Publications by authors named "M Carla Piazzon"

33 Publications

Infection in European Sea Bass: Revealing a Long Misunderstood Relationship.

Front Immunol 2021 11;12:645607. Epub 2021 Mar 11.

Fish Pathology Group, Institute of Aquaculture Torre de la Sal - Consejo Superior de Investigaciones Científicas (IATS-CSIC), Castellón, Spain.

(Cymothoidea, Isopoda) is a generalist crustacean parasite that negatively affects the economic sustainability of European sea bass () aquaculture in the North-East Mediterranean. While mortalities are observed in fry and fingerlings, infection in juvenile and adult fish result in approximately 20% growth delay. A transcriptomic analysis (PCR array, RNA-Seq) was performed on organs (tongue, spleen, head kidney, and liver) from infected vs. -free sea bass fingerlings. Activation of local and systemic immune responses was detected, particularly in the spleen, characterized by the upregulation of cytokines (also in the tongue), a general reshaping of the immunoglobulin (Ig) response and suppression of T-cell mediated responses. Interestingly, starvation and iron transport and metabolism genes were strongly downregulated, suggesting that the parasite feeding strategy is not likely hematophagous. The regulation of genes related to growth impairment and starvation supported the growth delay observed in infected animals. Most differentially expressed (DE) transcripts were exclusive of a specific organ; however, only in the tongue, the difference between infected and uninfected fish was significant. At the attachment/feeding site, the pathways involved in muscle contraction and intercellular junction were the most upregulated, whereas the pathways involved in fibrosis (extracellular matrix organization, collagen formation, and biosynthesis) were downregulated. These results suggest that parasite-inflicted damage is successfully mitigated by the host and characterized by regenerative processes that prevail over the reparative ones.
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http://dx.doi.org/10.3389/fimmu.2021.645607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7991915PMC
March 2021

Experimental Horizontal Transmission of (Microsporea: Enterocytozoonidae) in Gilthead Sea Bream ().

Animals (Basel) 2021 Feb 1;11(2). Epub 2021 Feb 1.

Fish Pathology Group, Instituto de Acuicultura Torre de la Sal (IATS-CSIC), 12595 Ribera de Cabanes, Spain.

is a microsporidian enteroparasite that infects mainly the intestine of gilthead sea bream (), leading to an emaciative syndrome. Thus far, the only available information about this infection comes from natural outbreaks in farmed fish. The aim of the present study was to determine whether . could be transmitted horizontally using naturally infected fish as donors, and to establish an experimental in vivo procedure to study this host-parasite model without depending on natural infections. Naïve fish were exposed to the infection by cohabitation, effluent, or intubated either orally or anally with intestinal scrapings of donor fish in four different trials. We succeeded in detecting parasite in naïve fish in all the challenges, but the infection level and the disease signs were always milder than in donor fish. The parasite was found in peripheral blood of naïve fish at 4 weeks post-challenge (wpc) in oral and effluent routes, and up to 12 wpc in the anal transmission trial. Molecular diagnosis detected in other organs besides intestine, such as gills, liver, stomach or heart, although the intensity was not as high as in the target tissue. The infection tended to disappear through time in all the challenge routes assayed, except in the anal infection route.
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http://dx.doi.org/10.3390/ani11020362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7912876PMC
February 2021

Genetic selection for growth drives differences in intestinal microbiota composition and parasite disease resistance in gilthead sea bream.

Microbiome 2020 11 23;8(1):168. Epub 2020 Nov 23.

Nutrigenomics and Fish Endocrinology Group, Instituto de Acuicultura Torre de la Sal (IATS-CSIC), Castellón, Spain.

Background: The key effects of intestinal microbiota in animal health have led to an increasing interest in manipulating these bacterial populations to improve animal welfare. The aquaculture sector is no exception and in the last years, many studies have described these populations in different fish species. However, this is not an easy task, as intestinal microbiota is composed of very dynamic populations that are influenced by different factors, such as diet, environment, host age, and genetics. In the current study, we aimed to determine whether the genetic background of gilthead sea bream (Sparus aurata) influences the intestinal microbial composition, how these bacterial populations are modulated by dietary changes, and the effect of selection by growth on intestinal disease resistance. To that aim, three different groups of five families of gilthead sea bream that were selected during two generations for fast, intermediate, or slow growth (F3 generation) were kept together in the same open-flow tanks and fed a control or a well-balanced plant-based diet during 9 months. Six animals per family and dietary treatment were sacrificed and the adherent bacteria from the anterior intestinal portion were sequenced. In parallel, fish of the fast- and slow-growth groups were infected with the intestinal parasite Enteromyxum leei and the disease signs, prevalence, intensity, and parasite abundance were evaluated.

Results: No differences were detected in alpha diversity indexes among families, and the core bacterial architecture was the prototypical composition of gilthead sea bream intestinal microbiota, indicating no dysbiosis in any of the groups. The plant-based diet significantly changed the microbiota in the intermediate- and slow-growth families, with a much lower effect on the fast-growth group. Interestingly, the smaller changes detected in the fast-growth families potentially accounted for more changes at the metabolic level when compared with the other families. Upon parasitic infection, the fast-growth group showed significantly lower disease signs and parasite intensity and abundance than the slow-growth animals.

Conclusions: These results show a clear genome-metagenome interaction indicating that the fast-growth families harbor a microbiota that is more flexible upon dietary changes. These animals also showed a better ability to cope with intestinal infections. Video Abstract.
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http://dx.doi.org/10.1186/s40168-020-00922-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686744PMC
November 2020

Passive Immunization Delays Disease Outcome in Gilthead Sea Bream Infected With (Myxozoa), Despite the Moderate Changes in IgM and IgT Repertoire.

Front Immunol 2020 11;11:581361. Epub 2020 Sep 11.

Fish Pathology Group, Institute of Aquaculture Torre de la Sal (IATS-CSIC), Castellón, Spain.

