Healthy Turbot Research Articles

Blood Transcriptomics of Turbot Scophthalmus maximus: A Tool for Health Monitoring and Disease Studies.

Simple SummaryThe analysis of blood gene expression is emerging as a relevant source of information about the health status of an organism. While these investigations are increasingly performed in human and terrestrial animals, their potential is still underexplored in fish pathology. The aim of this work was to analyze the blood transcriptional profile of a commercially important flatfish species, turbot (Scophthalmus maximus), in healthy and diseased specimens. The analysis of the most expressed genes in healthy fish indicated that turbot red blood cells have important immunological functions. In diseased fish, parasitized by a myxozoan, the blood analysis reflected a broad inhibition of the immune response followed by intense inflammatory activation in heavy infections. The results showed that turbot response appears delayed, dysregulated and ineffective in stopping the infection. Particularly, a proper development of the adaptive immune response was lacking. This study points out that blood gene expression profiling is a reliable tool for health monitoring, as well as to advance in the knowledge of fish immunity and diseases.Blood transcriptomics is emerging as a relevant tool to monitor the status of the immune system and assist in diagnosis, prognosis, treatment and pathogenesis studies of diseases. In fish pathology, the potential of transcriptome profiling of blood is still poorly explored. Here, RNA sequencing was applied to analyze the blood transcriptional profile of turbot (Scophthalmus maximus), the most important farmed flatfish. The study was conducted in healthy specimens and specimens parasitized by the myxozoan Enteromyxum scophthalmi, which causes one of the most devastating diseases in turbot aquaculture. The blood of healthy turbot showed a transcriptomic profile mainly related to erythrocyte gas transportation function, but also to antigen processing and presentation. In moderately infected turbot, the blood reflected a broad inhibition of the immune response. Particularly, down-regulation of the B cell receptor signaling pathway was shared with heavily parasitized fish, which showed larger transcriptomic changes, including the activation of the inflammatory response. Turbot response to enteromyxosis proved to be delayed, dysregulated and ineffective in stopping the infection. The study evinces that blood transcriptomics can contribute to a better understanding of the teleost immune system and serve as a reliable tool to investigate the physiopathological status of fish.

Characterization of antibacterial activities and the related mechanisms of a β-defensin in turbot (Scophthalmus maximus)

β-defensins (BDs) are small-molecule cysteine-rich cationic antimicrobial peptides that play an important role in innate immunity against microbial invasion. In this study, a β-defensin (SmBD) was identified and characterized from turbot (Scophthalmus maximus). SmBD has an open reading frame containing 192 bp and encodes a protein of 63 amino acids. It has a predicted signal peptide consisting of 20 amino acids and a mature peptide with six conserved cysteine residues forming three disulfide bonds (C1- C5, C2-C4 and C3-C6). The 3D structure prediction shows an N-terminal α-helix and three reverse parallel β-folds. SmBD showed high sequence identity (70.45% - 95.24%) with β-defensin 2 group members from other teleost species. SmBD had the highest expression in skin of healthy turbot and was significantly up-regulated in mucosal tissues (skin, gill, intestine) after bacteria challenge. SmBD showed antibacterial activity against Vibrio anguillarum, Edwardsiella tarda, Escherichia coli and Streptococcus iniae, and inhibitory effect on the formation of bacterial biofilms. SmBD changed bacterial membrane permeability and affected bacterial morphology, as shown in fluorescence microscopy and scanning electron microscopy analyses, respectively, which indicated that SmBD might exercise antibacterial behaviors via “membrane-attack” mechanism.

