Journal of Fish Diseases 2012, 35, 153167

doi:10.1111/j.1365-2761.2011.01331.x

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
H L Macpherson1, Bergh2,3 and T H Birkbeck1
1 Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK 2 Institute of Marine Research, Bergen, Norway 3 Department of Biology, University of Bergen, Norway

Abstract

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 haemolysinnegative mutants were obtained. Both had lost virulence for larval sh, 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 wildtype 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
Correspondence T H Birkbeck, University Marine Biological Station, Millport, Isle of Cumbrae, Scotland KA28 0EG, UK (e-mail: h.birkbeck@bio.gla.ac.uk) DNA sequences have been deposited with EMBL under accession number AM157713.

aerolysin causing cyclic AMP-dependent chloride secretion in cells of the gastrointestinal tract. Damage to the intestinal tract of marine sh larvae could occur in a similar way, and it is possible that several Vibrio spp. found in the developing bacterial ora of the larval sh 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. Keywords: aerolysin, Gadus morhua, gut microora, larval rearing, Scophthalmus maximus, Vibrio splendidus, vibrioaerolysin.
Introduction

The worldwide demand for sh cannot now be met from wild sheries alone, and aquaculture has expanded rapidly in the past three decades to meet this demand (FAO 2006). Although the majority of aquaculture production is from freshwater, production of sh in marine environments now exceeds 17 million tonnes per annum (FAO 2006). There is increasing interest in culturing other species such as turbot, Scophthalmus maximus (L.), halibut, Hippoglossus hippoglossus (L.), and cod, Gadus morhua L., but this requires a signicant increase in the sand, Ottera & Taranger supply of juvenile sh (Sva 2004). Large losses still occur in larval rearing (Vadstein, Mo & Bergh 2004), and bacteria are considered to play a major role in such losses because administration of antibiotics can enhance

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H L Macpherson et al. Aerolysin-like toxin from Vibrio splendidus

survival (Gatesoupe 1982, 1989; Verner-Jeffreys, Shields, Bricknell & Birkbeck 2004) and culture of turbot larvae in a bacteria-free rearing system can produce very high survival (Munro, Barbour & Birkbeck 1995). Bacterial species linked with mortalities in larval turbot rearing include Vibrio splendidus (Gatesoupe, Lambert & Nicolas 1999; Thomson, Macpherson, Riaza & Birkbeck 2005), Vibrio pelagius (Villamil, Figueras, Toranzo, Planas & Novoa 2003) and Vibrio harveyi (Sun, Zhang, Tang, Wang, Zhong, Chen & Austin 2007). Of these, V. splendidus has been isolated from several different batches of turbot larvae over a number of years (Gatesoupe et al. 1999; Thomson et al. 2005). The pathogenic mechanisms of sh-pathogenic vibrios have so far been largely restricted to an analysis of Vibrio anguillarum (Weber, Croxatto, Chen & Milton 2008) and V. harveyi (Sun et al. 2007). Vibrio anguillarum has been studied extensively, and virulence determinants such as an ironsequestering system (Crosa 1980), metalloprotease (Milton, Norqvist & Wolf-Watz 1992) and agella rstedt & Wolf-Watz 1996; (Milton, OToole, Ho rstedt & Wolf-Watz 1997) OToole, Milton, Ho have been identied, yet the causes of mortalities by this organism are still not completely understood. V. harveyi has been shown to produce a lethal, haemolytic phospholipase, and site-directed mutagenesis to inactivate this enzyme causes loss of lethality in turbot (Sun et al. 2007). Proteases produced by Vibrio alginolyticus (Nottage & Birkbeck 1987) and metalloprotease produced by V. splendidus (Le Roux, Binesse, Saulnier & Mazel 2007; Binesse, Delsert, Saulnier, Champomier`s, Zagorec, Munier-Lehmann, Mazel & Le Verge
Table 1 Bacterial strains used in this study
Bacterial strains and characteristics Escherichia coli S17-1kpir Escherichia coli SM10kpir Vibrio splendidus DMC-1 Vibrio splendidus DMC-1-M2, DMC-1-M3 and DMC-1-M4 Vibrio splendidus DMC-2, DTC-5, DTR-2, DTY-1, LTS-3, LTS-4, LMS-1, LMS-2, LMS-3, LMS-4, LTH-1, LTH-3, LTH-4, HNF-8 Vibrio anguillarum 91079 Roseobacter sp. HNF-1

Roux 2008) have been shown to be lethal to oyster larvae. Routine culture of larval turbot on a large scale without recourse to the use of antibiotics could be achieved if the microbial ora was regulated (Munro et al. 1995; Verschuere, Rombaut, Sorgeloos & Verstraete 2000; Birkbeck 2004; Gram & Ring 2004) and growth of pathogens prevented. With this in mind, we have begun a study of the mechanisms of pathogenesis of V. splendidus in larval turbot and cod. Here, we have used transposon mutagenesis to prepare V. splendidus mutants decient in haemolysin production. We show that a haemolysin-decient mutant is not virulent to larval turbot and does not cause the enteritis associated with infections caused by the wild-type organism. In addition, the DNA sequence of the haemolysin gene has been determined, and this shows the haemolysin to be unrelated to those known to be produced by Vibrio species but to be closely related to aerolysin (Parker, van der Goot & Buckley 1996), the rst time that expression of this type of toxin has been detected outside of the Aeromonas genus.

Materials and methods

Bacteria and transposon Tn10 The bacteria used in this study are shown in Table 1. V. splendidus DMC-1 was of biovar 1 (Thomson et al. 2005) and was resistant to ampicillin (25 lg), tetracycline (25 lg) and streptomycin (10 lg) but sensitive to chloramphenicol (50 lg) and kanamycin (30 lg) when screened with Mastring S discs (Mast Diagnostics Ltd).

