Jesús Lamas

Effect of oral administration of glucans on the resistance of gilthead seabream to pasteurellosis

Abstract The present study evaluated the effect of oral administration of three types of glucan on the resistance of gilthead seabream to Photobacterium damselae subsp. piscicida and on the activity of its phagocytes. Groups of fish were fed with a basal diet (controls) or with the basal diet supplemented with glucan for two different periods of time before being bath challenged with the bacterium. Groups of fish fed with 1 and 10 g kg −1 of glucan for a short period (2 weeks with glucan and 1 week with the basal diet) showed a higher degree of protection against pasteurellosis than the control group. This was especially pronounced in groups of fish fed with the higher concentrations of glucan. The respiratory burst and phagocytic activity of spleen phagocytes varied with time but no significant differences were found between groups in the third week when the challenge was carried out. The group of fish fed with 1 g kg −1 of glucan for longer periods of time (2 weeks with glucan, 1 week with the basal diet, 2 weeks with glucan, and 1 week with the basal diet) also showed enhanced protection against P. damselae . However, the group fed with 5 g kg −1 had mortality rates comparable to the control group, and protection in the group fed with 10 g kg −1 of glucan was significantly lower than in the control group. These results suggest that glucans can be used in the diet to prevent or reduce mortalities in gilthead seabream due to pasteurellosis, and they show the importance of the concentration and the period of administration of glucan to obtain optimal protection against this disease.

Efficacy of intraperitoneal and immersion vaccination against Enterococcus sp. infection in turbot

In this study the effectiveness of a toxoid-enriched whole-cell bacterin (ET-2) in cultured turbot (Scophthalmus maximus) against Enterococcus sp. was evaluated by bath immersion and intraperitoneal (i.p.) injection. In addition, the influences of fish size as well as enrichment of the standard semimoist diet with the commercial preparation Trouvitol plusR, which contains β-glucan from yeast, were also investigated. Regardless of fish size, type of diet, and bacterial dose employed in the experimental challenges, our Enterococcus bacterin ET-2 proved to be very effective when it was delivered by injection. The RPS (relative percent of survival) values achieved by this route ranged from 89 to 100 (for 45-g turbot) and from 67 to 86 (for 150-g turbot), depending on the bacterial levels and time of experimental challenge. Moreover, this strong degree of protection lasted for at least 1 year. No circulating antibodies were detected in the vaccinated turbot. However, the injected bacterin, irrespective of the diet utilized, elicited a significant (P < 0.01) increase of the phagocytic activity in the spleen. Although the glucan alone also induced an enhancement of the non-specific defence mechanisms, this stimulation was not correlated with the protection of turbot against Enterococcus infection.

Water-soluble seaweed extracts modulate the respiratory burst activity of turbot phagocytes

Abstract In this study, we investigated the effects of water-soluble extracts (WSE) from seaweed species Ulva rigida C. Agardh, Enteromorpha sp., Codium tomentosum (Huds) Stackh., Fucus vesiculosus L., Pelvetia canaliculata (L.) Decne et Thur, Dictyota dichotoma (Huds) Lamour, Chondrus crispus Stackh and Porphyra umbilicalis (L.) J. Agardh, on the respiratory burst of turbot phagocytes, in search of biologically active substances with immunostimulating capacities. The stimulatory capacities of the extracts varied greatly, depending on their origin, the concentrations used and the time of incubation. The best responses were induced by the extracts obtained from U. rigida , Enteromorpha sp. and C. crispus . Pre-incubation of the phagocyte cells with U. rigida and C. crispus extracts and subsequent incubation with phorbol myristate acetate (PMA) showed that these cells responded to further stimulation by this substance and, at some concentrations, they showed higher respiratory burst activity than control cells stimulated by PMA alone, suggesting that the extracts had a priming effect. We also tested the effects of protein-free water-soluble extracts (PF-WSE) and of the polysaccharide fractions (PSF) obtained from the PF-WSE of U. rigida and C. crispus . These extracts induced an increase in the respiratory burst activity of turbot phagocytes, suggesting that most of the stimulatory capacity of the water-soluble extracts was associated with polysaccharides.

The anti-inflammatory activity of the polyphenol resveratrol may be partially related to inhibition of tumour necrosis factor-α (TNF-α) pre-mRNA splicing