Passive immunization constitutes an emerging field of interest in aquaculture, particularly with the restrictions for antibiotic use. is a myxozoan intestinal parasite that invades the paracellular space of the intestinal epithelium, producing a slow-progressing disease, leading to anorexia, cachexia and mortalities. We have previously demonstrated that gilthead sea bream (GSB, ) that survive . infection become resistant upon re-exposure, and this resistance is directly related to the presence of high levels of specific IgM in serum. Thus, the current work was aimed to determine if passive immunization could help to prevent enteromyxosis in GSB and to study in detail the nature of these protective antibodies. Serum from a pool of resistant (SUR) or naïve (NAI) animals was intracoelomically injected 24 h prior to the . -effluent challenge and at 9 days post-challenge (dpc). Effluent challenge lasted for 23 days, and then the injected groups were allocated in separate tanks with clean water. A non-lethal parasite diagnosis was performed at 56 dpc. At the final sampling (100 dpc), blood, serum and tissues were collected for histology, molecular diagnosis and the detection of circulating antibodies. In parallel, we performed an immunoglobulin repertoire analysis of the fish generating SUR and NAI sera. The results showed that, fish injected with parasite-specific antibodies (spAbs) became infected with the parasite, but showed lower disease signs and intensity of infection than the other groups, indicating a later establishment of the parasite. Repertoire analysis revealed that induced a polyclonal expansion of diverse IgM and IgT subsets that could be in part an evasion strategy of the parasite. Nonetheless, GSB was able to produce sufficient levels of parasite-spAbs to avoid re-infection of surviving animals and confer certain degree of protection upon passive transfer of antibodies. These results highlight the crucial role of spAb responses against and set the basis for the development of effective treatment or prophylactic methods for aquaculture.
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http://dx.doi.org/10.3389/fimmu.2020.581361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7516018PMC
September 2020

Transcriptome sequencing supports a conservation of macrophage polarization in fish.

Sci Rep 2020 08 10;10(1):13470. Epub 2020 Aug 10.

Aquaculture and Fisheries Group, Wageningen University and Research, De Elst 1, 6708 WD, Wageningen, The Netherlands.

Mammalian macrophages can adopt polarization states that, depending on the exact stimuli present in their extracellular environment, can lead to very different functions. Although these different polarization states have been shown primarily for macrophages of humans and mice, it is likely that polarized macrophages with corresponding phenotypes exist across mammals. Evidence of functional conservation in macrophages from teleost fish suggests that the same, or at least comparable polarization states should also be present in teleosts. However, corresponding transcriptional profiles of marker genes have not been reported thus far. In this study we confirm that macrophages from common carp can polarize into M1- and M2 phenotypes with conserved functions and corresponding transcriptional profiles compared to mammalian macrophages. Carp M1 macrophages show increased production of nitric oxide and a transcriptional profile with increased pro-inflammatory cytokines and mediators, including il6, il12 and saa. Carp M2 macrophages show increased arginase activity and a transcriptional profile with increased anti-inflammatory mediators, including cyr61, timp2b and tgm2b. Our RNA sequencing approach allowed us to list, in an unbiased manner, markers discriminating between M1 and M2 macrophages of teleost fish. We discuss the importance of our findings for the evaluation of immunostimulants for aquaculture and for the identification of gene targets to generate transgenic zebrafish for detailed studies on M1 and M2 macrophages. Above all, we discuss the striking degree of evolutionary conservation of macrophage polarization in a lower vertebrate.
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http://dx.doi.org/10.1038/s41598-020-70248-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418020PMC
August 2020

(Microsporidia) in Gilthead Sea Bream (): Pathological Effects and Cellular Immune Response in Natural Infections.

Vet Pathol 2020 07 12;57(4):565-576. Epub 2020 Jun 12.

Instituto de Acuicultura Torre de la Sal, Castellón, Spain.

is a microsporidian responsible for an emaciative disease in gilthead sea bream (). Its intranuclear development and the lack of and models hinder its research. This study investigated the associated lesions, its detection by quantitative polymerase chain reaction, and the cellular immune response of naturally infected fish. The intensity of infection in the intestine was correlated with stunted growth and reduced body condition. At the beginning of the outbreaks, infection prevalence was highest in intestine and stomach, and in subsequent months, the prevalence decreased in the intestine and increased in hematopoietic organs and stomach. In heavy infections, the intestine had histologic lesions of enterocyte hypercellularity and proliferation of rodlet cells. Infected enterocytes had spores in the cytoplasm, and a pyknotic nucleus, karyorhexis or karyolysis. Lymphocytes were present at the base of the mucosa, and eosinophilic granule cells were located between the enterocytes. In intestinal submucosa, macrophage aggregates containing spores were surrounded by lymphocytes and granulocytes, with submucosal infiltration of granulocytes. Macrophage aggregates appeared to develop into granulomata with necrotic areas containing parasite remnants. Immunohistochemistry revealed mast cells as the main type of granulocyte involved. Abundant IgM and IgT cells were identified by hybridization in the submucosa when intracytoplasmic stages were present. This study describes the lesions of in gilthead sea bream, an important aquaculture species.
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http://dx.doi.org/10.1177/0300985820927707DOI Listing
July 2020

Effect of a functional feed additive on mitigation of experimentally induced gilthead sea bream Sparus aurata enteromyxosis.

Dis Aquat Organ 2020 Feb;138:111-120

Fish Pathology Group, Institute of Aquaculture Torre de la Sal (IATS-CSIC), 12595 Castellón, Spain.

In gilthead sea bream Sparus aurata, infection by Enteromyxum leei produces a cachectic syndrome with anorexia, weight loss, severe epaxial muscle atrophy and, eventually, death. Currently, there are neither vaccines nor effective prescription medicines to control this infection. Nutraceutical approaches are raising interest in the aquaculture industry, responding to the lack of therapeutic tools for the management of insidious chronic losses due to parasites. In this study, the effect of a commercially available health-promoting feed additive (SANACORE® GM) at 2 different doses was tested in comparison with a basal diet without the additive during a laboratory-controlled challenge with E. leei. Group performance and biometrical values were monitored, and an in-depth parasitological diagnosis, quantification of parasite loads and histopathological examination were carried out at the end of the trial. Supplemented diets mitigated the anorexia and growth arrestment observed in challenged fish fed the basal diet. This mitigation was maximum in the highest dose group, whose growth performance was not different from that of unchallenged controls. Treated groups also presented lower prevalence of infection and a lower parasite load, although the differences in the mean intensity of infection were not statistically significant. Although the decrease in parasite levels was similar with both doses of additive tested, the pathogeny of the infection was mostly suppressed with the higher dose, while only mitigated with the lower dose. The mechanisms involved in the effects obtained remain to be investigated, but the results point to a modulation of the immunopathological response to the infection.
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http://dx.doi.org/10.3354/dao03453DOI Listing
February 2020

Water temperature, time of exposure and population density are key parameters in Enteromyxum leei fish-to-fish experimental transmission.

J Fish Dis 2020 Apr 25;43(4):491-502. Epub 2020 Feb 25.

Fish Pathology Group, Instituto de Acuicultura Torre de la Sal, Consejo Superior de Investigaciones Científicas, Ribera de Cabanes, Spain.