References Tp Sm recA thi pro hsd R M RP4-2-Tc::Mu::Km Tn7 kpir thiL thrL leuB6 supE44 tonA21 lacY1 recA::RP4-2-Tc::Mu Kmr Pathogen of larval turbot isolated from a hatchery in Spain Haemolysin-negative Tn10- transposon mutants of DMC-1 Isolated from larval turbot in Spain
r r +

Simon, Priefer & Puhler (1983) Simons, Houman & Kleckner (1987) Thomson et al. (2005) This study Thomson et al. (2005)

Isolated from moribund juvenile turbot in the U.K. Isolated from turbot larvae from a hatchery in Spain

Horne, Richards, Roberts & Smith (1977) Thomson et al. (2005)

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The transposon Tn10 located in the plasmid vector pBSL181 (Alexeyev & Shokolenko 1995) was kindly supplied by Dr J.B. Andersen, Biocentrum, Danish Technical University, Denmark, in the host cell Escherichia coli S17-1kpir, and puried pBSL181 was transferred by electroporation into Escherichia coli Sm10kpir to provide a more convenient host strain for use in mutagenesis based on antibiotic selection. Cultures were stored at )85 C on Protect beads (Technical Service Consultants) with 100 lL sterile 20% NaCl solution added to each vial to increase the salinity to 3% (w/v). Culture of bacteria Vibrio splendidus DMC-1 was cultured in 50 mL Zobell 2216E marine broth (Difco) in bafed 250mL Erlenmeyer asks, shaken at 150 oscillations per min at 20 C for 16 h in a cooled orbital incubator. The culture supernatant was obtained by centrifugation at 9000 g for 10 min. Concentrated culture supernatant was prepared from eight 500 mL marine broth cultures in bafed 2-L Erlenmeyer asks cultured as above, and the supernatant was concentrated to 90 mL using a peristaltic pump and Filtron Omega (VWR) minisette unit with a 10-kDa Omega membrane cassette. In this way, the haemolysin titre was increased from 128 to 4096 HU mL)1. Haemolysin assays Sheep, horse and rabbit blood were obtained from commercial sources (E and O Laboratories). Turbot and salmon blood were collected freshly into an equal volume of 3.8% sodium citrate from adult turbot or salmon reared at local sh farms. Mammalian erythrocytes were washed twice in phosphate-buffered saline (PBS; Dulbecco & Vogt 1954), and sh erythrocytes were washed twice in PBS + 2% NaCl before resuspension to 1% v/v in PBS or PBS + 2% NaCl, respectively. Haemolysin titrations were carried out in a doubling dilution series (1/21/64) in 100 lL volumes in 96-well microtitre plates in PBS or PBS + 2% NaCl; 100 lL of erythrocyte suspension was added to each well including PBS as a negative control and 1% saponin as a positive control to produce 100% haemolysis. Plates were incubated at 20 C for 1 h, and the titration end point was taken as the highest dilution to yield approximately 50% lysis of
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erythrocytes. One haemolytic unit (HU) was dened as the amount of haemolysin that caused 50% haemolysis in 200 lL of a 0.5% suspension of erythrocytes. Osmotic protection studies on haemolysintreated erythrocytes To determine whether haemolysin-treated erythrocytes underwent colloid osmotic lysis, the method of Weiner, Schneider, Haest, Deuticke, Benz & Frimmer (1985), as modied by Knowles & Ellar (1987), was used. If pores (channels) are formed in a cell membrane, the cells become permeable to ions and will swell and lyse when suspended in an isotonic solution of a solute that can permeate the pores. The rate of lysis is a function of the rate at which the solute can enter the cell but solutes that are too large to penetrate the pores will act as osmotic protectants, inhibiting cell swelling and lysis. The size of the pores is measured by comparison with the reciprocal of the half-time of lysis of cells suspended in solutes of known viscometric radius. Briey, 2.5 mL volumes of a 0.4% suspension of washed sheep erythrocytes were mixed with equal volumes of 30 mm solutions of polyethylene glycol (PEG 300, PEG 400, PEG 600, PEG 1000 and PEG 1500) in PBS and allowed to equilibrate at room temperature for 10 min before addition of 100 lL concentrated culture supernatant (titre 1024 HU mL)1) to give a nal haemolysin concentration of approximately 100 HU mL)1. A duplicate set of erythrocyte/ PEG solution mixtures was used as controls, and 100 lL PBS was added to each of these tubes. After gentle vortexing to mix the suspensions, A600 was measured for each tube at 2-min intervals for 20 min and then every 10 min thereafter for 1 h. From graphs of A600 nm vs. time, the time to reach 50% lysis of erythrocytes (t50) was measured for each solution of PEG, and a graph of 1/t50 against the viscometric radius of each PEG fraction was plotted. Extrapolation of this line to the viscometric radius axis allowed the radius of the pore formed in the membrane to be estimated. Viscometric radius values were obtained from Kuga (1981) (PEG 300, 0.48 nm; PEG 400, 0.56 nm; PEG 600, 0.69 nm; PEG 1000, 0.89 nm and PEG 1500, 1.1 nm, respectively). Results were the mean values from three separate experiments. Puried proaerolysin, a kind gift from Professor T. Buckley, was activated by treatment

