Antibacterial effect of medium-chain fatty acid: caprylic acid on gnotobiotic <Emphasis Type="Italic">Artemia franciscana</Emphasis> nauplii against shrimp pathogens <Emphasis Type="Italic">Vibrio harveyi</Emphasis> and <Emphasis Type="Italic">V. parahaemolyticus</Emphasis>

The present study was carried out to determine the antibacterial effect of caprylic acid in the culture system of Artemia franciscana nauplii inoculated with shrimp pathogens Vibrio harveyi and V. parahaemolyticus. To begin with, the antibacterial effect of different concentrations (1, 10 and 100 mM) of caprylic acid against V. harveyi and V. parahaemolyticus was assessed through bacterial growth study. This indicated that at 100 mM concentration, the growth of the pathogens was completely inhibited within 5 h, whereas, in 1.0 and 10 mM concentrations, effective inhibition was observed with the extension of time. Subsequently, the influence of pH variation on the growth inhibitory effect of 10 mM caprylic acid against V. harveyi and V. parahaemolyticus at different pH (5–7) was also made through bacterial growth study. At pH 5, the pathogen growth was very less, compared with pH 6 and 7. The Artemia nauplii (instar II) reared in 10 mM caprylic acid supplemented medium were challenged with V. harveyi and V. parahaemolyticus, and the mortality was recorded at an interval of 6 h up to 60 h. In this study, the mortality of Artemia nauplii reduced to 20.61 and 16.30% in V. parahaemolyticus and V. harveyi challenged groups, respectively. The present results provide evidence for the potential antibacterial activity of caprylic acid in aquaculture against luminescent vibrios.     

Spatial learning-induced <Emphasis Type="Italic">egr</Emphasis>-<Emphasis Type="Italic">1</Emphasis> expression in telencephalon of gold fish <Emphasis Type="Italic">Carassius auratus</Emphasis>

The immediate-early gene (egr-1) expression was used to examine the neuron’s response in telencephalon of goldfish during spatial learning in small space. Fishes were pre-exposed in the experimental apparatus and trained to pick food from the tray in a rectangular-shaped arena. The apparatus was divided into identical compartments comprising three gates to provide different spatial tasks. After the fish learned to pass through the gate one, two more gates were introduced one by one. Fish made more number of attempts and took longer time (P < 0.05) to pass through the first gate than the gate two or three. This active learning induces the expression of egr-1 in telencephalon as established by western blot analysis. Subsequently, the fish learn quickly to cross the similar type of second and third gate and make fewer errors with a corresponding decline in the level of egr-1 expression. As the fish learned to pass through all the three gates, third gate was replaced by modified gate three. Interestingly, the level of egr-1 expression increased again, when the fish exhibit a high exploratory behavior to cross the modified gate three. The present study shows that egr-1 expression is induced in the telencephalon of goldfish while intensively acquiring geometric spatial information to pass through the gates.     

Dietary supplementation of curcumin augments heat stress tolerance through upregulation of <Emphasis Type="Italic">nrf-2</Emphasis>-mediated antioxidative enzymes and <Emphasis Type="Italic">hsps</Emphasis> in <Emphasis Type="Italic">Puntius sophore</Emphasis>

Heat stress is one of the major environmental concerns in global warming regime and rising temperature has resulted in mass mortalities of animals including fishes. Therefore, strategies for high temperature stress tolerance and ameliorating the effects of heat stress are being looked for. In an earlier study, we reported that Nrf-2 (nuclear factor E2-related factor 2) mediated upregulation of antioxidative enzymes and heat shock proteins (Hsps) provide survivability to fish under heat stress. In this study, we have evaluated the ameliorative potential of dietary curcumin, a potential Nrf-2 inducer in heat stressed cyprinid Puntius sophore. Fishes were fed with diet supplemented with 0.5, 1.0, and 1.5% curcumin at the rate 2% of body weight daily in three separate groups (n = 40 in each group) for 60 days. Fishes fed with basal diet (without curcumin) served as the control (n = 40). Critical thermal maxima (CTmax) was determined for all the groups (n = 10, in duplicates) after the feeding trial. Significant increase in the CTmax was observed in the group fed with 1.5% curcumin- supplemented fishes whereas it remained similar in groups fed with 0.5%, and 1% curcumin-supplemented diet, as compared to control. To understand the molecular mechanism of elevated thermotolerance in the 1.5% curcumin supplemented group, fishes were given a sub-lethal heat shock treatment (36 °C) for 6 h and expression analysis of nrf-2, keap-1, sod, catalase, gpx, and hsp27, hsp60, hsp70, hsp90, and hsp110 was carried out using RT-PCR. In the gill, expression of nrf-2, sod, catalase, gpx, and hsp60, hsp70, hsp90, and hsp110 was found to be elevated in the 1.5% curcumin-fed heat-shocked group compared to control and the basal diet-fed, heat-shocked fishes. Similarly, in the liver, upregulation in expression of nrf-2, sod, catalase, and hsp70 and hsp110 was observed in 1.5% curcumin supplemented and heat shocked group. Thus, this study showed that supplementation of curcumin augments tolerance to high temperature stress in P. sophore that could be attributed to nrf-2-induced upregulation of antioxidative enzymes sod, catalase, gpx, and the hsps.     

