Skeletal anomalies in reared Senegalese sole Solea senegalensis juveniles: a radiographic approach.

Reared Senegalese sole Solea senegalensis Kaup show a high incidence of vertebral anomalies; however, little is known about its skeletal anomaly profile in the later farming phases. The purpose of this study was to provide a detailed description and quantification of the most common skeletal anomalies in reared Senegalese sole in the juvenile stage by means of computed radiography. A total of 374 Senegalese sole were classified according to the external morphology of the fish as normal or altered and then radiographed in latero-lateral and in dorso-ventral projections. Radiographic evaluation of anomalies focused especially on vertebral body anomalies (VBA) and vertebral column deviations (VCD). The 2 orthogonal projections provided a more complete visualization of the skeleton. Approximately 75% of the individuals showed at least 1 anomaly, while VBA and/or VCD were detected in 48.9% of the specimens. Regarding external morphology, 88% of the fish were categorized as normal, although about 72% of these normal fish displayed abnormalities in radiographies. The most frequent anomalies consisted of deformations of the caudal complex plates (hypurals, parhypural and epural), preurals and caudal vertebrae. Scoliosis was the most prevalent among VCD, affecting the caudal area in almost 15% of the individuals. The anomaly profile at the juvenile stages showed some differences compared to what has been reported previously in earlier stages of development. In light of these results, further investigation into the progression of skeletal anomalies over time and the causative factors at later stages is required.

New insight on vertebral anomalies in cultured Senegalese sole (Solea senegalensis, Kaup) at early stages of development.

Senegalese sole (Solea senegalensis, Kaup) is a promising flatfish species in aquaculture. However, skeletal anomalies are still a great concern in sole farming. Investigation of this issue is crucial to improving larval quality and optimizing production. The aim of this study was to thoroughly assess anomalies in the rachis of reared sole at early developmental stages. Sole (n = 507) were sampled at 31 or 32 days after hatching (dah). The specimens were stained with alcian blue and alizarin red and evaluated for the detection of vertebral deformities. Most fish presented 9:34:3 vertebrae in abdominal, caudal and caudal complex regions, respectively. Remarkably, all specimens showed at least one spinal anomaly. Alterations of neural/haemal elements, as well as deformities of hypurals, parhypural and epural, were recurrent. Vertebral body anomalies and/or vertebral column deviations were identified in 52% of the individuals. Vertebral deformations and fusions were common, especially in caudal complex. 'Minor' anomalies were predominant, and some of the detected disorders might be a result of non-/low-pathological processes. These results contribute a new insight into the main skeletal anomalies affecting cultured sole larvae. Further research is required to determine their impact on fish welfare and external appearances at commercial stages.

Immunohistochemical diagnosis of tenacibaculosis in paraffin-embedded tissues of Senegalese sole Solea senegalensis Kaup, 1858.

A sensitive and specific immunohistochemical technique was developed to improve the diagnosis of tenacibaculosis and to better understand its pathogenesis. Senegalese sole Solea senegalensis Kaup, 1858 were inoculated subcutaneously with a bacterial suspension of Tenacibaculum maritimum, and samples were taken at different hours post-inoculation. Sections from different organs were used as positive controls. In addition, a total of 128 field samples from different organs collected from tenacibaculosis outbreaks were used. Tenacibaculum maritimum antigens were detected in several organs of experimentally infected Senegalese sole and in at least one of the tissues from fish suffering from natural tenacibaculosis previously confirmed by culture and PCR-based methods. In fish collected during outbreaks, a strong positive reaction was detected in ulcerative skin areas. Moreover, bacterial antigen was identified inside scale pockets and in sites of the skin with mild lesion. In kidney and spleen, evident immunostaining of bacterial antigen was detected in both naturally and experimentally infected fish. Besides, the presence of T. maritimum in the intestinal tract without associated histological changes suggests that this organ may act as a reservoir for T. maritimum. The results of this study confirm the usefulness of IHC for the diagnosis of tenacibaculosis in paraffin-embedded tissues.

Morphopathological features of a severe ulcerative disease outbreak associated with Tenacibaculum maritimum in cultivated sole, Solea senegalensis (L.).

This study describes morphopathologic changes in naturally infected farmed Senegalese sole affected by tenacibaculosis caused by Tenacibaculum maritimum. Macroscopic observation, in addition to light microscopy and scanning electron microscopy, was used to study the lesions. Main lesions were characterized by complete loss of epidermis and dermis, as well as extensive necrosis of muscle layers. Mild-to-moderate inflammatory response with the presence of macrophages was noted around hyaline degenerated muscle cells. Gram-negative filamentous bacteria could be detected only at the dermis. Under scanning electron microscopy, filamentous bacteria located over the scales without epithelium could be observed. These findings together with the isolation and PCR detection of the bacteria in kidney and skin tissues suggest that once the bacteria reach the dermis, probably through eroded epidermis, they are able to proliferate and produce enzymes that are responsible for the damage in the underlying tissues.

Chemical degradation of PCB in the contaminated soils slurry: direct Fenton oxidation and desorption combined with the photo-Fenton process.