Enteromyxum leei is a myxozoan histozoic parasite that infects the intestine of several teleost fish species. In gilthead sea bream (Sparus aurata), it provokes a chronic disease, entailing anorexia, delayed growth, reduced marketability and mortality. Direct fish-to-fish transmission, relevant in aquaculture conditions, has been demonstrated for E. leei via effluent, cohabitation, and oral and anal routes. However, the minimum time of exposure for infection has not been established, nor the possible effect on the fish immune response. Two effluent trials were performed at different temperatures (high: average of 25.6°C; and low: constant at 18°C), different times of exposure to the effluent (1, 3, 5 and 7 weeks) and different population densities. The results showed that 1 week was enough to infect 100% of fish at high temperature and 58.3% at low temperature. High temperature not only increased the prevalence of infection in posterior intestine, but also induced a higher production of specific antibodies, limiting the progression of the infection along the intestine. Longer time of exposure to the parasite and higher fish densities facilitated E. leei infection. These results show that effective diagnosis, lowering animal density and removal of infected fish are key aspects to manage this disease in aquaculture facilities.
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http://dx.doi.org/10.1111/jfd.13147DOI Listing
April 2020

Sex, Age, and Bacteria: How the Intestinal Microbiota Is Modulated in a Protandrous Hermaphrodite Fish.

Front Microbiol 2019 31;10:2512. Epub 2019 Oct 31.

Nutrigenomics and Fish Growth Endocrinology Group, Institute of Aquaculture Torre de la Sal (CSIC), Castellón, Spain.

Intestinal microbiota is key for many host functions, such as digestion, nutrient metabolism, disease resistance, and immune function. With the growth of the aquaculture industry, there has been a growing interest in the manipulation of fish gut microbiota to improve welfare and nutrition. Intestinal microbiota varies with many factors, including host species, genetics, developmental stage, diet, environment, and sex. The aim of this study was to compare the intestinal microbiota of adult gilthead sea bream () from three groups of age and sex (1-year-old males and 2- and 4-year-old females) maintained under the same conditions and fed exactly the same diet. Microbiota diversity and richness did not differ among groups. However, bacterial composition did, highlighting the presence of and starting at 2 years of age (females) and a higher presence of and in 1-year-old males. The core microbiota was defined by 14 Operational Taxonomic Units (OTUs) and the groups that showed more OTUs in common were 2- and 4-year-old females. Discriminant analyses showed a clear separation by sex and age, with bacteria belonging to the phyla Firmicutes, Proteobacteria and Actinobacteria driving the separation. Pathway analysis performed with the inferred metagenome showed significant differences between 1-year-old males and 4-year-old females, with an increase in infection-related pathways, nitrotoluene degradation and sphingolipid metabolism, and a significant decrease in carbohydrate metabolism pathways with age. These results show, for the first time, how intestinal microbiota is modulated in adult gilthead sea bream and highlight the importance of reporting age and sex variables in these type of studies in fish.
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http://dx.doi.org/10.3389/fmicb.2019.02512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6834695PMC
October 2019

Disruption of gut integrity and permeability contributes to enteritis in a fish-parasite model: a story told from serum metabolomics.

Parasit Vectors 2019 Oct 16;12(1):486. Epub 2019 Oct 16.

Associated Unit of Marine Ecotoxicology (IATS-IUPA), Castellon, Spain.

Background: In the animal production sector, enteritis is responsible for serious economic losses, and intestinal parasitism is a major stress factor leading to malnutrition and lowered performance and animal production efficiency. The effect of enteric parasites on the gut function of teleost fish, which represent the most ancient bony vertebrates, is far from being understood. The intestinal myxozoan parasite Enteromyxum leei dwells between gut epithelial cells and causes severe enteritis in gilthead sea bream (Sparus aurata), anorexia, cachexia, growth impairment, reduced marketability and increased mortality.

Methods: This study aimed to outline the gut failure in this fish-parasite model using a multifaceted approach and to find and validate non-lethal serum markers of gut barrier dysfunction. Intestinal integrity was studied in parasitized and non-parasitized fish by immunohistochemistry with specific markers for cellular adhesion (E-cadherin) and tight junctions (Tjp1 and Cldn3) and by functional studies of permeability (oral administration of FITC-dextran) and electrophysiology (Ussing chambers). Serum samples from parasitized and non-parasitized fish were analyzed using non-targeted metabolomics and some significantly altered metabolites were selected to be validated using commercial kits.

Results: The immunodetection of Tjp1 and Cldn3 was significantly lower in the intestine of parasitized fish, while no strong differences were found in E-cadherin. Parasitized fish showed a significant increase in paracellular uptake measured by FITC-dextran detection in serum. Electrophysiology showed a decrease in transepithelial resistance in infected animals, which showed a diarrheic profile. Serum metabolomics revealed 3702 ions, from which the differential expression of 20 identified compounds significantly separated control from infected groups in multivariate analyses. Of these compounds, serum inosine (decreased) and creatine (increased) were identified as relevant and validated with commercial kits.

Conclusions: The results demonstrate the disruption of tight junctions and the loss of gut barrier function, a metabolomic profile of absorption dysfunction and anorexia, which further outline the pathophysiological effects of E. leei.
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http://dx.doi.org/10.1186/s13071-019-3746-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6796429PMC
October 2019

Acquired protective immune response in a fish-myxozoan model encompasses specific antibodies and inflammation resolution.

Fish Shellfish Immunol 2019 Jul 5;90:349-362. Epub 2019 May 5.

Fish Pathology Group, Instituto de Acuicultura Torre de la Sal (IATS-CSIC), Castellón, Spain. Electronic address:

The myxozoan parasite Enteromyxum leei causes chronic enteritis in gilthead sea bream (GSB, Sparus aurata) leading to intestinal dysfunction. Two trials were performed in which GSB that had survived a previous infection with E. leei (SUR), and naïve GSB (NAI), were exposed to water effluent containing parasite stages. Humoral factors (total IgM and IgT, specific anti-E. leei IgM, total serum peroxidases), histopathology and gene expression were analysed. Results showed that SUR maintained high levels of specific anti-E. leei IgM (up to 16 months), expressed high levels of immunoglobulins at the intestinal mucosa, particularly the soluble forms, and were resistant to re-infection. Their acquired-type response was complemented by other immune effectors locally and systemically, like cell cytotoxicity (high granzyme A expression), complement activity (high c3 and fucolectin expression), and serum peroxidases. In contrast to NAI, SUR displayed a post-inflammatory phenotype in the intestine and head kidney, characteristic of inflammation resolution (low il1β, high il10 and low hsp90α expression).
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http://dx.doi.org/10.1016/j.fsi.2019.04.300DOI Listing
July 2019

Acting locally - affecting globally: RNA sequencing of gilthead sea bream with a mild Sparicotyle chrysophrii infection reveals effects on apoptosis, immune and hypoxia related genes.