The present study shows for the first time that the polyphenol resveratrol (RESV) blocks processing of tumour necrosis factor-alpha (TNF-alpha) pre-mRNA in mature mRNA. This study was carried out in turbot (Psetta maxima (L.)), a fish species that we are using to evaluate the effects of RESV on the inflammatory response in vertebrates. Treatment of turbot head kidney leucocytes with polysaccharides from the seaweed Ulva rigida (ulvan) resulted in an increase in TNF-alpha expression. RESV did not inhibit transcription but almost completely inhibited the production of mRNA in ulvan-induced cells and caused a notable increase in the level of unspliced TNF-alpha pre-mRNA. RESV also induced accumulation of IL-1beta pre-mRNA at the expense of mature mRNA, although the effects on IL-1beta were less evident than those on TNF-alpha. However, the housekeeping gene was not affected by RESV. We also evaluated the effects of RESV in vivo under an inflammatory stimulus and found an inhibitory effect on TNF-alpha and IL-1beta pre-mRNA splicing in turbot head kidney at 24 and 48h post-injection. In addition, RESV also reduced migration of cells to the peritoneal cavity under the same inflammatory stimulus. The results show that this fish species may be a useful model for analysing the effects of RESV on TNF-alpha and IL-1beta expression, and suggest that RESV could be used to decrease the levels of pro-inflammatory cytokines in vivo and to reduce inflammatory reactions in certain inflammatory diseases.

Intraspecific variability in several isolates of Philasterides dicentrarchi (syn. Miamiensis avidus), a scuticociliate parasite of farmed turbot

Research on intraspecific variation in ciliates is scarce, and in scuticociliate parasite of fish, virtually nonexistent. In this study, seven isolates obtained from turbots affected by scuticociliatosis in different parts of the Iberian Peninsula (northwest Spain and southwest Portugal) were morphologically and genetically characterized to investigate the intraspecific divergence in these amphizoic ciliates. The isolates were stained with ammoniacal silver carbonate and examined in an optical microscope; all were found to have the typical morphological characteristics described for Philasterides dicentrarchi (syn. Miamiensis avidus). Sixteen biometric characteristics of the seven isolates were used in a canonical discrimination analysis (CDA) to select a subset of those that best identified each isolate. Discriminant analysis indicated that the OPK3 width, length of the PM2, length of the buccal field, the body width, L:W ratio, the body length, the OPK1 width and the distance between OPK2 and OPK3 were the most important morphological variables for discriminating the isolates. The first three canonical functions accounted for 86% of the total variance. The scatter plots of the first two canonical variables grouped and separated the P. dicentrarchi isolates into five clusters. Flow cytometry analysis of isolates also indicated intraspecific polymorphisms among P. dicentrarchi isolates. Nuclear markers (a 349-bp and a 390-bp fragment of 18S rRNA and β-tubulin genes) and a 398-bp of the mitochondrial cytocrome oxidase subunit I (Cox1) gene were then used to investigate the intraspecific genetic variation in P. dicentrarchi. Haplotype analysis and neighbour-joining phylogenies of nucleotide sequences of seven isolates revealed a high degree of intraspecific genetic variation among the isolates. Analysis of Cox1 and β-tubulin genes revealed six haplotypes (and clusters) in both cases; however, analysis of the 18S rRNA gene revealed only two haplotypes. The results show clear intraspecific variation at morphological and genetic levels in the scuticociliate P. dicentrarchi, and verify the suitability of mitochondrial (Cox1) and nuclear (β-tubulin) genes for detecting intraspecific genetic variation within populations of scuticociliates that infect cultured turbot. The existence of this intraspecific variation must be taken into account in the design of an effective vaccine to control scuticociliatosis.

Vaccination of turbot, Psetta maxima (L.), against the protozoan parasite Philasterides dicentrarchi: effects on antibody production and protection

The efficacy of a vaccine against the fish pathogen Philasterides dicentrarchi was evaluated in turbot by measuring the production of specific antibodies and duration of protection. Four groups of turbot were vaccinated twice, on days 0 and 30, with phosphate-buffered saline, mineral oil adjuvant, antigen or antigen plus adjuvant. Specific serum antibodies were determined on day 0 and 1 month after the first and the second vaccinations. Protection was evaluated 1 month after the first vaccination and 1 and 5 months after the second vaccination. Serum antibody titres, measured by enzyme-linked immunosorbent assay, and protection, assessed by challenges, increased significantly 1 month after the second vaccination in the group injected with antigen plus adjuvant and the protection lasted for at least a further 5 months in this group. The relative protection was 77% and 66% 1 and 5 months after the second vaccination, respectively. Administration of antigen or adjuvant separately had no effect on antibody response or protection. The results indicate that emulsion containing antigen plus adjuvant induced durable protection against P. dicentrarchi after the administration of the two vaccinations, and that this preparation can be used as a vaccine against the pathogen.