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with trypsin (Howard & Buckley 1985a) and was tested similarly. Turbot tissue culture cell assays The turbot tissue culture cell line TV1-S4 (Fernndez-Puentes, Figueras & Novoa 1993) was kindly a supplied by Dr C. Dopazo, University of Santiago, Spain, and cells were maintained in Eagles Minimal Essential Medium (Invitrogen) with 10% new born calf serum and 2 mm glutamine at 15 C. Concentrated lter-sterilized supernatant from a 24-h culture of V. splendidus DMC-1 (haemolysin titre of 1024 HU mL)1) was diluted from 1/2 to 1/ 2560 in the above medium and added to monolayer cultures of TV1-S4 cells in a 24-well cell culture plate (Nunc, Fisher Scientic). Assay plates were examined using an inverted microscope, and cells were assessed visually hourly for the rst 4 h and again after 24 h for cytopathic effects. SDS-PAGE SDS-PAGE was carried out by the method of Laemmli (1970) using Novex 12% acrylamide BisTris 8 8 cm mini-gels (Invitrogen). Transposon mutagenesis Vibrio splendidus DMC-1 was subjected to mutagenesis with the transposon Tn10 located in the plasmid vector pBSL181 in E. coli Sm10kpir. Optimization of conditions for transposon mutagenesis led to the following protocol. The donor strain E. coli SM10kpir was grown overnight in 50 mL LB broth in a bafed 250-mL Erlenmeyer ask at 37 C and V. splendidus DMC-1 was grown overnight in 50 mL marine broth in a bafed 250mL Erlenmeyer ask at 20 C. The optical density of each culture was measured to estimate cell concentrations and appropriate dilutions spread on LB or marine agar plates to establish actual cell concentrations. The donor strain, E. coli SM10kpir, was spotted on to the surface of agar plates (LB agar containing 1.5% NaCl and 10 mm MgSO4 (LB15 + MgSO4) in 20 lL volumes and the plates dried in a laminar ow hood. The recipient strain, V. splendidus DMC-1, was then spotted on top of the donor strain in the same volume and the plates dried again. Several ratios were set up based on estimated cell concentrations to obtain an actual ratio as close as possible to 1:1. After overnight
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incubation at 20 C and once the viable counts for each strain had been established, only the plates with a viable count ratio of 1:1 were processed further. The bacterial growth was scraped from the plate surfaces and resuspended in microfuge tubes in Nine Salts Solution (NSS, Sambrook, Fritsch & Maniatis 1989) +5 mm MgSO4. The bacterial suspension was spread on selective plates, marine agar containing 5% sheep blood, 50 lg mL)1 chloramphenicol and 100 lg mL)1 ampicillin, and incubated at 20 C. Plates were inspected daily for 5 days, and non-haemolytic colonies were picked, Gram-stained, inoculated on to thiosulphate citrate bile salts sucrose agar, subjected to the oxidase test and tested with antiserum to V. splendidus to conrm their identity. Conrmation of transposon insertion in haemolysin-negative mutants Genomic DNA was obtained from bacteria using a Wizard Genomic DNA Purication Kit (Promega). The presence of transposon Tn10 in mutants was conrmed by PCR on genomic DNA with E. coli Sm10kpir (pBSL181:miniTn10Cm) and V. splendidus DMC-1 acting as positive and negative controls, respectively, with cat forward (5GCGTGTTACGGTGAAAACCT-3) and cat reverse (5- ATCACAAACGGCATGATGAA-3) primers (Alton & Vapnek 1979) specic for the chloramphenicol resistance cassette in Tn10. To identify the site of transposon insertion in the V. splendidus mutants, puried genomic DNA was digested with SphI and the DNA fragments ligated into SphI-digested and dephosphorylated pUC18 (Sambrook et al. 1989). The ligated plasmid was transformed into E. coli TOP10 cells using the manufacturers instructions (Invitrogen), and bacteria were cultured on LB agar plates containing 50 lg mL)1 chloramphenicol and 100 lg mL)1 ampicillin, to select only clones containing pUC18 with Tn10 inserts. Plasmid purication and DNA sequencing Plasmids were puried using a Quiaprep Purication Kit (Qiagen), and DNA sequencing was performed by DBS Genomics at the University of Durham, using the PRIDM DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems) and an Applied Biosystems 373A. Primers used were initially M13F (5-GTAAAACGACGGCCAGT-

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H L Macpherson et al. Aerolysin-like toxin from Vibrio splendidus

3) and M13R (5-AACAGCTATGACCATG-3) (Messing 1983), but to extend the V. splendidus sequences surrounding the transposon insertions primers were designed based on the sequences determined so far. DNA sequences were analysed and contigs aligned using the Genebuilder sequence analysis programme. Sequence alignments The vibrioaerolysin amino acid sequence was aligned with other aerolysin amino acid sequences using the DIALIGN program from the MRC RFCGR Bioinformatics Applications. Evolutionary analyses were conducted in MEGA4 (Tamura, Dudley, Nei & Kumar 2007), and the evolutionary history was inferred by using the maximum likelihood method based on the JTT matrix-based model (Jones, Taylor & Thornton 1992). Incidence of the vibrioaerolysin gene in isolates of Vibrio splendidus In addition to V. splendidus DMC-1, 14 other V. splendidus strains (Table 1), V. anguillarum strain 91079 (Table 1), and Roseobacter strain HNF-1 (Table 1) were selected and their chromosomal DNA extracted using a commercial kit (Biorad). PCR and agarose gel electrophoresis were carried out as described above using primers specic for the vibrioaerolysin gene (DMC-1 Hly forward, 5-CAACTCGAATCGGAAGCTCT-3 and DMC-1 Hly reverse, 5-AGCCGAAGAGCAAAAGAGTG-3) to amplify an 800-nt product from V. splendidus DMC-1. Effect of bacteria on larval turbot Three trials were carried out at the Institute of Marine Research, Bergen, in which larval turbot were exposed to V. splendidus or the transposon mutants derived from strain DMC-1. In the rst two trials on turbot yolk-sac larvae, eggs were obtained from the hatchery of Stolt Sea Farms, Kvinesdal, Norway The challenge procedure was as described by Sandlund & Bergh (2008) and Sandlund, Rdseth, Knappskog, Fiksdal & Bergh (2010), with adaptations (temperature and bacterial concentrations). Briey, eggs were acclimatized to 16 C and distributed individually into the wells of 24-well polystyrene dishes (Nunc) containing 2 mL autoclaved sea water (30& salinity). Immediately
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after distributing the eggs, 100 lL sterile sea water or bacterial suspension (1 106 or 1 108 mL)1 for trial 1 and 1 105 or 1 107 mL)1 for trial 2) was added to each well for control and test groups, respectively. Seventy-two larvae were assigned to each group (three multi-well dishes). Survival or death of each larva was assessed daily for 6 days post-hatch. As the survival of each larva was independent of the survival of larvae in other wells, it was assumed that the data were binomially distributed and differences in mortalities on individual days were tested by the Chi-square contingency table test using the Minitab program. For the third trial, turbot larvae (1 day posthatch) were obtained from the above hatchery, and approximately 1500 larvae were introduced into each of six 160-L tanks containing 42 L of fullstrength (32& salinity) sea water containing Nannochloropsis (Reed Mariculture) to create a green water system (Alderson & Howell 1973) aerated with two airstones (1 4 cm). The water temperature was maintained between 15.7 and 17.1 C, and 0.5 g wet paste of Nannochloropsis was added to each tank on days 13 and 68. A rotifer (Brachionus plicatilis) suspension (13 L containing 600 rotifers mL)1) supplied by Julie Skadal, University of Bergen, was cultured in 80-L conical tanks containing 42 L of 22& salinity sea water at 24 C. Rotifers were maintained by daily feeding with 1 g fresh yeast per million rotifers and 0.1 g DC DHA Selco (INVE) g)1 of yeast with numbers of rotifers counted and recorded daily. For bacterial challenges, V. splendidus DMC-1 and the haemolysin-negative mutants DMC-1-M2 and DMC-1-M3 were precultured for 24 h in marine broth and 0.4 mL was used to inoculate 20 mL marine broth in conical asks at 20 C shaken at 100 oscillations per min for 24 h. Bacteria were harvested by centrifugation, washed twice with sterile sea water and resuspended in sterile sea water to a cell density of approximately 2 108 cfu mL)1 (A600 nm = 0.3). Cell concentrations were veried by plate counts on marine agar plates at 18 C. Rotifers from the growth vessel were rinsed with 22& salinity sea water, and the suspension was adjusted for daily feeding to turbot larvae from day 2 post-hatch. When required, bacteria were added to the rotifer suspension to a concentration of 1 107 cfu mL)1 (from plate counts, the actual concentrations of bacteria added were DMC1 = 5 107 and 7 107, DMC-1-M2 = 7.5