BMC Genomics 2019 Mar 11;20(1):200. Epub 2019 Mar 11.

Fish Pathology Group, Institute of Aquaculture Torre de la Sal (IATS-CSIC), Ribera de Cabanes, Castellón, Spain.

Background: Monogenean flatworms are the main fish ectoparasites inflicting serious economic losses in aquaculture. The polyopisthocotylean Sparicotyle chrysophrii parasitizes the gills of gilthead sea bream (GSB, Sparus aurata) causing anaemia, lamellae fusion and sloughing of epithelial cells, with the consequent hypoxia, emaciation, lethargy and mortality. Currently no preventive or curative measures against this disease exist and therefore information on the host-parasite interaction is crucial to find mitigation solutions for sparicotylosis. The knowledge about gene regulation in monogenean-host models mostly comes from freshwater monopysthocotyleans and almost nothing is known about polyopisthocotyleans. The current study aims to decipher the host response at local (gills) and systemic (spleen, liver) levels in farmed GSB with a mild natural S. chrysophrii infection by transcriptomic analysis.

Results: Using Illumina RNA sequencing and transcriptomic analysis, a total of 2581 differentially expressed transcripts were identified in infected fish when compared to uninfected controls. Gill tissues in contact with the parasite (P gills) displayed regulation of fewer genes (700) than gill portions not in contact with the parasite (NP gills) (1235), most likely due to a local silencing effect of the parasite. The systemic reaction in the spleen was much higher than that at the parasite attachment site (local) (1240), and higher than in liver (334). NP gills displayed a strong enrichment of genes mainly related to immune response and apoptosis. Processes such as apoptosis, inflammation and cell proliferation dominated gills, whereas inhibition of apoptosis, autophagy, platelet activation, signalling and aggregation, and inflammasome were observed in spleen. Proteasome markers were increased in all tissues, whereas hypoxia-related genes were down-regulated in gills and spleen.

Conclusions: Contrasting forces seem to be acting at local and systemic levels. The splenic down-regulation could be part of a hypometabolic response, to counteract the hypoxia induced by the parasite damage to the gills and to concentrate the energy on defence and repair responses. Alternatively, it can be also interpreted as the often observed action of helminths to modify host immunity in its own interest. These results provide the first toolkit for future studies towards understanding and management of this parasitosis.
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http://dx.doi.org/10.1186/s12864-019-5581-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6416957PMC
March 2019

Gene expression analysis of Atlantic salmon gills reveals mucin 5 and interleukin 4/13 as key molecules during amoebic gill disease.

Sci Rep 2018 09 12;8(1):13689. Epub 2018 Sep 12.

Fish Pathology Group, Instituto de Acuicultura Torre de la Sal (IATS-CSIC), Castellón, 12595, Spain.

Amoebic gill disease (AGD) is one of the main diseases affecting Atlantic salmon (Salmo salar L.) mariculture. Hallmarks of AGD are hyperplasia of the lamellar epithelium and increased production of gill mucus. This study investigated the expression of genes involved in mucus secretion, cell cycle regulation, immunity and oxidative stress in gills using a targeted 21-gene PCR array. Gill samples were obtained from experimental and natural Neoparamoeba perurans infections, and sampling points included progressive infection stages and post-freshwater treatment. Up-regulation of genes related to mucin secretion and cell proliferation, and down-regulation of pro-inflammatory and pro-apoptotic genes were associated with AGD severity, while partial restoration of the gill homeostasis was detected post-treatment. Mucins and Th2 cytokines accoun ted for most of the variability observed between groups highlighting their key role in AGD. Two mucins (muc5, muc18) showed differential regulation upon disease. Substantial up-regulation of the secreted muc5 was detected in clinical AGD, and the membrane bound muc18 showed an opposite pattern. Th2 cytokines, il4/13a and il4/13b2, were significantly up-regulated from 2 days post-infection onwards, and changes were lesion-specific. Despite the differences between experimental and natural infections, both yielded comparable results that underline the importance of the studied genes in the respiratory organs of fish, and during AGD progression.
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http://dx.doi.org/10.1038/s41598-018-32019-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6135806PMC
September 2018

Hints on T cell responses in a fish-parasite model: Enteromyxum leei induces differential expression of T cell signature molecules depending on the organ and the infection status.

Parasit Vectors 2018 Jul 31;11(1):443. Epub 2018 Jul 31.

Fish Pathology Group, Institute of Aquaculture Torre de la Sal (IATS-CSIC), Ribera de Cabanes, Castellón, Spain.

Backgroud: Enteromyxum leei is a myxozoan parasite that produces a slow-progressing intestinal disease. This parasite invades the paracellular space of the intestinal epithelium and progresses from the posterior to the anterior intestine. The aim of the present study was to gain insights into fish T cell responses in the gilthead sea bream-E. leei infection model using a PCR-array with 30 signature molecules for different leukocyte responses in head kidney, spleen, anterior and posterior intestine.

Results: The PCR-array results suggest that E. leei induced migration of T cells from head kidney to intestines where T, CTL and T profiles were activated and kept in balance by the upregulation of regulatory cytokines. These results were partially validated by the use of cross-reacting antibodies and BrdU immunostaining to monitor proliferation. Zap70 immunostaining supported the increased number of T cells in the anterior intestine detected by gene expression, but double staining with BrdU did not show active proliferation of this cell type at a local level, supporting the migration from lymphohaematopoietic tissues to the site of infection. Global analyses of the expression profiles revealed a clear separation between infected and exposed, but non-infected fish, more evident in the target organ. Exposed, non-infected animals showed an intermediate phenotype closer to the control fish.

Conclusions: These results evidence a clear modulation of the T cell response of gilthead sea bream upon E. leei infection. The effects occurred both at local and systemic levels, but the response was stronger and more specific at the site of infection, the intestine. Altogether, this research poses a promising basis to understand the response against this important parasite and establish effective preventive or palliative measures.
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http://dx.doi.org/10.1186/s13071-018-3007-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6069777PMC
July 2018

Under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets.

Microbiome 2017 12 28;5(1):164. Epub 2017 Dec 28.

Nutrigenomics and Fish Growth Endocrinology Group, Instituto de Acuicultura Torre de la Sal (IATS-CSIC), Castellón, Spain.

