toxic shock syndrome isolate) by a combination of heparin-agarose and hydroxylapatite chromatography. Both strains produced two haemolytic components, designated gamma 1 and gamma 2. Though each component was weakly haemolytic they acted synergistically to potentiate haemolysis on rabbit, sheep and human blood. Rabbit and sheep erythrocytes were more sensitive to lysis by gamma-lysin than human erythrocytes. The molecular mass of gamma 1 was 32 kDa and its pI value was 9.4. gamma 2 had a molecular mass of 36 kDa and a pI value of 9.3. While both trypsin and papain acted synergistically with gamma 2 to induce increased haemolysis, no such synergism was seen with gamma 1. Also, protease inhibitors acted to inhibit synergism between gamma 1 and gamma 2. These findings suggest that gamma 1 could be a protease.