Molecular cloning and expression analyses of immunoglobulin tau heavy chain (IgT) in turbot, Scophthalmus maximus

Immunoglobulins (Igs) are humoral mediators playing the prevailing role in the innate and adaptive immunities of jawed vertebrates and provide obligatory duties to protect the organism from a wide variety of pathogens. In the present study, the membrane-bound and secretory immunoglobulin T (mIgT and sIgT) genes of turbot (Scophthalmus maximus) were cloned for the first time with the intention of better understanding the IgT functions. The mIgT cDNA is 2, 049 bp in length including a leader region, a variable region, four constant regions and a transmembrane region (TM), while the 1, 932 bp sIgT cDNA lacks the transmembrane region. In healthy turbot, the total IgT was highly expressed in gill, spleen and liver followed by peripheral blood leucocytes (PBL), skin and hindgut, and then in stomach, head kidney, trunk kidney, midgut and foregut. The expression levels of sIgT in PBL, gill, skin and spleen were much higher than mIgT. Furthermore, the expression profiles of turbot mIgT and sIgT were investigated post vaccination with formalin-inactivated Vibrio anguillarum via intraperitoneal injection and immersion, and the results showed that the expressions of mIgT and sIgT were both significantly induced by two administration routes, whereas intraperitoneal injection mostly induced mIgT expression in systematic organs including head kidney, spleen and trunk kidney, and the immersion vaccination elicited a much stronger response of sIgT in mucosa-associated tissues including gills, liver, hindgut and skin. Taken together, these results demonstrated that mIgT and sIgT were differentially expressed in different tissues and both responded positively to the vaccinations in turbot, and indicated that IgT-secreting plasma cells are abundant in mucosa-associated tissues and played important roles in mucosal immunity of turbot.

Identification and expression analysis of toll-like receptor genes (TLR8 and TLR9) in mucosal tissues of turbot (Scophthalmus maximus L.) following bacterial challenge

Mucosal immune system is one of the most important components in the innate immunity and constitutes the front line of host defense against infection, especially for teleost, which are living in the pathogen-rich aquatic environment. The pathogen recognition receptors (PRRs), which can recognize the conserved pathogen-associated molecular patterns (PAMPs) of bacteria, are considered as one of the most important component for pathogen recognition and immune signaling pathways activation in mucosal immunity. In this regard, we sought to identify TLR8 and TLR9 in turbot (Scophthalmus maximus), as well as their mucosal expression patterns following different bacterial infection in mucosal tissues for the first time. The full-length TLR8 transcript consists of an open reading frame (ORF) of 3108 bp encoding the putative peptide of 1035 amino acids. While the TLR9 was 6730 bp long, containing a 3168 bp ORF that encodes 1055 amino acids. The phylogenetic analysis revealed both TLR8 and TLR9 showed the closest relationship to large yellow croaker. Moreover, both TLR8 and TLR9 could be detected in all examined healthy turbot tissues, with the lowest expression level in liver and a relatively moderate expression pattern in healthy mucosal tissues. Distinct expression patterns of TLR8 and TLR9 were comparatively observed in the mucosal tissues (intestine, gill and skin) following Vibrio anguillarum and Streptococcus iniae infection, suggesting their different roles for mucosal immunity. Further functional studies are needed to better characterize TLR8 and TLR9 and their family members, to better understand the ligand specificity and to identify their roles in different mucosal tissues in protecting fish from the pathogenically hostile environment.

Histological study as indicator of juveniles farmed turbot, Scophthalmus maximus L. health status

Several tools can be used to evaluate the health status of fish. Histology is one of them because it assesses pathological changes induced by environment, pathogens and/or nutritional factors. The histological condition of macroscopically healthy turbot juveniles in the beginning of the growing phase in an intensive fish farm was evaluated. The gill, skin, liver, kidney and intestine were histologically processed by standard techniques and their histological status determined semi-quantitatively. The skin, the gills and the liver of examined turbot were in a good overall condition or presented slight alterations frequently observed in farmed fish. The kidney and the intestine of some fish presented pronounced histological alterations caused mainly by the occurrence of nephrocalcinosis in the kidney, and necrotic areas in intestinal lamina propria of the mucosa. The possible causes of these findings are discussed. Statement of relevance“This is a very first histology based survey conducted in an intensive farm to evaluate the health status of turbot juveniles. The results showed that i) the histological analysis is a useful tool for assessing the fish health and, ii) most of the fish were in good health condition”

Tissue Localization of Lymphocystis Disease Virus (LCDV) Receptor-27.8 kDa and Its Expression Kinetics Induced by the Viral Infection in Turbot (Scophthalmus maximus).