This paper reports the results of the direct treatment of PCBs sorbed to solid particles (glass beads and sand) employing the Fenton process. The results obtained with contaminated sandy soils show 98% removal of the original PCB structure and 82% dechlorination, all within a reaction time of 72 hours. The degree of removal was observed to be dependent on the level of congener chlorination. The optimized conditions were: 5% H(2)O(2); 100 ppm Fe(3+); sandy soil mass/volume of oxidizing solution ratio (m/V) of 1/3 g/mL, vigorous agitation and dispensed with the need for heat. Results obtained by applying an integrated desorption treatment followed by photo-Fenton oxidation to sandy soils contaminated with PCBs are also present in this paper. Desorption of PCBs with surfactant solutions took place to an extent of around 90% (92.2% with K-perfluoroalkyl sulfonate, FT800, and 87% with Lineal Alkyl benzene Sulfonate, LAS), while photo-Fenton oxidation at 254 nm achieved degradation percentages close to 100% in 30 minutes for PCBs in solution, both with FT800 (98%) and LAS (97%) surfactants.

Optimization of the solution parameters during the degradation of Orange II in a photo-reactor mediated by Fe-Nafion membranes.

The Fenton and photo-Fenton mediated degradation process of Orange II was investigated in a flow photo-reactor. The degradation was monitored as a function of the wavelength of the applied light, recirculation flow rate, amount of H(2)O(2) and the initial concentration of Orange II. Optimization of the photo-Fenton degradation mediated by Fe-Nafion membranes indicated that an Orange II (0.25 M) solution discolored above 95% within 2.5 hours at an H(2)O(2)/Orange II ratio of 20. A concomitant mineralization of 40% of Orange II was observed after 5 h reaction. Homogeneous photo-Fenton processes were able to fully discolore Orange II within 1 hour and concomitantly fully mineralize the dye in the presence of Fe(III) (2 ppm) and an H(2)O(2)/Orange II ratio of 20. Surfactants such as linear alkylbenzene sulphonates (LAS) and K-perfluoroalkyl sulphonate (FT 800) slowed down the Orange II abatement in photo-Fenton processes.

Comparative gene expression of gonadotropins (FSH and LH) and peptide levels of gonadotropin-releasing hormones (GnRHs) in the pituitary of wild and cultured Senegalese sole (Solea senegalensis) broodstocks.

The Senegalese sole (Solea senegalensis) is a valuable flatfish for aquaculture, but it presents important reproductive problems in captivity. Spawning is achieved by wild-caught breeders but cultured broodstocks fail to spawn spontaneously and, when they do, eggs are unfertilized. To gain knowledge on the physiological basis underlying this reproductive dysfunction, this study aimed at analyzing comparative hormone levels between wild and cultured broodstocks at the spawning season. The Senegalese sole gonadotropin (GTH) subunits, FSHbeta, LHbeta and GPalpha, were cloned and qualitative (in situ hybridization) and quantitative (real-time PCR) assays developed to analyze pituitary GTH gene expression. In females, FSHbeta and GPalpha mRNA levels were higher in wild than in cultured broodstocks, whereas in males all three subunits were highest in cultured. By ELISA, three GnRH forms were detected in the pituitary, displaying a relative abundance of GnRH2>GnRH1>GnRH3. All GnRHs were slightly more abundant in wild than cultured females, whereas no differences were observed in males. Plasma levels of vitellogenin and sex steroids were also analyzed. Results showed endocrine differences between wild and cultured broodstocks at the spawning period, which could be related to the endocrine failure of the reproductive axis in cultured breeders.

Protective acquired immunity to Enteromyxum scophthalmi (Myxozoa) is related to specific antibodies in Psetta maxima (L.) (Teleostei).

The acquired protection of three groups of turbot that had survived enteromyxosis outbreaks was tested by challenging with E. scophthalmi in three different experiments. The relation of such a response with the kinetics and duration of antibody production (determined by an ELISA) was studied. The progression of the infection was evaluated by PCR. In experiments 1 and 2, in which turbot had cohabited with highly infected fish during outbreaks, parasite prevalence and mortality were very low or null, and there was a progressive and statistically significant increase in the mean antibody production up to 350 and 152 days post-exposure respectively. By contrast, in experiment 3, fish (coming from non-infected tanks during the initial outbreak), both infection prevalence and cumulative mortality reached 92.8%, and specific antibodies were detected only in two fish. The observed differences in mortality after challenge appear to be related to the production of specific antibodies and it is probably accompanied by a repertoire of mechanisms of innate immunity. The exploitation of the immune system through breeding selection programmes as a possible strategy to control the disease is discussed.

Risk factors associated with Enteromyxum scophthalmi (Myxozoa) infection in cultured turbot, Scophthalmus maximus (L.).