Effect of <Emphasis Type="Italic">Cratoxylum formosum</Emphasis> on innate immune response and disease resistance against <Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> in tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis>

The effect of aqueous extract of Cratoxylum formosum on innate immune response and disease resistance in tilapia Oreochromis niloticus was investigated. The fish were fed diets containing 0% (control), 0.5% (diet 1), 1% (diet 2), and 1.5% (diet 3) of C. formosum aqueous extract for 30 days. At the end of the experimental period, parameters of innate immune response including phagocytosis of blood leukocytes, lysozyme activity in plasma, and respiratory bust activity were examined. Feeding the fish with diet 2 and diet 3 for 20 days enhanced phagocytic activity and for 30 days stimulated lysozyme and respiratory burst activities. Diet 1 increased the phagocytic activity at 30 days, but did not affect the other measured parameters. All parameters were not significantly changed (P > 0.05) in the control group throughout the experiment. Following 30 days of feeding, fish were infected with S. agalactiae. The cumulative mortalities of bacterial-infected tilapia that were fed diet 1, diet 2, and diet 3 were 56, 12, and 10%, respectively, compared with 85% in the control group. These results indicate that the aqueous extract of C. formosum may elevate the innate immune response and enhance disease resistance in tilapia.     

Growth and fatty acid composition of <Emphasis Type="Italic">Octopus vulgaris</Emphasis> paralarvae fed with enriched <Emphasis Type="Italic">Artemia</Emphasis> or co-fed with an inert diet

The rearing of Octopus vulgaris paralarvae during its planktonic life stage is a major challenge, as mortality is currently very high and unpredictable. In this study, we examined the survival and growth rates, as well as the fatty acid composition, of O. vulgaris paralarvae fed on three different dietary treatments: group ArDHA was offered juvenile Artemia enriched with a lipid emulsion (Easy DHA-Selco^®); group ArMA was fed with juvenile Artemia enriched with a mixture of microalgae (Rhodomonas lens and Isochrysis galbana); and group ArMA+ID received the same Artemia as group ArMA complemented with an inert diet. Dietary treatments were tested in triplicate with homogenous groups of paralarvae (25 individuals l^?1) established in 50-l tanks, and the experiment was conducted for 15 days. The survival rate of 15-day post-hatch (-dph) paralarvae from groups ArMA (20 ± 8%) and ArMA+ID (17 ± 4%) tended to be higher than in group ArDHA (13 ± 5%), though these differences were not statistically different. The dry weight (DW) of 15-dph paralarvae increased by almost 60% in groups ArMA and ArMA+ID, and nearly 40% in group ArDHA, with respect to hatchlings. The fatty acid (FA) composition of paralarvae revealed a remarkable drop of docosahexaenoic acid (22:6n-3, DHA) from hatchlings to 15-dph paralarvae of all groups (P < 0.05). However, paralarvae from group ArDHA contained higher levels of DHA than those from ArMA and ArMA+ID (P < 0.05). Despite Artemia enriched with DHA-Selco^® contained three-times more DHA than Artemia enriched with microalgae, no beneficial effects of this dietary treatment were observed on the performance of paralarvae.     