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H L Macpherson et al. Aerolysin-like toxin from Vibrio splendidus

107 and 4.4 107, respectively, on the two challenge days) and were incubated with rotifers for 1 h at 20 C. Bacteria-inoculated rotifers were fed to larvae at a concentration of 2 rotifers mL)1 on days 4 and 5 post-hatch, and thereafter, the density of rotifers was maintained at 46 rotifers mL)1 by daily addition. Although six tanks were set up initially, one tank crashed on day 2 and only one control group was present in this trial. Effect of bacteria on larval cod As turbot larvae were not readily available, the fourth trial was carried out in Glasgow with cod larvae as these are also susceptible to V. splendidus DMC-1 (Reid, Treasurer, Adam & Birkbeck 2009). Larvae (14 days post-hatch) were obtained from Viking Sea Farms, Ardtoe, and were challenged with bacteria as described by Reid et al.(2009). Briey, larvae were dispensed individually into the wells of 6-well multidishes in 10 mL sea water. Rotifer suspensions were ltered, rinsed with 22& salinity sea water and adjusted to a concentration of 50 rotifers mL)1 with 1 107 cfu mL)1 added bacteria, which were prepared as described above (actual concentrations of DMC-1 and DMC-1-M4 = 12 107 on the three challenge days). Each well received 1 mL of rotifer suspension containing no added bacteria (control), DMC-1 or DMC-1-M4 on days 1, 2 and 3 of the trial with all wells receiving rotifers with no added bacteria on days 4 and 5. Ten 6-well plates were set up for control larvae and 5 for V. splendidus DMC-1 and DMC-1-M4. Larvae were inspected daily and mortalities recorded when the trial was terminated on day 6 post-infection. Immunohistochemistry In the third trial, samples of larvae were taken daily and processed for immunohistochemistry as described by Engelsen, Sandlund, Fiksdal & Bergh (2008). Briey, larvae were xed in 3.7% phosphate-buffered formaldehyde at pH 7.0 for at least 24 h, dehydrated in ethanol, embedded in parafn and serially sectioned at 3 lm. Sections were dewaxed in xylene and rehydrated in an ethanol bath series before washing in running water. As primary antibody, a rabbit antiserum made against V. splendidus DMC-1, diluted to 1:2500, was used. Positive staining was visualized with the ABC complex reaction kit by DAKO (New Fuchsin
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Substrate System), and haematoxylin was used for counterstaining. Sections were examined using a Leica DMBE microscope and photographs taken using a Leica Wild MPS52 phototube attached to the microscope.

Results

Haemolytic and cytotoxic activities in Vibrio splendidus DMC-1 culture supernatant uid Haemolytic activity in V. splendidus DMC-1-ltered bacterial culture supernatant from marine broth was detected against turbot (>1/64), salmon (1/128), sheep (1/128), horse (1/128) and rabbit (1/256) erythrocytes, and activity was destroyed by treatment at 100 C for 10 min. Culture supernatant was concentrated to a titre of 1024 HU mL)1 and this was highly cytotoxic towards turbot TV1S4 tissue culture cells. At a dilution of 1/160, it caused dendritic elongations in the cells, cell rounding, clustering of the rounded cells and, nally, detachment of cells from the plastic surface. Preparation of transposon mutants of Vibrio splendidus After optimization of conditions for mutagenesis, over 10 000 transconjugants were screened for loss of haemolytic activity and 3 hly- mutants (termed DMC-1-M2, DMC-1-M3 and DMC-1-M4) were obtained. All 3 hly- mutants, but not the parent strain, were shown by PCRs to contain the chloramphenicol resistance cassette associated with Tn10, showing the presence of the transposon in genomic DNA. Biochemical and physiological properties of the haemolysin-negative mutants The growth rate of the mutants in marine broth was indistinguishable from that of the wild-type parent strain over 48 h, and the protein contents of the culture supernatants of wild-type DMC-1 and the mutants DMC-1-M2, DMC-1-M3 and DMC-1M4 were similar at 58, 58, 66 and 64 lg mL)1, respectively, with haemolysin titres of 1/256 for the wild type and none detected for the mutants. Against monolayer cultures of TVS1-S4 turbot tissue culture cells, the culture supernatants of the hly- mutants lacked cytotoxicity, even when tested at a dilution of 1/2. When concentrated bacterial