The 27.8 kDa membrane protein expressed in flounder (Paralichthys olivaceus) gill cells was proved to be a receptor mediating lymphocystis disease virus (LCDV) infection. In this study, SDS-PAGE and Western blotting demonstrated that 27.8 kDa receptor (27.8R) was shared by flounder and turbot (Scophthalmus maximus). Indirect immunofluorescence assay (IIFA) and immunohistochemistry showed that 27.8R was widely expressed in tested tissues of healthy turbot. The indirect enzyme-linked immunosorbent assay indicated that 27.8R expression was relatively higher in stomach, gill, heart, and intestine, followed by skin, head kidney, spleen, blood cells, kidney and liver, and lower in ovary and brain in healthy turbot, and it was significantly up-regulated after LCDV infection. Meanwhile, real-time quantitative PCR demonstrated that LCDV was detected in heart, peripheral blood cells, and head kidney at 3 h post infection (p.i.), and then in other tested tissues at 12 h p.i. LCDV copies increased in a time-dependent manner, and were generally higher in the tissues with higher 27.8R expression. Additionally, IIFA showed that 27.8R and LCDV were detected at 3 h p.i. in some leukocytes. These results suggested that 27.8R also served as a receptor in turbot, and LCDV can infect some leukocytes which might result in LCDV spreading to different tissues in turbot.

Immunolocalization of tumor necrosis factor alpha in turbot (Scophthalmus maximus, L.) tissues

Tumor necrosis factor alpha (TNFα) is a cytokine involved in a broad spectrum of cellular and organismal responses. Its main function, as a potent pro-inflammatory mediator, has been demonstrated in numerous teleost species and there are many reports on the modulation of TNFα gene expression under pathological conditions. Nevertheless, there is still scarce knowledge about the tissue distribution and type of cells that express this cytokine in fish species, which would help to further investigate its biological activities. These studies are hampered by the lack of molecular markers for teleost that hinder the development of morphological techniques, like immunohistochemistry. The aim of this work was to develop an immunohistochemical technique for the detection of TNFα in paraffin-embedded organs from healthy turbot (Scophthalmus maximus), an economically-important marine fish species. A commercial anti-human TNFα antibody, whose specificity was confirmed by western blot analysis, was used. Immunoreactive cells were observed in higher numbers in the lymphohematopoietic organs, kidney, spleen and thymus, although TNFα-positive cells were also present in the digestive tract, liver, heart, gills and skin. Similarly to non-fish species, monocytes/macrophages appeared to be the main producers of this cytokine; nevertheless, the presence of immunoreactive rodlet cells in different tissues was also reported. The nature and distribution of the labeled cells appeared to be related with a strategic localization for defense response to antigenic challenge. The relative abundance of TNFα-positive cells in the lymphohematopoietic organs also suggests that this cytokine may have a broader role in the normal physiology of those organs. The immunohistochemical technique allowed the in-situ characterization of TNFα expression, representing a valid tool to investigate the immune response of turbot.

Increased survival of juvenile turbot Scophthalmus maximus by using bacteria associated with cultured oysters.

Preventing vibriosis in juvenile cultured Turbot Scophthalmus maximus caused by Vibrio anguillarum frequently requires the use of feed supplemented with antibiotics in addition to vaccines. Whether the use of probiotics instead of antibiotics in juvenile Turbot is a safer strategy requires more study. The antibacterial potential of 148 Vibrio spp. strains (mostly isolated from cultures of healthy oysters, clams, and Turbot) was analyzed in vitro against V. anguillarum and other pathogens by means of an agar diffusion assay. A wide spectrum of inhibitory activity was shown by 9 strains. Based on their easy phenotypic differentiation from V. anguillarum, we selected two strains (S1 and S2, both isolated from the European flat oyster Ostrea edulis) for testing in juvenile Turbot (3 g). None of the strains were virulent by intraperitoneal or bath challenges, and all were susceptible to the antibiotics most frequently used in aquaculture. Three different stocks of Turbot, which were assayed separately, were significantly protected from infection with V. anguillarum. The final survival rates of fish treated in mixed challenges with S1 or S2 and V. anguillarum were 44% and 66%, respectively, whereas only 17% of the fish treated with only the pathogenic strain survived. The application of probiotic strains also increased the survival time of juvenile Turbot after infection with V. anguillarum. Both strains persisted in the epidermal mucus layer of the fish for 30 d, and they were not displaced by the pathogen. These data prove the efficacy of using bacteria well adapted to the dynamics of culture production as a way to provide juvenile Turbot immediate protection against infection by V. anguillarum. Moreover, the epidermal mucus sampling was useful for investigating the persistence of both probiotic strains when exposed to the pathogen.