Antigenic and cross-protection studies on two turbot scuticociliate isolates

The protection induced in turbot by inactivated vaccines containing either of two isolates (I(1) and C(1)) of the scuticociliate parasite Philasterides dicentrarchi, which causes important mortalities in turbot cultures, was evaluated in the present study. The results obtained after challenging the fish with the two isolates show that vaccination protected fish only against the homologous isolate, but did not confer cross-protection. The two isolates constitute two serotypes, as shown in the immobilization tests with mouse and turbot anti-I(1) and anti-C(1) antisera, in which only the homologous antisera immobilized the ciliates. ELISA assays, using total antigen free of proteases (TAWP), cytosolic antigens (CYA), ciliar antigens (CA) or membrane protein fraction (MPF), were also carried out. Differences in the levels of antibodies produced in mouse against the homologous and heterologous antigens were observed; these differences were significantly different when the antigen preparations used in the ELISA were TAWP, CYA or CA. Nevertheless, ELISA assays using turbot sera against TAWP did not show significant differences in the levels of antibodies against the homologous and heterologous antigens. Antigenic cross-reactivity was also detected in the Western blot assays, as well as significant differences in the patterns of antigenic recognition in the two isolates - in both reduced and non-reduced TAWP antigens, but which was noteworthy when mouse antisera were used. The results obtained in the present study demonstrate for the first time the existence of serotypes of the ciliate parasite of turbot Philasterides dicentrarchi that display clear antigenic differences, which must be taken into consideration in the future development of a vaccine against scuticociliatosis.

Interactions between peritoneal exudate cells (PECs) of gilthead seabream (Sparus aurata) and Pasteurella piscicida. A morphological study

Abstract Peritoneal exudate cells from gilthead seabream (Sparus aurata) weighing 20–30 g and 0.5 g were studied using transmission electron microscopy at several time intervals after intraperitoneal injection with a virulent Pasteurella piscicida strain. Fish of 20–30 g were injected with 0.3 ml of a bacterial suspension containing 1 × 109 cells/ml of P. piscicida. Fish of 0.5 g were injected with 0.1 ml of a suspension of 1 × 106 cells/ml. The microscopic findings showed that peritoneal exudate cells (PECs) of the bigger fish were capable of phagocytosing and killing the bacteria during the first 24 h following injection and, in consequence, of controlling the infection. PECs of seabream of 0.5 g also phagocytosed P. piscicida; however, macrophages appeared to be unable to kill the microorganism. As a result, P. piscicida was able to establish an infection and kill the fish within 5 days. We conclude that the resistance of gilthead seabream to P. piscicida is dependant on the size of the fish and may be related to the efficiency of its phagocytes.

Characterisation of γ-interferon responsive promoters in fish

Reporter constructs of three interferon (IFN)-gamma-induced rainbow trout genes were generated to examine specificity to type I or type II IFN. Constructs included gammaIP-10, LMP2 and TAP2 and were used to transfect trout fibroblast cells (RTG-2) which were then exposed to rainbow trout rIFNs. The gammaIP-10 construct showed high reporter activity even in the absence of rIFNs. The LMP2 promoter contained one GAS element and two double ISRE elements, of four constructs made, only those with ISRE elements showed significant reporter activity following rIFN-gamma stimulation. The TAP2 regulatory region contained two GAS, two ISRE and one C/EBP element from which four constructs were made. Reporter expression for the construct containing all five elements showed an 11- and 2-fold increase in response to rIFN-gamma and type I rIFN, respectively. Constructs containing only the GAS elements did not respond to rIFNs. The TAP2 construct with two ISRE and the C/EBP gave the greatest dose-dependent reporter response to rIFN-gamma, with no significant response to type I rIFN. These data suggest that the ISRE elements, or elements nearby, are essential for the induction of type II IFN responsive genes in trout. The TAP2 construct is a candidate to develop a IFN-gamma reporter stable cell line.

Hydrogenosome Metabolism Is the Key Target for Antiparasitic Activity of Resveratrol against Trichomonas vaginalis

ABSTRACT Metronidazole (MDZ) and related 5-nitroimidazoles are the recommended drugs for treatment of trichomoniasis, a sexually transmitted disease caused by the protozoan parasite Trichomonas vaginalis. However, novel treatment options are needed, as recent reports have claimed resistance to these drugs in T. vaginalis isolates. In this study, we analyzed for the first time the in vitro effects of the natural polyphenol resveratrol (RESV) on T. vaginalis. At concentrations of between 25 and 100 μM, RESV inhibited the in vitro growth of T. vaginalis trophozoites; doses of 25 μM exerted a cytostatic effect, and higher doses exerted a cytotoxic effect. At these concentrations, RESV caused inhibition of the specific activity of a 120-kDa [Fe]-hydrogenase (Tvhyd). RESV did not affect Tvhyd gene expression and upregulated pyruvate-ferredoxin oxidoreductase (a hydrogenosomal enzyme) gene expression only at a high dose (100 μM). At doses of 50 to 100 μM, RESV also caused overexpression of heat shock protein 70 (Hsp70), a protective protein found in the hydrogenosome of T. vaginalis. The results demonstrate the potential of RESV as an antiparasitic treatment for trichomoniasis and suggest that the mechanism of action involves induction of hydrogenosomal dysfunction. In view of the results, we propose hydrogenosomal metabolism as a key target in the design of novel antiparasitic drugs.