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culture supernatants were compared by SDSPAGE, no differences in banding patterns could be seen (results not shown), and identical results were obtained for the hly- mutants and DMC-1 for the enzymes detected using the API-ZYM system (alkaline phosphatase, C4 esterase, C8 esterase lipase, leucine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, b-galactosidase, a-glycosidase and N-acetyl-b-d-glucosaminidase). The only difference detected between the wildtype and mutant strains was in production of cytotoxin active against tissue culture cells and erythrocytes. Identication of the transposon insertion site in the mutants DMC-1-M2, DMC-1-M3 and DMC-1-M4 The Tn10-containing fragments cloned from DMC-1-M2 and DMC-1-M3 were of 4.5 kB and for mutant DMC-1-M4, 3.5 kB. Sequencing of these inserts showed Tn10 insertion sites in mutants M2 and M3 to be identical and, therefore, most likely originating from the same mutagenesis event, with the insertion occurring in a ToxR-like gene adjacent to a gene with high homology to a sodium/ alanine symporter from Vibrio spp. In contrast, the transposon insertion site in mutant DMC-1-M4 was in a gene with no signicant identity to genes of Vibrio spp. but with high homology with the aerolysin gene from several Aeromonas spp. As the haemolysin appeared to be a novel protein unrelated to known Vibrio haemolysins, it was named vibrioaerolysin. DNA sequence surrounding the transposon insertion site in Vibrio splendidus DMC-1 mutants DMC-1-M2 and DMC-1-M4 The DNA sequence surrounding the transposon insertion sites was determined, and this showed that the insertion sites in DMC-1-M2 and DMC-1-M4 were in adjacent genes. In total, 8.7-kb DNA was sequenced and six signicant open reading frames were identied (see EMBL sequence AM157713), four of which showed closest matches with genes common to a range of vibrios (Fig. 1), the remaining two being the putative ToxR-like regulator and vibrioaerolysin. Figure 1 shows the gene arrangement in V. splendidus in comparison with the genes published for V. splendidus LGP32 (Le Roux, Zouine, Chakroun, Binesse, Saulnier, Bouchier,
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Zidane, Ma, Rusniok, Lajus, Buchrieser, Medigue, Polz & Mazel 2009) and V. harveyi, indicating a possible DNA insertion involving the vibrioaerolysin and ToxR-like genes into a common framework of Vibrio genes. The transposon insertion sites identied in the DMC-1-M2 and DMC-1-M4 mutants are both within the region of inserted DNA, in the ToxR-like gene and vibrioaerolysin genes, respectively, resulting in haemolysin-negative phenotypes. To identify the possible sites at which DNA insertion had occurred in the V. splendidus genome, the sequences prior to and after the ToxR and the haemolysin genes were analysed using the EMBOSS program palindrome to identify inverted repeats in the nucleotide sequence. Four pairs of inverted repeats were identied. Of these, inverted repeat two spanned the putative insertion of vibrioaerolysin and the ToxR-like gene (Fig. 1). Incidence of the vibrioaerolysin gene in isolates of Vibrio splendidus Of the 15 V. splendidus isolates from a turbot hatchery isolated by Thomson et al. (2005), 9 of the 13 biotype 1 isolates yielded PCR products for the vibrioaerolysin gene, but neither of the two V. splendidus biotype 2 isolates gave a PCR product. Comparison of the vibrioaerolysin and aerolysin amino acid sequences The vibrioaerolysin amino acid sequence was aligned with 12 aerolysin amino acid sequences using the DIALIGN program. For the aerolysin group of sequences, all except Aeromonas salmonicida 17-2 and Aeromonas punctata possess a putative signal peptide cleavage site between alanine residues 23 and 24; the equivalent residues in vibrioaerolysin are asparagine and alanine that may represent the vibrioaerolysin signal peptide site (results not shown; see EMBL sequence AM157713). Phylogenetic comparison of vibrioaerolysin and aerolysin A maximum likelihood consensus tree comparing the vibrioaerolysin amino acid sequence with six selected aerolysin sequences from Aeromonas spp. and four sequences from genome sequences of Vibrio spp. currently being completed is shown in Fig. 2. The sequences fall into four groups with vibrioaerolysin from V. splendidus DMC-1 being

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V. harveyi ATCCBAA-1116 (CP000789)

VH02469 VH02473 VH02474

VH2470

VH02471

VH02472

V. splendidus LGP32 (FM954972)

VS1294

VS1291

VS1290

VS1289

V. splendidus DMC-1 (AM157713)

VS1 Hypothetical protein VS2 Putative Na/ala symporter VS5 Putative Thr VS6 Putative carboxypeptidase (incomplete) efflux protein

IR2

VS3 Tox R-like regulator

VS4 vibrioaerolysin

IR2

9kb

Figure 1 Schematic diagram showing homologous genes in Vibrio splendidus strain DMC-1, V. splendidus strain LGP32 and Vibrio harveyi strain ATCC BAA-1116. Homologous genes in the different bacteria are shown with the same shading pattern. The genes covered are V. harveyi: VH02469, putative sodium/alanine symporter; VH02470 hypothetical protein; VH02471, hypothetical protein; VH02472, aerolysin-like protein; VH02473, hypothetical protein; and VH02474, putative carboxypeptidase. V. splendidus LGP32 VS1289, carboxypeptidase; VS1290, hypothetical protein; VS1291, sodium/alanine symporter; and VS1294, hypothetical protein. VS1292 and VS1293 (non-coding RNA, glycine riboswitch) are not shown. V. splendidus DMC-1: VS1, hypothetical protein; VS2, putative sodium/alanine symporter; VS3, putative transcription activator ToxR; VS4, vibrioaerolysin; VS5, putative threonine efux protein; VS6, putative carboxypeptidase. The gene insertions that appear to have occurred in V. harveyi and V. splendidus DMC-1 in the region common to V. splendidus LGP32 and vibrios such as Vibrio parahaemolyticus (AP005079) and Vibrio vulnicus (AE004220) are shown in lled arrows. The locations of inverted repeats IR2 are shown. EMBL accession numbers for nucleotide sequences are shown in parentheses.

similar to sequences identied in the genomes of V. splendidus 12B01-1 and V. harveyi, whereas most of the Aeromonas aerolysins form a separate group (Fig. 2). Effect of Vibrio splendidus DMC-1-M2 and DMC-1-M3 on turbot larvae In the rst two trials, yolk-sac larvae were exposed to wild-type V. splendidus DMC-1 and to the mutants DMC-1-M2 and DMC-1-M3. Even when exposed to 108 cfu bacteria mL)1, mortalities were not signicantly different from those of the control groups (Fig. 3). In subsequent experiments, rstfeeding turbot larvae were exposed to V. splendidus DMC-1 or strain DMC-1-M2, which were incorporated in the rotifer live food. The parent strain DMC-1 gave signicantly higher mortalities than the control and DMC-1-M2 groups, with DMC-1 2012 Blackwell Publishing Ltd

M2 behaving similarly to the control group (Fig. 4). Immunohistochemistry showed that DMC-1-M2 colonized the gut epithelium and was present in the lumen, but did not damage or invade the gut cells (Fig. 5, a12) to cause the cellular destruction that was seen in larvae infected with V. splendidus DMC-1 (Fig. 5, b2, arrow 5). Effect of Vibrio splendidus DMC-1-M4 on cod larvae Turbot larvae were not available in the later stages of this work, and as V. splendidus DMC-1 has been shown to be lethal to cod larvae (Reid et al. 2009) as well as turbot larvae, rst-feeding cod larvae were challenged with V. splendidus DMC-1 or the haemolysin-decient DMC-1-M4. V. splendidus DMC-1 gave signicantly greater mortalities than the control group from day 3 onwards (P < 0.05).