Background: The constant increase of aquaculture production and wealthy seafood consumption has forced the industry to explore alternative and more sustainable raw aquafeed materials, and plant ingredients have been used to replace marine feedstuffs in many farmed fish. The objective of the present study was to assess whether plant-based diets can induce changes in the intestinal mucus proteome, gut autochthonous microbiota and disease susceptibility of fish, and whether these changes could be reversed by the addition of sodium butyrate to the diets. Three different trials were performed using the teleostean gilthead sea bream (Sparus aurata) as model. In a first preliminary short-term trial, fish were fed with the additive (0.8%) supplementing a basal diet with low vegetable inclusion (D1) and then challenged with a bacteria to detect possible effects on survival. In a second trial, fish were fed with diets with greater vegetable inclusion levels (D2, D3) and the long-term effect of sodium butyrate at a lower dose (0.4%) added to D3 (D4 diet) was tested on the intestinal proteome and microbiome. In a third trial, the long-term effectiveness of sodium butyrate (D4) to prevent disease outcome after an intestinal parasite (Enteromyxum leei) challenge was tested.

Results: The results showed that opposed forces were driven by dietary plant ingredients and sodium butyrate supplementation in fish diet. On the one hand, vegetable diets induced high parasite infection levels that provoked drops in growth performance, decreased intestinal microbiota diversity, induced the dominance of the Photobacterium genus, as well as altered the gut mucosal proteome suggesting detrimental effects on intestinal function. On the other hand, butyrate addition slightly decreased cumulative mortality after bacterial challenge, avoided growth retardation in parasitized fish, increased intestinal microbiota diversity with a higher representation of butyrate-producing bacteria and reversed most vegetable diet-induced changes in the gut proteome.

Conclusions: This integrative work gives insights on the pleiotropic effects of a dietary additive on the restoration of intestinal homeostasis and disease resilience, using a multifaceted approach.
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http://dx.doi.org/10.1186/s40168-017-0390-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5745981PMC
December 2017

Bromodeoxyuridine DNA labelling reveals host and parasite proliferation in a fish-myxozoan model.

J Fish Dis 2018 Apr 19;41(4):651-662. Epub 2017 Dec 19.

Fish Pathology Group, Instituto de Acuicultura Torre de la Sal, Consejo Superior de Investigaciones Científicas, Castellón, Spain.

Enteromyxum leei is a myxozoan parasite responsible for enteritis in gilthead sea bream (Sparus aurata). The parasite proliferates in the paracellular space of the intestinal epithelium and induces an inflammatory reaction. To assess intestinal cell turnover and parasite proliferation, fish were infected with the parasite by anal intubation; after 17 and 64 days, the cell proliferative marker bromodeoxyuridine (BrdU) was administered; and after 24 hr, tissue samples were taken for immunohistochemical detection. Parasite exposure induced increased epithelial and immune cell proliferation in all intestinal segments at all time points, even before parasite establishment. This increased turnover was triggered early after intubation and mainly at a local level, as shown by an increased proliferating cell nuclear antigen (pcna) gene expression only at the posterior intestine after 17 days (not found in lymphohaematopoietic organs). Incorporation of BrdU in parasite secondary and tertiary daughter cells indicated that parasite endogeny is not by schizogonial division, which uses de novo synthesis pathway of pyrimidines. Altogether, BrdU immunolabelling and pcna gene expression showed the rapid proliferative response of the fish intestines upon a myxozoan infection and how this response is effectively triggered even before the parasite reaches or establishes in the site.
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http://dx.doi.org/10.1111/jfd.12765DOI Listing
April 2018

Sodium salt medium-chain fatty acids and -based probiotic strategies to improve growth and intestinal health of gilthead sea bream ().

PeerJ 2017 4;5:e4001. Epub 2017 Dec 4.

Nutrigenomics and Fish Growth Endocrinology Group, Institute of Aquaculture Torre de la Sal, CSIC, Castellón, Spain.

Background: The increased demand for fish protein has led to the intensification of aquaculture practices which are hampered by nutritional and health factors affecting growth performance. To solve these problems, antibiotics have been used for many years in the prevention, control and treatment against disease as well as growth promoters to improve animal performance. Nowadays, the use of antibiotics in the European Union and other countries has been completely or partially banned as a result of the existence of antibiotic cross-resistance. Therefore, a number of alternatives, including enzymes, prebiotics, probiotics, phytonutrients and organic acids used alone or in combination have been proposed for the improvement of immunological state, growth performance and production in livestock animals. The aim of the present study was to evaluate two commercially available feed additives, one based on medium-chain fatty acids (MCFAs) from coconut oil and another with a -based probiotic, in gilthead sea bream (GSB, ), a marine farmed fish of high value in the Mediterranean aquaculture

Methods: The potential benefits of adding two commercial feed additives on fish growth performance and intestinal health were assessed in a 100-days feeding trial. The experimental diets (D2 and D3) were prepared by supplementing a basal diet (D1) with MCFAs in the form of a sodium salt of coconut fatty acid distillate (DICOSAN; Norel, Madrid, Spain), rich on C-12, added at 0.3% (D2) or with the probiotic CECT 5940, added at 0.1% (D3). The study integrated data on growth performance, blood biochemistry, histology and intestinal gene expression patterns of selected markers of intestinal function and architecture.

Results: MCFAs in the form of a coconut oil increased feed intake, growth rates and the surface of nutrient absorption, promoting the anabolic action of the somatotropic axis. The probiotic (D3) induced anti-inflammatory and anti-oxidant effects with changes in circulating cortisol, immunoglobulin M, leukocyte respiratory burst, and mucosal expression levels of cytokines, lymphocyte markers and immunoglobulin T.

Discussion: MCFA supplementation showed positive effects on GSB growth and intestinal architecture acting mainly in the anterior intestine, where absorption takes place. The probiotic CECT 5940 exhibited key effects in the regulation of the immune status inducing anti-inflammatory and anti-oxidant effects which can be potentially advantageous upon infection or exposure to other stressors. The potential effects of these feed additives in GSB are very promising to improve health and disease resistance in aquaculture.
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http://dx.doi.org/10.7717/peerj.4001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5719961PMC
December 2017

Conserved Fever Pathways across Vertebrates: A Herpesvirus Expressed Decoy TNF-α Receptor Delays Behavioral Fever in Fish.