An epidemiological cohort study of Enteromyxum scophthalmi in cultured turbot was performed on a farm in North Western Spain. Four different ongrowing stocks (A, B, C, D) were monitored monthly until market size. Fish from stocks C and D were divided into 2 subgroups, receiving filtered (CF and DF) or unfiltered (CUF and DUF) water. The lack of water filtration was positively associated with infection prevalence, as all fish kept in filtered water remained uninfected. Parasite abundance varied seasonally (P<0.05) in stock B and subgroup CUF. Infection was also associated (P<0.05) with host weight, and the highest prevalences and intensities were detected in 101-200 g and 201-300 g fish. Distribution pattern of E. scophthalmi in subgroups CUF and DUF had a variance higher than the mean, indicating overdispersion. The minimum period necessary for the first detection of the parasite and for the appearance of disease symptoms and mortality, varied depending on the stock and introduction date, although a long pre-patent period was always observed. Several factors, such as host density, parasite recruitment and parasite-induced fish mortality can contribute to the observed distribution pattern. Risk factors found to be associated with E. scophthalmi infection, including water quality and accumulation of infective stages in the culture tanks, should be considered when designing control strategies to prevent the introduction and spread of infective stages in the facilities.

Innate and adaptive immune responses of turbot, Scophthalmus maximus (L.), following experimental infection with Enteromyxum scophthalmi (Myxosporea: Myxozoa).

The innate and adaptive immune responses against Enteromyxum scophthalmi was studied in turbot (Scopthalmus maximus (L.)) experimentally exposed to the parasite by cohabitation. Haematological, histopathological, cellular and humoral factors were determined in samples taken from control (CTRL) and recipient (RCPT, naïve fish cohabited with donor infected fish) animals at 0, 20, 29, 40 and 43 days post exposure (p.e). Infection was first detected at day 20 p.e. and prevalence reached 100% at 40 days p.e, when first mortalities occurred. A significant reduction in weight and condition factor was found in RCPT, though no significant differences in haematocrit or serum protein levels were detected between CTRL and RCPT. Some immune effectors were clearly activated in RCPT: the percentage of circulating granulocytes was significantly increased, as well as the number of blood cells positive in the respiratory burst assay; leucocyte infiltration in intestine was found mainly on days 20 and 29 p.e.; total serum antiproteases and alpha-2-macroglobulin levels were higher in most of the samplings, with significant differences on the last sampling. Other effectors were clearly down regulated in RCPT: haematopoietic depletion appeared in head kidney from day 29 p.e. onwards, and the number of apoptotic cells and MMC increased in head kidney and spleen; the percentage of lymphocytes decreased progressively and significantly; a clear, but not statistically significant, drop in serum complement was registered at 40 days p.e.; also, a significant decrease occurred in serum lysozyme at 29 days p.e. No specific antibodies against the parasite were detected in any sampling.

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

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

Efficacy of furunculosis vaccines in turbot, Scophthalmus maximus (L.): evaluation of immersion, oral and injection delivery.

The commercial furunculosis vaccine Aquavac Furovac 5 and an autogenous vaccine, based on the challenge strain, induced immune protection in turbot, Scophthalmus maximus (L.), as shown in challenge tests 120 days post-immunization by injection (relative percentage of survival, RPS = 72-99%). This protective effect lasted for at least 6 months post-immunization at appreciable levels (RPS = 50-52%). Neither the autogenous vaccine nor the commercial vaccine was able to induce significant levels of protection against Aeromonas salmonicida in turbot when administered by immersion. Antibody levels were high or moderate in fish vaccinated by injection with the different vaccines and very low in fish vaccinated by immersion. The field results show that delivering an oral boost after the primary vaccination by injection did not enhance protection of turbot against furunculosis and that water-based (autogenous vaccine) and oil adjuvanted (Alpha Ject 1200) vaccines administered by injection conferred similar levels of protection (RPS > 80%) in turbot.

Development of immunohistochemistry and enzyme-linked immunosorbent assays for the detection of circulating antibodies against Enteromyxum scophthalmi (Myxozoa) in turbot (Scophthalmus maximus L.).

Immunohistochemistry and enzyme-linked immunosorbent assays were developed for the detection of specific antibodies against the myxosporean parasite Enteromyxum scophthalmi in turbot (Scophthalmus maximus L.). Fish which had survived a previous epizootic were exposed to the parasite by cohabitation with infected animals, and 83 days later the plasma was tested for the presence of antibodies. Plasma of non-exposed fish was used as negative control. Immunohistochemistry (IHC) using rabbit anti-turbot IgM antibody was first used to detect these antibodies, and to study to which parasite structures they were directed against. Also, an antibody-ELISA using whole cell lysates of the parasite as antigen, and a monoclonal antibody anti-turbot IgM, was developed. All the exposed fish were found to have specific antibodies against the parasite, and none of them developed signs of disease or died during the experiment. Primary cells were the main parasite stage immunolabelled, and the staining was distinctly located on the cytoplasm and the cytoplasmic membrane. IHC was more sensitive than ELISA, as the endpoint was two to four fold higher with the former technique. Although there was great individual variation, the antibody titres found can be considered high, reaching up to 1:32,000 with ELISA and 1:64,000 with IHC. The results suggest that turbot showing acquired immunity against E. scophthalmi, could develop resistance against new infections.