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V splendidusDMC-1 Vsplendidus12B01-1 Vharveyi Vsplendidus12BO1-2 VcorallyticusATCCBAA450 AveroniA8QIT4 AhydrophilaNLEP Q93CE6 AhydrophilaATCC7966 AtrotaAB3 AtrotaATCC49659 Aenteropelogenes O85370

0.1

Figure 2 Molecular phylogenetic analysis by the maximum likelihood method. The evolutionary history was inferred by using the maximum likelihood method based on the JTT matrix-based model (Jones et al. 1992). The tree with the highest log likelihood ()5340.5625) is shown. Initial tree(s) for the heuristic search were obtained automatically as follows. When the number of common sites is <100 or less than one-fourth of the total number of sites, the maximum parsimony method was used; otherwise, the BIONJ method with MCL distance matrix was used. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 11 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 483 positions in the nal data set. Evolutionary analyses were conducted in MEGA4 (Tamura et al. 2007).

The mutant strain DMC-1-M4 gave signicantly fewer mortalities than the parent strain DMC-1 (P < 0.01) and fewer mortalities than the control group although the latter was not statistically signicant (Fig. 6). When the experiment was terminated, the bacterial content of homogenates of the larvae was analysed. The control group contained 1.9 104 cfu per larva of a diverse bacterial ora, none of which resembled V. splendidus DMC-1. Larvae exposed to DMC-1 contained 3.3 105 cfu per larva over 90% of which resembled DMC-1 in colony morphology. Five representative colonies were conrmed as V. splendidus by reaction with antiserum to V. splendidus DMC1. The group challenged with DMC-1-M4 contained 9 104 cfu per larva, of which ca 30% resembled the colony morphology of V. splendidus and all isolates tested reacted with antiserum to V. splendidus. Comparison of the action of vibrioaerolysin and Aeromonas hydrophila aerolysin on sheep erythrocytes Vibrioaerolysin gave a doseresponse curve for lysis of sheep erythrocytes that was indistinguishable from that caused by aerolysin of A. hydrophila (Buckley, Halasa, Lund & Macintyre 1981) (results not shown). As solutes such as PEG 1500 inhibited lysis, it was concluded that haemolysis occurred by colloid osmotic lysis as is known to occur with
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aerolysin (Buckley 1999). The diameters of the channels induced in sheep erythrocytes, as measured in osmotic protection experiments, were 1.86 nm for vibrioaerolysin (95% CL 1.821.90) and 1.93 nm for aerolysin (95% CL 1.862.00), respectively.
Discussion

Several studies have reported that V. splendidus is involved in infections in turbot (Myhr, Larsen, stein 1991; Pazos, Lillehaug, Gudding, Heum & Ha os, Bandin, Nu n ez & Toranzo Santos, Magarin 1993; Angulo, Lopez, Vicente & Saborido 1994; Gatesoupe et al. 1999; Thomson 2001; Thomson et al. 2005) and oysters (Gay, Renault, Pons & Le Roux 2004; Le Roux et al. 2007). In isolates from moribund turbot, Gatesoupe et al. (1999) and Thomson et al. (2005) were only able to distinguish between pathogenic and non-pathogenic isolates of V. splendidus by their effects on larval turbot and not from physiological and biochemical properties. As cytolytic toxins have been found to be important virulence determinants in many bacterial pathogens (Alouf & Freer 1999), the properties of the haemolysin of V. splendidus biotype 1 were investigated. Mutagenesis with transposon Tn10 resulted in identication of three V. splendidus mutants completely lacking in production of haemolysin and virulence towards marine sh larvae with all other properties tested being the same. This

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Trial 1 100
Control DMC-1 10e6

100
Tank 2 Control

75 % Mortalities

DMC-1 10e8 M2 10e6 M2 10e8

75 % Mortalities

Tank 3 DMC-1 Tank 4 DMC-1 Tank 5 M2

50

M3 10e6 M3 10e8

50

Tank 6 M2

25

25
0 Day 2 Day 3 Day 4 Post-hatch
Trial 2

Day 5

Day 6

0 4 5 6 7 8 9 Days Post-hatch

100
Control DMC-1 10e5 DMC-1 10e7 M2 10e5 M2 10e7 M3 10e5 M3 10e7

75 % Mortalities

50

Figure 4 Turbot larvae rst-feeding trials with wild-type Vibrio splendidus DMC-1 and haemolysin-negative mutant V. splendidus DMC-1-M2. Turbot larvae were challenged on days 4 and 5, with challenge bacteria added to the live food rotifers to give a nal bacterial concentration of 3 104 cfu mL)1 in each challenge tank.

25

80 70

Day 2

Day 3

Day 4 Post-hatch

Day 5

Day 6

% Total mortalities

60 50 40 30 20 10 0 1 2 3 4 Days post infection 5 6

Figure 3 Turbot yolk-sac larval trials with wild-type Vibrio splendidus DMC-1 and haemolysin-negative mutants V. splendidus DMC-1-M2 and V. splendidus DMC-1-M3. Two separate trials were conducted, and the concentration of bacteria to which larvae were exposed is shown in the legends.

suggests that the haemolysin plays a role in the virulence of this organism. Sequence determination of a region of the chromosome of V. splendidus DMC-1 showed a gene arrangement almost identical to those in other Vibrio species; the only major difference between V. splendidus DMC-1 and Vibrio cholerae (Genbank AE004220), Vibrio vulnicus (AE016806) and Vibrio parahaemolyticus (AP005079) sequences was the section of the genome that contained the vibrioaerolysin and ToxR-like genes, and this provided strong evidence that there had been a recombination event in the V. splendidus chromosome with the insertion of foreign DNA. The complete genome sequence of V. splendidus strain LGP32, a pathogen of oysters, has been reported recently by Le Roux et al. (2009), and this lacks the
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Figure 5 Cod larvae rst-feeding trials with wild-type Vibrio splendidus DMC-1 and haemolysin-negative mutant V. splendidus DMC-1-M4. Cod larvae were challenged on days 1 and 2, with challenge bacteria added to the live food rotifers to give a nal bacterial concentration of 2 106 cfu mL)1 in each challenge tank (--, control; -j-, DMC-1; -m-, DMC-1-M4).

apparent insertion containing the ToxR-like and vibrioaerolysin genes. The sequences of V. splendidus LGP32 and DMC-1 correspond except that a 231-bp sequence of the V. splendidus LGP32 genome between the sodium/alanine symporter and thermostable carboxypeptidase genes is absent in V. splendidus DMC-1 and is replaced by a 3767bp section with the ToxR-like and vibrioaerolysin

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(a1) 100 magnification.