Monitoring of <Emphasis Type="Italic">Francisella noatunensis</Emphasis> subsp. <Emphasis Type="Italic">orientalis</Emphasis> in farmed Nile tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>) in Brazil

Francisella noatunensis orientalis is a bacterium that causes emerging bacteriosis in Nile tilapia (Oreochromis niloticus) in many parts of the world, including Brazil. It is a non-motile, Gram-negative, strictly aerobic, facultative intracellular coccobacillus. This species of bacteria is responsible for low to high mortality in fish farms, causing economic losses for fish farmers. This study aimed to detect the presence of F. noatunensis orientalis using qPCR (real-time polymerase chain reaction) and to describe lesions caused by the bacterium in O. niloticus in Brazilian aquaculture. For this purpose, 360 fish from six fish farms (30 per farm) were sampled at two time points (n = 180 per sampling). Necropsies and histopathology were performed for lesion observation, in addition to qPCR and sequencing for detection and identification of Francisella species. Environmental data were collected using a multiparameter sonde YSI EXO2. All measured limnological variables were within the optimum range for cultivation of Nile tilapia. The major lesions present were melanization of the skin, splenomegaly, granulomas, and inflammatory cell responses. The prevalence of francisellosis varied from 0 to 86.66% between time periods and fish farms analyzed, and an outbreak was observed during the second sampling period. This study describes the prevalence of francisellosis in O. niloticus and reports that the lesions found are not exclusively associated with this bacterial disease.     

Stocking density for the intensive rearing of juvenile crayfish, <Emphasis Type="Italic">Pacifastacus leniusculus</Emphasis> (Astacidae), using <Emphasis Type="Italic">Artemia</Emphasis> nauplii to supplement a dry diet from the onset of exogenous feeding

Recent advances in intensive rearing of astacid juvenile crayfish have greatly improved the results. This challenges the current application possibilities of the studies performed previously, and new research on density is required. A 100-day experiment was carried out under controlled conditions to evaluate density effects on survival and growth rates of juvenile crayfish in optimal conditions of feeding. Juvenile stage 2 Pacifastacus leniusculus were stocked in fibreglass tanks (1 m², 200 l water) at 20 ± 1°C and fed a dry diet for salmonids supplemented with restricted amounts of Artemia nauplii. Stocking densities were 100, 300, 600 and 1,000 crayfish m⁻². Mean survival rate was reduced significantly with increased stocking density, ranging from 86.33% (100 m⁻²) to 39.13% (1,000 m⁻²). All checks showed that at the lowest initial density (100 m⁻²) animals grew significantly faster those at higher densities, recording a final carapace length of 15.28 mm and weight of 1.08 g. Among the treatments of 300, 600 and 1,000 m⁻² no differences were found either in carapace length or in weight throughout the experimental period, with a final mean growth of 14 mm carapace length and 0.72 g weight. The final proportion of animals with chelae autotomy rose significantly with increasing stocking density, ranging from 14.44% (100 m⁻²) to 41.45% (1,000 m⁻²). This study shows that diet is a decisive factor for stocking successfully high densities under controlled conditions and provides useful information to set adequate densities in accordance with the production objectives.     

Low-salinity tolerance of juvenile fugu <Emphasis Type="Italic">Takifugu rubripes</Emphasis>

ABSTRACT:   To study the adaptability of juvenile fugu Takifugu rubripes to low-salinity environments, fish were transferred from full-strength seawater (100% SW) to freshwater (FW) and 25, 50, 75 and 100% SW, and checked for mortality over 3 days. No mortality was observed in 25–100% SW, whereas all fish died in FW. In fish transferred to 25–100% SW, blood osmolality was maintained within a physiological range. To further explore the lower limit of salinity that fugu could tolerate, fish were transferred from 100% SW to FW and 1, 5, 10, 15 and 25% SW. All fish survived in 5–25% SW, but fish died in FW and 1% SW. In fish surviving transfer to FW and 1 and 5% SW, blood osmolality was decreased to a near sublethal level of approximately 300 mOsm/kg·H₂O. Therefore, the lower limit of salinity tolerance is estimated to lie between 5 and 10% SW. Preacclimation in 25% SW for 7 days did not essentially affect the survival salinity range. Although survival rates and blood osmolality were slightly improved by preacclimation in 25% SW, blood osmolality was markedly decreased in salinities less than 10% SW, as was seen in the direct transfer. Neither chloride cell morphology nor sodium-potassium adenosinetriphosphatase activity in the gills showed a significant change following transfer to low salinities. These findings indicate that fugu can be adapted to hypoosmotic environments to some extent, exerting hyperosmoregulatory ability, although chloride cells are less likely to absorb ions in hypoosmotic environments.     