Molecular cloning and characterization of secretory and membrane-bound IgM of turbot

In recent years, increasing diseases especially bacterial diseases have brought a host of losses with the expansive cultivation of turbot (Scophthalmus maximus). In order to do more research about the immune system of turbot for better understanding the mechanism of resisting diseases, the immunoglobulin genes related to secretory and membrane-bound IgM (s-IgM and m-IgM) of turbot were cloned using homology sequences cloning and SMART RACE PCR method. The heavy chain of s-IgM cDNA is 1900 bp in length including a leader region, a variable region, four constant regions (CH1, CH2, CH3 and CH4) and a C-terminal while the cDNA of m-IgM is 1795 bp with the same leader region, variable region, three constant regions (CH1, CH2 and CH3) and two transmembrane regions (TM1 and TM2). The sequence of IgM gene was also obtained and the structure consisted of V–CH1–CH2–CH3–CH4-TM1-TM2 is similar to other fishes. The highest level of s-IgM expression was observed in spleen, followed by kidney, gills, eyes, skin of the healthy turbot whereas the same profile of m-IgM expression is found with low level. And s-IgM takes up dominant proportion of total IgM expression. Also the relative expressions of s-IgM and m-IgM were analyzed in turbot vaccinated with the live attenuated vaccine Vibrio anguillarum. Not only the transcriptions of both s-IgM and m-IgM in liver, spleen and kidney of turbot injected with V. anguillarum MVAV6203 were up-regulated but also the expressions of s-IgM and m-IgM in spleen, kidney, gut, skin and gills of bath-vaccinated turbot were increased. Comparing the ratio changes of relative expression of m-IgM and s-IgM in vaccinated turbot, we found that the proportion of m-IgM were increasing in both administration routes, which probably indicated that the increasing expression of m-IgM strengthen the phagocytic ability of B cells.

Cloning and expression analysis of interferon regulatory factor 7 (IRF-7) in turbot, Scophthalmus maximus

Interferon regulatory factor (IRF) 7 is known as the master regulator of type I interferon (IFN)-dependent immune responses in mammals. In this study, the cDNA and genomic sequences of turbot ( Scophthalmus maximus) IRF-7 ( SmIRF-7) were cloned and found to encode a putative protein of 439 amino acids. The gene is composed of 10 exons and 9 introns similar to known IRF-7 genes of fish. The SmIRF-7 shows the highest amino acid identity of 49.0–80.3% to fish IRF-7 and possesses a DNA-binding domain (DBD), an IRF association domain (IAD) and a serine-rich domain (SRD) of vertebrate IRF-7. In addition, the tryptophan cluster of SmIRF-7 DBD consists of only four tryptophans, which is a characteristic unique to all fish IRF-7 members. The SmIRF-7 transcripts were expressed constitutively in all analyzed tissues of healthy turbot, with higher levels observed in immune relevant tissues. Gene expressions of SmIRF-7 and Mx were monitored over a 7-day time course by quantitative real time PCR in head kidney and muscle of turbot challenged with turbot reddish body iridovirus (TRBIV), which is a prevalent viral pathogens in farmed turbot in China. Both genes were up-regulated by TRBIV although their inducibility was much weaker in the muscle. The peak levels of SmIRF-7 transcripts were detected at day 2 post-infection in the two organs with a 12- and 4.5-fold increase, respectively. Further, the Mx showed two waves of induced expression and the maximum expression of SmIRF-7 arose earlier than the second wave of the Mx expression in both organs. These findings contribute to an understanding of functions of SmIRF-7 in antiviral response.