(a2) 1000 magnification.

4
(b1) 100 magnification. (b2) 1000 magnification.

(b3) 1000 magnification.

Figure 6 Immunohistochemistry of sections from rst-feeding larval turbot (trial 3, IMR Bergen). (a1 and a2) Vibrio splendidus vibrioaerolysin-negative mutant DMC-1-M2; (b1, b2 and b3) vibrioaerolysin-producing V. splendidus DMC-1-challenged rst-feeding larvae sampled on day 8. Arrows 1 and 3 show stained cells in the gut of larvae challenged with V. splendidus DMC-1-M2 and DMC-1, respectively; arrows 2 and 4 show individual bacterial cells, arrows 5 and 6 indicate damage to the brush border of larvae challenged with V. splendidus DMC-1. In addition to individual bacterial cells, dense red-stained material can be seen, representing either clusters of bacteria or extracellular material from bacteria. The blue-stained globule-like structures in the lumen are the remains of rotifers.

genes, terminating 122 bp past the vibrioaerolysin gene. The V. harveyi genome sequence is currently under completion, and this also contains a similar insertion with an aerolysin-like gene (Genbank CP000789). Several short inverted repeat sequences were identied before and after the vibrioaerolysin gene, and inverted repeat pair two spanned the difference in sequence between strains LGP32 and DMC-1, consistent with this section of DNA having undergone a genetic mobilization event during the organisms evolution, perhaps through a transposon insertion or phage integration. Mobilization events leading to acquisition of haemolysin or toxin genes are common in Vibrio species such as V. cholerae (Faruque, Albert & Mekalanos 1998), V. parahaemolyticus (Terai, Baba, Shirai, Yoshida, Takeda & Nishibuchi 1991; Nishibuchi & Kaper 1995), Vibrio pommerensis (Jorres, Appel & Lewin 2003)
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as well as in V. anguillarum for acquisition of the pJM1 genes involved in the anguibactin ironuptake system (Tolmasky & Crosa 1995; Di Lorenzo, Stork, Tolmasky, Actis, Farrell, Welch, Crosa, Wertheimer, Chen, Salinas, Waldbeser & Crosa 2003). Motile aeromonads are widely distributed in the aquatic environment and are the causative agents of haemorrhagic septicaemia of sh, reptiles and amphibians (Austin & Austin 2007). Several extracellular toxins are recognized from Aeromonas spp. including haemolysins, enterotoxin, cytotoxin, acetylcholinesterase, phospholipid cholesterol acyltransferase and proteases. Phylogenetic analysis of vibrioaerolysin with other aerolysin sequences indicates that the aerolysin toxins cluster into two different groups with Aeromonas trota, A. hydrophila and Aeromonas enteropelogenes forming one group of the conventional aerolysins and those from vibrios

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forming a second broad group with Aeromonas veroni. The V. splendidus DMC-1 aerolysin shows high homology with database sequences from similar genes of V. splendidus 12B01-1 and V. harveyi. A further sequence from V. splendidus (12B012) appears quite distinct from all other aerolysin genes. Aerolysin is a well-characterized pore-forming toxin identied over 30 years ago (Bernheimer & Avigad 1974), and the crystal structure has been solved (Parker et al. 1996). Conversion of inactive A. hydrophila proaerolysin into haemolytically active aerolysin occurs by cleavage at a site between arginine and leucine residues near the carboxy terminus (Howard & Buckley 1985a), and in vibrioaerolysin, an arginineisoleucine bond in a similar position may serve as the equivalent site. Aerolysin is synthesized as a preprotoxin containing an N-terminal signal sequence and a C-terminal activation peptide (Howard & Buckley 1985a), where it is subsequently cleaved to form proaerolysin (Howard & Buckley 1985b) Proaerolysin binds to glycosylphosphatidylinositol-anchored proteins of the cell membrane such as Thy-1 (Nelson, Raja & Buckley 1997), contactin (Diep, Nelson, Raja, Pleshak & Buckley 1998) or erythrocyte aerolysin receptor (Cowell, Aschauer, Gruber, Nelson & Buckley 1997), and a carboxyterminal peptide of about 40 amino acids is cleaved by proteases to form active aerolysin (Howard & Buckley 1985b) that inserts into the membrane to form a heptameric transmembrane channel or pore leading to membrane depolarization and death of the cell. Estimates of the diameter of the transmembrane channel depend upon the measuring procedure employed. The pore size in erythrocytes in this study produced by vibrioaerolysin and aerolysin of A. hydrophila (Buckley et al. 1981) was 1.86 nm for vibrioaerolysin and 1.93 nm for A. hydrophila aerolysin, respectively, which are comparable with other estimates. Aeromonas hydrophila can cause gastroenteritis, deep wound infections and septicaemia (Buckley 1999), and Aeromonas sobria is a related species causing acute gastroenteritis in adults and children (Tanoue, Takahashi, Okamoto, Fujii, Taketani, Harada, Nakano & Nakaya 2005). Aerolysin is recognized as the major virulence factor for these organisms, and the aerolysin toxin of A. sobria has been shown to cause uid accumulation in the mouse ileal loop test and to activate cyclic AMP (cAMP)-dependent chloride secretion (Tanoue
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et al. 2005) via prostaglandin E2 stimulation (Fujii, Tsurumi, Sato, Takahashi & Okamoto 2008) causing diarrhoea in a manner similar to that of V. cholerae and E. coli. Thus, the mechanism by which aerolysin causes damage to cells of the gastrointestinal tract mucosa in humans and subsequent diarrhoea is well understood, and vibrioaerolysin may well have a similar, damaging effect when V. splendidus colonizes the intestinal tract of marine sh larvae in sufcient numbers. Unlike V. anguillarum, V. splendidus does not appear to be invasive. Turbot larvae seem unaffected when exposed to V. splendidus in water, and mortalities only occur when bacteria are associated with food such as rotifers and gain entry to the digestive tract. Also, damage appears to be localized to the gut epithelium as occurs for enteropathogens such as V. cholerae (Faruque et al. 1998). The nine V. splendidus biotype 1 strains shown by PCR to contain the vibrioaerolysin gene included all four isolates shown to be pathogenic to turbot larvae by Thomson et al. (2005) and four isolates found to be non-pathogenic. This suggests that in V. splendidus, as in most bacterial pathogens, virulence is multi-factorial (Smith 2000) and that other factors are also required for virulence in V. splendidus. The vibrioaerolysin gene was located immediately downstream of an ORF encoding a ToxR-like gene, and transposon insertion into this ORF (mutants DMC-1-M2 and DMC-1-M3) rendered V. splendidus unable to produce haemolysin, to cause damage to the intestinal tract or to kill larval turbot, which is possible evidence that this is a transcriptional factor involved in governing the level of haemolysin produced. Vibrio cholerae ToxR and similar molecules are transmembrane transcriptional activators that have a characteristic periplasmic domain acting as a sensor, e.g., for changes in pH or ionic conditions, a transmembrane domain and a winged helix-turn-helix motif associated with DNA binding and regulation of gene transcription (Crawford, Krukonis & DiRita 2003). Comparison of the V. splendidus ToxR homologue sequence with that of ToxR of E. coli shows that it contains both putative helix-turn-helix motif and transmembrane domains but lacks a periplasmic domain. This may not be essential in transmembrane signalling as the periplasmic domain is not required for transcriptional regulation of TcpP-dependent toxT, ompU and ompT genes in V. cholerae (Crawford et al. 2003). Thus,