Characterization of <Emphasis Type="Italic">kiss2</Emphasis> and <Emphasis Type="Italic">kissr2</Emphasis> genes and the regulation of kisspeptin on the HPG axis in <Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>

Reproduction allows organisms to produce offspring. Animals shift from immature juveniles into mature adults and become capable of sexual reproduction during puberty, which culminates in the first spermiation and sperm hydration or ovulation. Reproduction is closely related to the precise control of the hypothalamic–pituitary–gonadal (HPG) axis. Kisspeptin peptides are considered as the important regulator of HPG axis in mammalian. However, the current understanding of kisspeptin in flatfish is not comprehensive. In this study, we cloned and analyzed the kiss2 and kissr2 genes in Cynoglossus semilaevis. Interesting alternative splicing in the 5′-untranslated regions (UTR) of the Cskissr2 gene was found. The expression profiles of Cskiss2 and Cskissr2 showed relative high messenger RNA (mRNA) levels at the late gastrula stage during embryonic development, at total length = 40 mm during early gonadal differentiation, and in the brains and gonads of all investigated tissues. These results suggested that the kisspeptin system participated in embryogenesis and in the regulation of gonadal differentiation and development. Considering that the control and regulatory mechanisms of kisspeptin in the central reproductive axis are still unclear, we documented that the intramuscular injection of kisspeptin caused different sGnRH and cGnRH mRNA levels in a dose- and tissue-dependent manner. The mRNA expressions of FSH and LH were stimulated in the ovary and were inhibited in the testis under the kisspeptin treatments. These results provided foundations for understanding the roles of kisspeptin in the neuroendocrine system in fish. The manipulation of the kisspeptin system may provide new opportunities to control the gonadal development and even reproduction in fish.     

Growth and survival of grouper <Emphasis Type="Italic">Epinephelus</Emphasis><Emphasis Type="Italic">coioides</Emphasis> (Hamilton) larvae fed free-living nematode <Emphasis Type="Italic">Panagrellus redivivus</Emphasis> at first feeding

The free-living nematode, Panagrellus redivivus, was tested as live food for grouper Epinephelus coioides larvae during the first feeding stage. A series of experiments were conducted to determine the acceptability of the free-living nematodes in grouper larvae at first feeding, the optimum nematode density and the response of the larvae to nutritionally enriched nematode. All experiments were conducted in 200-L conical tanks filled with 150-L filtered seawater and stocked at 15 larvae L⁻¹. Duration of feeding experiments was up to day 21 (experiment 1) and 14 days (experiment 2 and 3). Brachionus plicatilis and Artemia (experiment 1) and Brachionus plicatilis alone (experiment 2 & 3) was used as the control treatment. Observations indicated that the grouper larvae readily fed on free-living nematodes as early as 3 days posthatching, the start of exogenous feeding. Optimum feeding density for the larvae was 75 nematodes ml⁻¹. The enrichment of cod liver oil or sunflower oil influenced the total lipids and n-3 highly unsaturated fatty acids of P. redivivus, which in turn influenced those of the grouper larvae, however, growth and survival of the larvae were not affected (P > 0.05). The results from this investigation showed that the nematode, P. redivivus, can be used as first live food for grouper larvae from the onset of exogenous feeding until they could feed on Artemia nauplii.     

Use of a <Emphasis Type="Italic">Noctiluca</Emphasis>-killing bacterium <Emphasis Type="Italic">Marinobacter salsuginis</Emphasis> strain BS2 to reduce shrimp mortality caused by <Emphasis Type="Italic">Noctiluca scintillans</Emphasis>

The ability of an algicidal bacterium Marinobacter salsuginis strain BS2, isolated from shrimp pond water, to reduce shrimp mortality was investigated under laboratory conditions. When two species of shrimp (Penaeus monodon and Litopenaeus vannamei) (body length 1.5–1.8 cm) were cultured together with the dinoflagellate Noctiluca scintillans, nearly 80 % of the shrimps died within 7 days. However, when bacterial strain BS2 was also added to the culture, N. scintillans was killed within 48 h, and shrimp survival rates on the 7th day improved from 23 to 87 % for both P. monodon and L. vannamei. The bacterium BS2 alone had no effect on shrimp condition. Under conditions of increased dissolved oxygen, the effect of using BS2 was greater, and shrimp survival rates improved even more dramatically, from 26 to 98 %. These studies provide the first evidence that the use of killing bacteria, isolated from shrimp culture water, can suppress harmful algal blooms (HABs) and thus restore the efficiency of shrimp production. The control of HABs in this way in shrimp culture farms would be a major benefit for shrimp production.     