Cell Host Microbe 2017 Feb;21(2):244-253

Immunology-Vaccinology, FARAH, Faculty of Veterinary Medicine, University of Liège, Liège 4000, Belgium. Electronic address:

Both endotherms and ectotherms (e.g., fish) increase their body temperature to limit pathogen infection. Ectotherms do so by moving to warmer places, hence the term "behavioral fever." We studied the manifestation of behavioral fever in the common carp infected by cyprinid herpesvirus 3, a native carp pathogen. Carp maintained at 24°C died from the infection, whereas those housed in multi-chamber tanks encompassing a 24°C-32°C gradient migrated transiently to the warmest compartment and survived as a consequence. Behavioral fever manifested only at advanced stages of infection. Consistent with this, expression of CyHV-3 ORF12, encoding a soluble decoy receptor for TNF-α, delayed the manifestation of behavioral fever and promoted CyHV-3 replication in the context of a temperature gradient. Injection of anti-TNF-α neutralizing antibodies suppressed behavioral fever, and decreased fish survival in response to infection. This study provides a unique example of how viruses have evolved to alter host behavior to increase fitness.
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http://dx.doi.org/10.1016/j.chom.2017.01.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5301049PMC
February 2017

Differential Modulation of IgT and IgM upon Parasitic, Bacterial, Viral, and Dietary Challenges in a Perciform Fish.

Front Immunol 2016 27;7:637. Epub 2016 Dec 27.

Instituto de Acuicultura Torre de la Sal, Consejo Superior de Investigaciones Científicas (IATS-CSIC) , Castellón , Spain.

Three different immunoglobulin (Ig) isotypes can be found in teleost fish, IgM, IgD, and the teleost-specific IgT. IgM is considered to have a systemic activity, and IgT is attributed a mucosal role, similar to mammalian IgA. In this study, the complete sequence of gilthead sea bream IgM and IgT in their membrane (m) and soluble (s) forms are described for the first time in a perciform fish. Their constitutive gene expression is analyzed in different tissues, and their regulation upon viral, bacterial, parasitic, mucosal vaccination and dietary challenges are studied. GCB IgM and IgT have the prototypical structure when compared to other fish Igs. The constitutive expression of was the highest overall in all tissues, whereas expression was highest in mucosal tissues, such as gills and intestine. and were differentially regulated upon infection. was highly upregulated locally upon infection with the intestinal parasite or systemically after Nodavirus infection. Long-term intestinal parasitic infections increased the serum titer of both isotypes. Mucosal vaccination against subsp. finely regulated the Ig response inducing a systemic increase of IgM titers in serum and a local IgT response in skin mucus when animals were exposed to the pathogen by bath challenge. Interestingly, plant-based diets inhibit IgT upregulation upon intestinal parasitic challenge, which was related to a worse disease outcome. All these results corroborate the mucosal role of IgT and emphasize the importance of a finely tuned regulation of Ig isotypes upon infection, which could be of special interest in vaccination studies.
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http://dx.doi.org/10.3389/fimmu.2016.00637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5186763PMC
December 2016

IL10, A Tale of an Evolutionarily Conserved Cytokine across Vertebrates.

Crit Rev Immunol 2016 ;36(2):99-129

Cell Biology and Immunology Group, Wageningen University, The Netherlands.

IL10 was discovered in 1989, and since then it has been the subject of intense investigation, which has revealed its potent anti-inflammatory and regulatory activities in most immune processes during infection and disease. In 2003, the first non-mammalian IL10 sequence was identified in teleost fish, followed in 2004 by the chicken IL10 sequence. In this review, we summarize the work performed in non-mammalian vertebrates in which the IL10, IL10 receptors (IL10Rs), and their signaling components have been identified. We review the genomic organization, genes, and protein structure of IL10(Rs); we focus on studies providing a functional characterization of their biological activities. In addition, we describe the activities of viral IL10s identified in viruses infecting non-mammalian hosts. Altogether, our analysis reveals remarkable conservation of the anti-inflammatory and regulatory activities of (viral) IL10 across vertebrates, confirming the crucial role of IL10 throughout evolution. Interestingly, in some teleost fish, the presence of multiple copies of IL10(Rs) adds an additional degree of complexity. In fact, the evidence suggests that gene duplication does not necessarily imply functional redundancy, and leaves teleosts with additional possibilities to fine tune IL10 activities. Finally, we discuss the use of zebrafish (Danio rerio) as a complementary animal model for the study of IL10 activities in non-mammalian vertebrates.
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http://dx.doi.org/10.1615/CritRevImmunol.2016017480DOI Listing
December 2017

Carp Il10a and Il10b exert identical biological activities in vitro, but are differentially regulated in vivo.

Dev Comp Immunol 2017 02 29;67:350-360. Epub 2016 Aug 29.

Cell Biology and Immunology Group, Department of Animal Sciences, Wageningen University, 6708 WD, Wageningen, The Netherlands. Electronic address:

We recently reported on the functional characterization of carp Il10. We showed that carp Il10 is able to downregulate proinflammatory activities by carp phagocytes and promote B cell proliferation, differentiation and antibody production as well as proliferation of memory T cells. Taking advantage of the recent annotation of the carp genome, we completed the sequence of a second il10 paralogue, named il10b, the presence of which was expected owing to the recent (8 million years ago) fourth round of whole genome duplication that occurred in common carp. In the present study we closely compared the two Il10 paralogues and show that Il10a and Il10b have almost identical gene structure, synteny, protein sequence as well as bioactivity on phagocytes. Although the two il10 paralogues show a large overlap in tissue expression, il10b has a low constitutive expression and is highly upregulated upon infection, whereas il10a is higher expressed under basal conditions but its gene expression remains constant during viral and parasitic infections. This differential regulation is most likely due to the observed differences in their promoter regions. Altogether our results demonstrate that gene duplication in carp, although recent, led to sub-functionalization and expression divergence rather than functional redundancy of the Il10 paralogues, yet with very similar protein sequences.
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http://dx.doi.org/10.1016/j.dci.2016.08.016DOI Listing
February 2017

Cyprinid Herpesvirus 3 Il10 Inhibits Inflammatory Activities of Carp Macrophages and Promotes Proliferation of Igm+ B Cells and Memory T Cells in a Manner Similar to Carp Il10.

J Immunol 2015 Oct 14;195(8):3694-704. Epub 2015 Sep 14.