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the V splendidus DMC-1 ToxR could well act as a sensor of environmental stimuli and a regulator of vibrioaerolysin and possibly other virulence genes. Kamisaka, Jordal, Edvardsen, Kryv, Otterlei & Rnnestad (2010) have recently described a syndrome, termed distended gut syndrome, that can affect various stages in larval cod and is characterized by low larval activity, reduced appetite and a gut that is distended and lled with uid and with an opaque lumen. The authors concluded that the pathophysiological basis of distended gut syndrome involves disturbed enteral water and electrolyte balance caused by toxins. Such an outcome is entirely compatible with damage caused by an aerolysin-like enterotoxin as described here. This does not indicate a role for V. splendidus in distended gut syndrome as many other vibrios could have acquired the genetic elements for aerolysin production. Based on the evidence here of how some haemolytic bacteria could be harmful to larval marine sh, it is advisable that hatcheries should attempt to reduce the concentration of haemolytic bacteria associated with live feed and the rearing system in general by routine screening on blood agar plates and enhanced hatchery hygiene procedures.
Acknowledgements

Alouf J. & Freer J.H. (1999) Bacterial Protein Toxins, 2nd edn. Academic Press, London. Alton N.K. & Vapnek D. (1979) Nucleotide sequence analysis of the chloramphenicol resistance transposon Tn9. Nature 282, 464469. Angulo L., Lopez J.E., Vicente J.A. & Saborido A.M. (1994) Haemorrhagic areas in the mouth of farmed turbot, Scophthalmus maximus (L.). Journal of Fish Diseases 17, 163169. Austin B.A. & Austin D.A. (2007) Bacterial Fish Pathogens: Diseases of Farmed and Wild Fish, 4th edn. Springer and Praxis Publishing Ltd., Chichester, UK, pp. 552. Bernheimer A.W. & Avigad L.S. (1974) Partial characterization of aerolysin, a lytic exotoxin from Aeromonas hydrophila. Infection and Immunity 9, 10161021. `s M.-C., Binesse J., Delsert C., Saulnier D., Champomier-Verge Zagorec M., Munier-Lehmann H., Mazel D. & Le Roux F. (2008) The metalloprotease vsm is the main toxic factor for Vibrio splendidus extracellular products. Applied and Environmental Microbiology 74, 71087117. Birkbeck T.H. (2004) Role of probiotics in sh disease prevention. In: Current Trends in Bacterial and Viral Fish and Shrimp Diseases (ed. by K.Y. Leung), pp. 390416. World Scientic, Hackensack, New Jersey. Buckley J.T. (1999) The channel-forming toxin aerolysin. In: Bacterial Protein Toxins, 2nd edn (ed. by J. Alouf & J.H. Freer), pp. 362372. Academic Press, London. Buckley J.T., Halasa L.N., Lund K.D. & Macintyre S. (1981) Purication and some properties of the haemolytic toxin aerolysin. Canadian Journal of Biochemistry 59, 430435. Cowell S., Aschauer W., Gruber H.J., Nelson K.L. & Buckley J.T. (1997) The erythrocyte receptor for the channelforming toxin aerolysin is a novel glycerophosphatidylinositol-anchored protein. Molecular Microbiology 25, 343350. Crawford J.A., Krukonis E.S. & DiRita V.J. (2003) Membrane localisation of the winged-helix domain is required for TcpPmediated virulence gene activation in Vibrio cholerae. Molecular Microbiology 47, 14591473. Crosa J.H. (1980) A plasmid associated with virulence in the marine sh pathogen, Vibrio anguillarum species an ironsequestering system. Nature 284, 566568. Di Lorenzo M., Stork M., Tolmasky M.E., Actis L.A., Farrell D., Welch T.J., Crosa L.M., Wertheimer A.A., Chen Q., Salinas P., Waldbeser L. & Crosa J.H. (2003) Complete sequence of the virulence plasmid pJM1 from the marine sh pathogen Vibrio anguillarum strain 775. Journal of Bacteriology 185, 58225830. Diep D.B., Nelson K.L., Raja S.M., Pleshak E.N. & Buckley J.T. (1998) Glycosylphosphatidylinositol anchors of membrane glycoproteins are binding determinants for the channelforming toxin aerolysin. Journal of Biological Chemistry 273, 23552360. Dulbecco R. & Vogt M. (1954) Plaque formation and isolation of pure lines with poliomyelitis viruses. Journal of Experimental Medicine 99, 167182. Engelsen A.R., Sandlund N., Fiksdal I.U. & Bergh . (2008) Immunohistochemistry of Atlantic cod larvae Gadus morhua

We thank the European Union for supporting this work through the PROBE project (Improved Procedures for Flatsh Larval Rearing through the use of Probiotic Bacteria: Q5RS-2000-31457) and through the Improving Human Potential Programme for funding an exchange visit by HLM to the Institute of Marine Research, Bergen. We also thank the UK Seash Industry Authority and the British Marine Finsh Association for their support, Hari Rudra for help with the challenge experiment and Ingrid Uglenes Fiksdal for help with immunohistochemistry.
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