Potential of cinnamon (<Emphasis Type="Italic">Cinnamomum verum</Emphasis>) oil to control <Emphasis Type="Italic">Streptococcus iniae</Emphasis> infection in tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>)

In this study, four essential oils—cinnamon oil, leech lime oil, lemongrass oil, and turmeric oil—were examined for their antimicrobial activities against Streptococcus iniae, a bacterium that is pathogenic in fish, in which it causes streptococcosis. Cinnamon oil was the most potent antimicrobial agent among these oils, with a minimal inhibitory concentration (MIC) of 40 μg/ml. By using gas chromatography–mass spectrometry (GC–MS), it was found that the major components of cinnamon oil were cinnamaldehyde (90.24), limonene (2.42%), cinnamyl acetate (2.03%), linalool (1.16%), and α-terpineol (0.87%). Of these compounds, only cinnamaldehyde exhibited antimicrobial activity against S. iniae, with an MIC of 20 μg/ml. In an in vivo trial, no mortality was apparent in fish fed on fish diets supplemented with 0.4% (w/w) of cinnamon oil and with 0.1% (w/w) of oxytetracycline 5 days prior to infection with S. iniae. These results indicate that cinnamon oil had a protective effect on experimental S. iniae infection in tilapia, and thus has the potential to replace the antibiotics used to control this disease.     

Recent expansion of Northeast Atlantic and Mediterranean populations of <Emphasis Type="Italic">Melicertus</Emphasis> (<Emphasis Type="Italic">Penaeus</Emphasis>) <Emphasis Type="Italic">kerathurus</Emphasis> (Crustacea: Decapoda)

We analysed the genetic diversity of Melicertus kerathurus (Penaeidae), a commercially valuable penaeid shrimp that is distributed in the Mediterranean Sea and eastern Atlantic Ocean. We examined the polymorphism of a 494 bp DNA segment of the mitochondrial COI region in 173 individuals, sampled in nine Mediterranean and two Atlantic samples, covering the whole range of the species from the tropical waters of the Gulf of Guinea to the eastern part of the Mediterranean Sea. The mean nucleotide and haplotype diversities were π = 0.00275 and h = 0.718, respectively, for the global data set, with the highest values occurring in the African samples and the lowest in the Adriatic Sea. A clear sample differentiation was found (F _st = 0.194), but this did not reflect a geographical pattern and there were only faint traces of an Atlantic–Mediterranean subdivision. Mismatch analysis and a high significant negative value of Tajima’s D suggested that M. kerathurus is not at mutation drift-equilibrium, but underwent a recent expansion after a period of low effective sample size. A postglacial recolonisation of the Mediterranean from an Atlantic refuge could be hypothesised based on these data.     

Effects of potential probiotic <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> on digestive enzymatic activities of live feed <Emphasis Type="Italic">Artemia franciscana</Emphasis>

In the present study, cellulase, protease, lipase and amylase activities were performed to investigate the effects of Lactococcus lactis subsp. lactis CF4MRS bioencapsulation of Artemia franciscana. Our results show that cellulase activities (total cellulase—FPase activity, exoglucanase and endoglucanase—CMCase activity) were significantly higher (P < 0.05) in A. franciscana tissue homogenates compared to those in the control group after 8 h of L. lactic bioencapsulation. Notably, an exception case was found in β-D-glucosidase activity, whereby the cellulase activity was not significantly different (P > 0.05) compared to the control group. Administrations of L. lactis at cell concentration of 10⁸ CFU mL^?1 showed considerable improvement of other important enzymatic activities such as amylase, protease and lipase in A. franciscana. The amylase/protease ratio in probiotic-treated A. franciscana was recorded at 0.343, approximately two times higher than those without probiotic administration (0.184). In contrary, amylase/lipase ratio showed half of a reduction (0.330) in L. lactis-administrated A. franciscana compared to the control (0.614). Our study suggests that important digestive enzymes, e.g., cellulase, amylase, protease and lipase, can be enhanced through bioencapsulation of A. franciscana with L. lactis subsp. lactis, which could in turn lead to further stimulation of endogenous enzymes in the fish and shrimp larvae.     