Cell Biology and Immunology Group, Department of Animal Sciences, Wageningen University, 6708WD Wageningen, the Netherlands; and

Cyprinid herpesvirus 3 (CyHV-3) is the causative agent of a lethal disease of carp and encodes for an Il10 homolog (ORF134). Our previous studies with a recombinant ORF134-deleted strain and the derived revertant strain suggested that cyprinid herpesvirus 3 Il10 (CyHV-3 Il10 [cyhv3Il10]) is not essential for viral replication in vitro, or virulence in vivo. In apparent contrast, cyhv3Il10 is one of the most abundant proteins of the CyHV-3 secretome and is structurally very similar to carp Il10 and also human IL10. To date, studies addressing the biological activity of cyhv3Il10 on cells of its natural host have not been performed. To address the apparent contradiction between the presence of a structurally conserved Il10 homolog in the genome of CyHV-3 and the lack of a clear phenotype in vivo using recombinant cyhv3Il10-deleted viruses, we used an in vitro approach to investigate in detail whether cyhv3Il10 exerts any biological activity on carp cells. In this study, we provide direct evidence that cyhv3Il10 is biologically active and, similarly to carp Il10, signals via a conserved Stat3 pathway modulating immune cells of its natural host, carp. In vitro, cyhv3Il10 deactivates phagocytes with a prominent effect on macrophages, while also promoting proliferation of Igm(+) B cells and memory T cells. Collectively, this study demonstrates a clear biological activity of cyhv3Il10 on cells of its natural host and indicates that cyhv3Il10 is a true viral ortholog of carp Il10. Furthermore, to our knowledge, this is the first report on biological activities of a nonmammalian viral Il10 homolog.
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http://dx.doi.org/10.4049/jimmunol.1500926DOI Listing
October 2015

Carp Il10 Has Anti-Inflammatory Activities on Phagocytes, Promotes Proliferation of Memory T Cells, and Regulates B Cell Differentiation and Antibody Secretion.

J Immunol 2015 Jan;194(1):187-99

Cell Biology and Immunology Group, Department of Animal Sciences, Wageningen University, 6708WD Wageningen, the Netherlands

In the current study, we investigated the effects of carp Il10 on phagocytes and lymphocytes. Carp Il10 shares several prototypical inhibitory activities on phagocytes with mammalian IL-10, including deactivation of neutrophils and macrophages, as shown by inhibition of oxygen and nitrogen radical production, as well as reduced expression of proinflammatory genes and mhc genes involved in Ag presentation. Similar to mammalian IL-10, carp Il10 acts through a signaling pathway involving phosphorylation of Stat3, ultimately leading to the early upregulation of socs3 expression. To our knowledge, this is the first study of the effects of Il10 on lymphocytes in fish. Although Il10 did not affect survival and proliferation of T cells from naive animals, it greatly promoted survival and proliferation of T cells in cultures from immunized animals, but only when used in combination with the immunizing Ag. Preliminary gene expression analysis suggests that, under these circumstances, carp Il10 stimulates a subset of CD8+ memory T cells while downregulating CD4+ memory Th1 and Th2 responses. In addition to the regulatory effect on T cells, carp Il10 stimulates proliferation, differentiation, and Ab secretion by IgM+ B cells. Overall, carp Il10 shares several prototypical activities with mammalian IL-10, including downregulation of the inflammatory response of phagocytes, stimulation of proliferation of subsets of memory T lymphocytes, and proliferation, differentiation, and Ab secretion by IgM+ B lymphocytes. To our knowledge, this is the first comprehensive analysis of biological activities of fish Il10 on both phagocytes and lymphocytes showing functional conservation of several properties of Il10.
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http://dx.doi.org/10.4049/jimmunol.1402093DOI Listing
January 2015

Reprint of "fish immunity to scuticociliate parasites".

Dev Comp Immunol 2014 Apr 2;43(2):280-9. Epub 2013 Dec 2.

Departamento de Biología Celular y Ecología, Universidad de Santiago de Compostela, Santiago de Compostela, Spain. Electronic address:

Some species of scuticociliates (Ciliophora) behave as facultative parasites and produce severe mortalities in cultured fish. Pathogenic scuticociliates can cause surface lesions and can also penetrate inside the body, where they feed on tissue and proliferate in the blood and most internal organs, killing the host in a few days. In this review, we describe the current knowledge on the protective role of fish cellular and humoral immune responses against these parasites. Immune humoral factors, especially complement, are of particular importance in defending fish against these ciliates. However, knowledge about how the fish immune system responds to scuticociliates is scant, and the cellular and molecular events that occur during the response are not known. We also describe the possible mechanisms used by scuticociliates to avoid or resist the defensive reaction of the host. For example, the release of proteases can help parasites enter fish tissues and impair the fish cellular and humoral responses. Several vaccine formulations containing scuticociliates have induced a good antibody response and protection in fish immunized and challenged with homologous strains of particular species. However, protection was not achieved in fish immunized and challenged with heterologous strains, and the antigens involved in protection and the antigenic differences between heterologous strains have not yet been determined.
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http://dx.doi.org/10.1016/j.dci.2013.11.015DOI Listing
April 2014

Fish immunity to scuticociliate parasites.

Dev Comp Immunol 2013 Oct 10;41(2):248-56. Epub 2013 Jun 10.

Cell Biology and Immunology Group, Department of Animal Sciences, Wageningen University, Wageningen PG 6709, Netherlands.

Some species of scuticociliates (Ciliophora) behave as facultative parasites and produce severe mortalities in cultured fish. Pathogenic scuticociliates can cause surface lesions and can also penetrate inside the body, where they feed on tissue and proliferate in the blood and most internal organs, killing the host in a few days. In this review, we describe the current knowledge on the protective role of fish cellular and humoral immune responses against these parasites. Immune humoral factors, especially complement, are of particular importance in defending fish against these ciliates. However, knowledge about how the fish immune system responds to scuticociliates is scant, and the cellular and molecular events that occur during the response are not known. We also describe the possible mechanisms used by scuticociliates to avoid or resist the defensive reaction of the host. For example, the release of proteases can help parasites enter fish tissues and impair the fish cellular and humoral responses. Several vaccine formulations containing scuticociliates have induced a good antibody response and protection in fish immunized and challenged with homologous strains of particular species. However, protection was not achieved in fish immunized and challenged with heterologous strains, and the antigens involved in protection and the antigenic differences between heterologous strains have not yet been determined.
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http://dx.doi.org/10.1016/j.dci.2013.05.022DOI Listing
October 2013

Are nested networks more robust to disturbance? A test using epiphyte-tree, comensalistic networks.

PLoS One 2011 May 11;6(5):e19637. Epub 2011 May 11.

Mediterranean Institute for Advanced Studies (IMEDEA, CSIC-UIB), C/Miquel Marquès 21, Esporles, Mallorca, Balearic Islands, Spain.