Identification of a marine antagonistic strain JG1 and establishment of a polymerase chain reaction detection technique based on the gyrB gene

A marine antagonistic bacterium, JG1, was isolated from rearing water of healthy turbot (Scophthalmus maximus) in Qingdao, China. Strain JG1 was Gram-negative, straight rod and motile by polar flagella. The colony, when cultured for 24 h under room temperature, produced a yellow pigment. On the basis of morphological, physiological and biochemical characteristics, along with 16S rDNA sequence analysis, JG1 was identified as Pseudoalteromonas flavipulchra. JG1 had good inhibitory effects on several bacterial pathogens of aquaculture in the genus Vibrio (V. anguillarum, V. alginolyticus, V. campbellii, V. harveyi, V. mimicus, V. parahaemolyticus and V. tubiashii) and Aeromonas (A. hydrophila and A. salmonicida). No mortality occurred 14 days after zebra fish and 7 days after mantis shrimp were intraperitoneally injected with JG1 at 106 CFU per animal, and 7 days after scallop and clam were immersed in JG1 at 107 CFU mL−1. A good antagonistic effect on several bacterial pathogens and nontoxicity to the above-mentioned animals make JG1 a potential probiotic in aquaculture. In addition, a fast detection technique based on polymerase chain reaction amplification of the gyrB gene was established to allow us to determine the fate of JG1 when it is applied in aquaculture in the future.

Histopathology of experimental scuticociliatosis in turbot Scophthalmus maximus

A scuticociliate strain (B-2), originally isolated from an outbreak in a turbot Scophthalmus maximus (= Psetta maxima) farm in Galicia (northwestern Spain) and maintained in axenic culture, was injected intracoelomically (lethal dose 80 equivalent, LD80) in healthy turbot (50 g). Ciliate-injected fish were kept under controlled conditions in a recirculating seawater system and sampled on Days 1 through 8, 10, 12 and 14 postinfection (PI). Necropsies were conducted and included blood collection from the caudal vein and samples of liver, spleen, heart, digestive tract, kidney, gills, abdominal wall and neurocranium taken for routine histology. Mortality occurred from Day 6 until Day 12 PI and reached 66.7% by the end of the experiment. Presence of ciliates in the coelomic fluid was scarce until Day 4 PI. Parasitaemia was only observed from Day 5 until Day 10 PI and its incidence was always low. Presence of scuticociliates in tissue sections followed a progressive pattern of diffusion, with ciliates showing preference for loose connective tissue and also a clear haematophagous activity. The most severely affected organs were the pancreas and digestive tract. No special tropism for nervous tissues was observed in this study. The inflammatory reaction was variable depending on the tissue. After 3 wk, survivors had apparently managed to extinguish the infection.

Genotypic diversity of Vibrio isolates associated with turbot ( Scophthalmus maximus) culture

One hundred environmental strains of Vibrio isolated from seawater and skin of healthy turbot in an aquaculture system were analyzed. Chromosomal DNA was digested with HindIII and MluI, and hybridized using a digoxigenin-labeled probe complementary to 16S and 23S rRNA of Escherichia coli. Nineteen riboclusters were defined by ribotyping analysis at a value of S D ⩾ 70 % , using the Dice coefficient ( S D ) and UPGMA. The phylogenetic position of each ribocluster was achieved by 16S rRNA gene sequencing of representative isolates. Both techniques were necessary and useful for identifying the isolates. V. parahaemolyticus, V. scophthalmi, V. splendidus– V. lentus related group, V. halioticoli, V. fischeri and V. ichthyoenteri were identified and clustered separately. Their ribocluster diversity was studied. Throughout the year, ribotypic profiles of corresponding strains isolated from both seawater and turbot skin appeared, indicating that environmental strains can easily colonize turbot. No correspondence between riboclusters and the season of isolation was found. Some ribotypes had been found in previous studies, demonstrating that ribotyping is a useful tool for monitoring environmental isolates and to finding strains that can colonize aquatic organisms and are able to produce outbreaks. The ribotype schemes defined here can be used as a ribotype database of environmental isolates of these species.