Homogeneity of <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> from farmed amberjack <Emphasis Type="Italic">Seriola dumerili</Emphasis> in Japan

Streptococcus dysgalactiae strains have been isolated from cultured amberjack Seriola dumerili and yellowtail Seriola quinqueradiata in Japan. To characterize the fish isolates, we performed genetic analysis and compared the biochemical properties of these isolates with those of the S. dysgalactiae subsp. dysgalactiae and S. dysgalactiae subsp. equisimilis strains isolated from mammals. The genetic analysis revealed that the fish isolates were genetically very similar to each other with high DNA–DNA relatedness (>95.4%) and sequence homology. Meanwhile, the DNA relatedness between mammalian isolates and the fish isolates was 73.4–82.6%. In biased sinusoidal gel electrophoresis (BSFGE) analysis, the restriction patterns of mammalian isolates were different from those of fish isolates. The fish isolates did not show streptokinase activity in plasminogen obtained from mammals. These characteristics enabled us to distinguish between the fish isolates and the Sdd and Sde strains isolated from mammals. In order to obtain epidemiological information on the fish isolates, BSFGE patterns from 284 S. dysgalactiae strains from fish in Japan were examined. Based on the results of BSFGE analysis, the fish isolates were classified into 16 groups (AP1–AP16) with restriction enzyme ApaI. The dendrogram based on BSFGE analysis indicated that all fish isolates using in this study were closely related.     

Random sequencing of genomic DNA of <Emphasis Type="Italic">Photobacterium damselae</Emphasis> subsp. <Emphasis Type="Italic">piscicida</Emphasis>

ABSTRACT:   Random sequencing of the genomic DNA of Photobacterium damselae subsp. piscicida was conducted. The sequences were assembled into 930 contigs. The total length of these contigs accounts for 37.5% of the genome of P. damselae subsp. piscicida. The contigs contain 2055 open reading frames (ORFs) that have homology with genes in the GenBank database. Furthermore, some of the ORFs have homology with reported virulence related genes, and are classified as encoding colonization factors, exotoxin, and lipopolysaccharide (LPS). Thirty-seven ORFs have homology with colonization factors. In those colonization factors, 27, three, and four ORFs have homology with polar flagellar-related genes, capsule-related genes, and others (accessory colonization factors, superoxide dismutase (Cu-Zn), MshA biogenesis protein, and heme receptor genes), respectively. Five ORFs have homology with exotoxin-related genes (2 hemolysin, phospholipase, hyaluronidase, lysophospholipase L2 genes). Further, six ORFs have homology with LPS-related genes.     

Immune response of sea bass <Emphasis Type="Italic">Dicentrarchus labrax</Emphasis> to <Emphasis Type="Italic">Tenacibaculum maritimum</Emphasis> antigens

ABSTRACT:   Seawater fishes are affected by a pathology commonly called 'myxobacteriosis', caused by Tenacibaculum maritimum (formerly Flexibacter maritimus ). The disease is characterized by fin erosion and necrotic ulcers of skin and muscle, and by low but constant mortality in cultured marine fish; in Italy is one of the most important and widely spread diseases affecting sea bass Dicentrarchus labrax , gilthead seabream Sparus aurata , sharp-snouted bream Diplodus puntazzo , white bream Diplodus sargus , and six-tooted bream Dentex dentex . In order to obtain an effective vaccine against the disease, formalin killed cells (FKC), extracellular products (ECPs) and crude lipopolysaccharide (LPS) preparations were obtained from the T. maritimum strain SPVId and injected intraperitoneally twice into the sea bass. The fish immune response to the preparations was studied: agglutinating antibody titer and in vitro phagocytosis were determined after the first and second injection in order to evaluate whether the preparations are immunogenic or not and if the booster effect took place. The results show that FKC and LPS preparations increased the antibody titer after the first injection when compared to the control sea bass. Moreover, all the preparations stimulated a secondary (booster) response. In vitro phagocytosis of the total blood was significantly higher for all the preparations when compared to the controls, but the crude LPS immunized sea bass showed the highest activity.