Recent research on ecological networks suggests that mutualistic networks are more nested than antagonistic ones and, as a result, they are more robust against chains of extinctions caused by disturbances. We evaluate whether mutualistic networks are more nested than comensalistic and antagonistic networks, and whether highly nested, host-epiphyte comensalistic networks fit the prediction of high robustness against disturbance. A review of 59 networks including mutualistic, antagonistic and comensalistic relationships showed that comensalistic networks are significantly more nested than antagonistic and mutualistic networks, which did not differ between themselves. Epiphyte-host networks from old-growth forests differed from those from disturbed forest in several topological parameters based on both qualitative and quantitative matrices. Network robustness increased with network size, but the slope of this relationship varied with nestedness and connectance. Our results indicate that interaction networks show complex responses to disturbances, which influence their topology and indirectly affect their robustness against species extinctions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0019637PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3092765PMC
May 2011

Turbot resistance to Philasterides dicentrarchi is more dependent on humoral than on cellular immune responses.

Fish Shellfish Immunol 2011 Jun 21;30(6):1339-47. Epub 2011 Mar 21.

Departamento de Biología Celular y Ecología, Biología Celular, Edificio CIBUS, Rúa Lope Gómez de Marzoa, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain.

Philasterides dicentrarchi is a ciliate that causes high mortalities in cultured turbot, Psetta maxima (L.). This pathogen displays high phagocytic activity and after entering the body it multiplies and feeds on host cells and tissue components. In previous studies, we found that complement, activated through the classical pathway, is a potent killer of P. dicentrarchi. Here, we compared the killing activity of turbot leucocytes and humoral factors against two virulent isolates of P. dicentrarchi, in order to determine the importance of leucocytes in the defence against this pathogen. Components of P. dicentrarchi (ciliary and membrane) stimulated turbot leucocytes, and increased the respiratory burst, degranulation and the expression of pro-inflammatory cytokines. We tested the susceptibility of ciliates to reactive oxygen and nitrogen species, by incubating them with different oxidative systems (H(2)O(2), Fe/ascorbate, which induces lipid peroxidation, an O(2)(-) donor (XOD/HX), an NO donor (SNAP) and an ONOO(-) donor (SIN-1)), for 24h. Both isolates were susceptible to high concentrations of H(2)O(2,) Fe/ascorbate, XOD/HX, and SIN-1 but were resistant to incubation with SNAP. Leucocytes became strongly activated when they were in contact with or were phagocytosed by the ciliate. Incubation of P. dicentrarchi with a combination of fresh serum and specific antibodies killed most of the ciliates, but the addition of leucocytes to ciliate cultures did not increase the toxicity to the ciliates. On the contrary, the number of ciliates increased when leucocytes were added to the culture because the ciliates fed on them. Despite being activated, leucocytes did not produce sufficiently high concentrations of toxic substances to kill the parasite. The most virulent isolate was that which induced greatest activation of leucocytes but was least susceptible to complement. We concluded that humoral factors such as complement (activated through the classical pathway) are critical for fish defence against P. dicentrarchi and that cellular responses appear to play a minor role, if any, in defence against this ciliate.
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http://dx.doi.org/10.1016/j.fsi.2011.02.026DOI Listing
June 2011

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

Mol Immunol 2010 Feb 27;47(5):1114-20. Epub 2009 Nov 27.

Instituto de Investigación e Análises Alimentarias, Universidade de Santiago de Compostela, Santiago de Compostela, Spain.

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.
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http://dx.doi.org/10.1016/j.molimm.2009.10.030DOI Listing
February 2010

Complement-mediated killing of Philasterides dicentrarchi (Ciliophora) by turbot serum: relative importance of alternative and classical pathways.

Parasite Immunol 2008 Oct 28;30(10):535-43. Epub 2008 Jul 28.

Institute of Tropical Medicine, School of Medicine, National Yang-Ming University, Peitou, Taipei, Taiwan.

The present study was carried out to elucidate the in vitro killing activity of turbot complement and specific antibodies against the ciliate parasite Philasterides dicentrarchi. Fresh serum from nonimmunized fish showed a moderate ability to kill the parasite, which indicates that P. dicentrarchi is able to activate the alternative complement pathway (ACP). Fresh serum from immunized fish, which contained high levels of specific antibodies, showed greater killing activity. Heat-inactivated serum, with or without antibodies, and antibodies alone did not have any effect on parasite viability, which indicates that serum mainly kills P. dicentrarchi through the antibody-mediated classical complement pathway (CCP). Ascitic fluid from infected fish, but containing low levels of specific antibodies, showed a low ability to kill the parasite, and fresh serum from nonimmunized infected fish did not kill the parasite. The latter serum contained some specific antibodies but lower levels of complement than serum from control and vaccinated fish, and the lack of ability of this serum to kill the parasite was probably related to low levels of complement. In addition, serum and ascitic fluid from infected turbot showed high proteolytic activity which degraded fish Igs. The proteolytic activity generated may favour survival of the parasite during infection.
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http://dx.doi.org/10.1111/j.1365-3024.2008.01052.xDOI Listing
October 2008

In vitro activity of the nonsteroidal anti-inflammatory drug indomethacin on a scuticociliate parasite of farmed turbot.

Vet Parasitol 2007 Sep 6;148(3-4):318-24. Epub 2007 Aug 6.

Departamento de Microbiología y Parasitología, Laboratorio de Parasitología, Instituto de Investigación y Análisis Alimentarios, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain.

The scuticociliatosis produced by the endoparasite Philasterides dicentarchi is a severe parasitic infection of farmed turbot (Scophthalmus maximus) characterized by several histopathological effects including extensive inflammation. Indomethacin is a nonsteroidal anti-inflammatory drug that specifically inhibits synthesis of the proinflammatory mediator prostaglandins. The effect of indomethacin on the in vitro growth of P. dicentrarchi was investigated. In vitro growth of the scuticociliate was significantly inhibited by treatment with 100 microM indomethacin for 48 h. Higher concentrations of indomethacin (mM levels) did not affect the gelatinolytic activity of the cysteine proteinases of P. dicentrarchi. In vitro treatment with 25, 50 or 100 microM indomethacin for 3 days did not significantly affect the enzymatic activity of cysteine proteinases, as assayed with p-nitroanilide as substrate. Immunoblot analysis with anti-cysteine proteinase antibodies revealed an increase in proteinase expression (molecular weights of 80, 32 and 40-45 kDa) in parasite lysates originating from in vitro cultures incubated with 25 microM indomethacin for 72 h. Degradation of genomic DNA of the ciliates was observed in cultures incubated with 100 microM indomethacin for 1, 3 and 7 days. The results suggest that indomethacin is capable of inhibiting in vitro growth of the scuticociliate P. dicentrarchi by a mechanism related to the induction of programmed cell death, without affecting the enzymatic activation of parasite proteinases, which demonstrates the potential therapeutic use of this drug in the control of turbot scuticociliatosis.
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http://dx.doi.org/10.1016/j.vetpar.2007.06.018DOI Listing
September 2007