Isolation in cell culture and detection by PCR‐based technology of IPNV‐like virus from leucocytes of carrier turbot, Scophthalmus maximus (L.)

A non-destructive procedure was utilized to determine the infectious pancreatic necrosis virus (IPNV) status of an apparently healthy turbot broodstock. Blood samples were used to detect IPNV by reverse transcriptase-polymerase chain reaction (RT-PCR), Southern blot hybridization and nested PCR. In addition, viral isolation from turbot leucocytes was performed. Around 22% of the fish were IPNV positive by RT-PCR, and this increased to close to 60% when nested PCR was performed. The present report supports the use of blood samples for the detection of IPNV-like viruses in brood fish. In addition, we demonstrate that it is possible to isolate the virus from the blood of carrier fish, as a non-lethal detection method, although it is much less sensitive than RT-PCR and nested PCR as a IPNV-like strain was isolated from only five of the 15 blood sample pools assayed. The viral isolate was identified as type Dry Mills (genogroup I) by means of restriction fragment length polymorphisms and DNA sequencing.

Colonization of Vibrio pelagius and Aeromonas caviae in early developing turbot (Scophthalmus maximus L.) larvae.

Polyclonal antisera made in rabbits against whole washed cells of Vibrio pelagius and Aeromonas caviae were used for detection of these bacterial species in the rearing water and gastrointestinal tract of healthy turbot (Scophthalmus maximus) larvae exposed to V. pelagius and/or Aer. caviae. The results demonstrated that this method is suitable for detection of V. pelagius and Aer. caviae in water samples and larvae at population levels higher than 10(3) ml-1 and 10(3) larva-1. Populations of aerobic heterotrophic bacteria present in the gastrointestinal tract of turbot larvae, estimated using the dilution plate technique, increased from approximately 4 x 10(2) bacteria larva-1 on day 3 post-hatching to approximately 10(5) bacteria fish-1 16 days post-hatching. Sixteen days after hatching, Vibrio spp. accounted for approximately 3 x 10(4) cfu larva-1 exposed to V. pelagius on days 2, 5 and 8 post-hatching. However, only 10(3) of the Vibrio spp. belonged to V. pelagius. When larvae were exposed to Aer. caviae on day 2 post-hatching, the gut microbiota of 5-day old larvae was mainly colonized by Aeromonas spp. (10(4) larva-1), of which 9 x 10(3) belonged to Aer. caviae. Later in the experiment, at the time when high mortality occurred, 9 x 10(5) Aer. caviae were detected. Introduction of V. pelagius to the rearing water seemed to improve larval survival compared with fish exposed to Aer. caviae and with the control group. It was therefore concluded that it is beneficial with regard to larval survival to introduce bacteria (V. pelagius) to the rearing water.

Siderophore production and probiotic effect ofVibriosp. associated with turbot larvae,Scophthalmus maximus

Resume The proportion of vibrio E, dominant in healthy turbot larvae, was artificially increased in the rotifer enrichment medium. The main effect of this vibrio enrichment was to improve the resistance of larval turbot challcngcd with a pathogenic strain of Vibrio sp1endidu.s. vibrio P. The improvement of the sumival rates was significant with 1.5 x 10' CFU of vibrio E added per ml of enrichment medium, and it was still maximum at the highest dose tested (5 x lu7 CFU.~~.'; 80 I 2% of survivors after 48 h of challenge, versus 50 I 2 '36 for the control group). Further in vitro characterisation showcd that vibrio E was able to grow in the presence of the chelating agents EDDHA and EDTA, while producing siderophore, as revealed by chrome azurol S dyeing. The pathogenic vibrio P did not grow on iron-depleted medium, and it did not seem to produce siderophore. Consequently, a second expcriment attempted to appraise the role of iron availability in the interaction between the pathogenic and probiotic vibrios. To this end, turbot larvae were fed with rotifers treated by the bacterial siderophore deferoxamine. This trcatment significantly increased the resistance of larvae challenged with vibrio P in comparison with the control group, but tu a Iesser extent than the group enriched with vibrio E. There was also an increase of the growth rate due to either the siderophore treatment or the vibrio enrichment, as well. No further improvement was observed when the rotifers were simultaneously both treated and enriched. It was concluded that the probiotic effect of vibrio E may be at least partly due to competition for iron with the pathogen.

A sandwich immunoassay to quantify low levels of turbot ( Scophthalmus maximus) immunoglobulins

We have developed an enzyme-linked immunosorbent assay for the quantification of turbot ( Scophthalmus maximus) immunoglobulin (Ig). The capture antibody is a rabbit polyclonal antiserum to turbot Ig, and the detector antibody a monoclonal antibody (UR3) to the turbot Ig heavy chain. Both antibodies bind nearly 100% of turbot Ig. The assay allows detection of turbot Ig in serum at concentrations as low as 0.16 μg ml −1 and takes less than 4 h. Precision is satisfactory, with intra-assay coefficients of variation (CVs) ranging from 2.1 to 16.6%, and inter-assay CVs ranging from 5.8 to 24.6%. We used the assay to determine Ig concentrations in the sera of healthy turbot of different weights. Mean serum Ig concentration was 3.35 ± 0.74 mg ml −1 for fish weighing 15–25 g and 11.14 ± 1.87 mg ml −1 for fish weighing 1000–2000 g.

Flavobacterium scophthalmum sp. nov., a Pathogen of Turbot (Scophthalmus maximus L.)

Fifty orange-pigmented, gram-negative, rod-shaped isolates were recovered from healthy and diseased turbot and from coastal waters (collected in Scotland). On the basis of the results of an examination of 125 phenotypic characteristics and the results of DNA-DNA and DNA-rRNA hybridization experiments, we concluded that these isolates are members of a new species in the genus Flavobacterium, for which the name Flavobacterium scophthalmum is proposed. The type strain is CCM 4109 (= LMG 13028).

Microflora associated with healthy and diseased turbot ( Scophthalmus maximus) from three farms in northwest Spain

A comparative analysis of the microbiological quality of three turbot ( Scophthalmus maximus) farms (A, B, and C) located in Galicia (northwest Spain) is given. The microbial load and types of bacteria in the internal organs (liver and kidney) of apparently healthy fish was monitored over a year, and all the disease problems occurring during this survey were analyzed. The percentage of healthy turbot in which positive bacterial growth was obtained was relatively high in the three ongrowing facilities. Farm A exhibited the poorest conditions of fish health with an average of 42% fish infected, while farm B showed the best microbiological quality with 27% of turbot harbouring bacteria in the internal organs. In all three farms, a wide range of bacteria was found in healthy turbot with Vibrio ( V. splendidus-V. pelagius, V.fisheri-V. harveyi and Vibrio spp.) and Pseudomonas spp. being the predominant groups comprising at least 80% of the total bacterial isolates in each farm. The highest number of pathological problems (22) with the most diverse bacterial flora occurred in farm A. Vibrio spp. and Pseudomonas spp. were the most prevalent bacteria recovered from diseased turbot. Haemorrhages in palate and jaws, tail and fins, and ulcerative lesions were the most frequent external clinical sings of diseased fish recorded in the three farms. However, it was not possible to associate a particular bacterial species with a specific pathology. Routine use in farm A of oxolinic acid and nitrofurantoin may have led to the development in the Vibrio strains of resistances to both chemotherapeutants (up to 25%).

Quantitative and qualitative studies of the bacterial microflora of turbot, Scophthalmus maximus L., gills

Populations of aerobic heterotrophic bacteria, occurring on the gills of healthy turbot, were estimated using a dilution plate technique. From a comparison of 18 media, the highest counts, i.e. 7.0 × l05 g−1, were obtained after incubation at 15–25°C on a specifically formulated medium which contained low quantities of beef extract, casein, tryptone and yeast extract. These bacteria were equated with Asticacaulis sp., Hyphomicrobium sp., Janthinobacterium lividum, Prosthecomicrobium sp., Pseudomonas fluorescens and Vibrio sp. Evidence from scanning electron microscopy pointed to a general lack of intimate colonization of exposed areas of the gill. Instead, micro‐organisms colonized protected niches, such as the clefts between lamellae and in secluded areas on the arches.