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

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.

Effects of Enteromyxum spp. (Myxozoa) infection in the regulation of intestinal E-cadherin: Turbot against gilthead sea bream.

Enteromyxoses are relevant diseases for turbot and gilthead sea bream aquaculture. The myxozoan parasites invade the intestinal mucosa, causing a cachectic syndrome associated with intestinal barrier alteration; nonetheless, their pathological impact is different. Turbot infected by Enteromyxum scophthalmi develop more severe intestinal lesions, reaching mortality rates of 100%, whereas in E. leei-infected gilthead sea bream, the disease progresses slowly, and mortality rates are lower. The mechanisms underlying the different pathogenesis are still unclear. We studied the distribution and expression changes of E-cadherin, a highly conserved protein of the adherens junctions, in the intestine of both species by immunohistochemistry and quantitative PCR, using the same immunohistochemical protocol and common primers. The regular immunostaining pattern observed in control fish turned into markedly irregular in parasitized turbot, showing an intense immunoreaction at the host-parasite interface. Nevertheless, E-cadherin gene expression was not significantly modulated in this species. On the contrary, no evident changes in the protein distribution were noticed in gilthead sea bream, whereas a significant gene downregulation occurred in advanced infection. The results contribute to the understanding of the different host-parasite interactions in enteromyxoses. Host and parasite cells appear to establish diverse relationships in these species, which could underlie the different pathological picture.

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

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 il1ß, high il10 and low hsp90α expression).

Comprehensive biometric, biochemical and histopathological assessment of nutrient deficiencies in gilthead sea bream fed semi-purified diets.

Seven isoproteic and isolipidic semi-purified diets were formulated to assess specific nutrient deficiencies in sulphur amino acids (SAA), n-3 long-chain PUFA (n-3 LC-PUFA), phospholipids (PL), P, minerals (Min) and vitamins (Vit). The control diet (CTRL) contained these essential nutrients in adequate amounts. Each diet was allocated to triplicate groups of juvenile gilthead sea bream fed to satiety over an 11-week feeding trial period. Weight gain of n-3 LC-PUFA, P-Vit and PL-Min-SAA groups was 50, 60-75 and 80-85 % of the CTRL group, respectively. Fat retention was decreased by all nutrient deficiencies except by the Min diet. Strong effects on N retention were found in n-3 LC-PUFA and P fish. Combined anaemia and increased blood respiratory burst were observed in n-3 LC-PUFA fish. Hypoproteinaemia was found in SAA, n-3 LC-PUFA, PL and Vit fish. Derangements of lipid metabolism were also a common disorder, but the lipodystrophic phenotype of P fish was different from that of other groups. Changes in plasma levels of electrolytes (Ca, phosphate), metabolites (creatinine, choline) and enzyme activities (alkaline phosphatase) were related to specific nutrient deficiencies in PL, P, Min or Vit fish, whereas changes in circulating levels of growth hormone and insulin-like growth factor I primarily reflected the intensity of the nutritional stressor. Histopathological scoring of the liver and intestine segments showed specific nutrient-mediated changes in lipid cell vacuolisation, inflammation of intestinal submucosa, as well as the distribution and number of intestinal goblet and rodlet cells. These results contribute to define the normal range of variation for selected biometric, biochemical, haematological and histochemical markers.

Effects of dietary NEXT ENHANCE®150 on growth performance and expression of immune and intestinal integrity related genes in gilthead sea bream (Sparus aurata L.).

Gilthead sea bream juveniles were fed different doses (0, 50, 100, 200, 300 ppm) of NEXT ENHANCE®150 (NE) for 9 weeks. Feed gain ratio (FGR) was improved by a 10% with all the doses, but feed intake decreased in a dose dependent manner. The optimum inclusion level to achieve maximum growth was set at 100 ppm. The hepatosomatic index did not vary and only at the highest dose, viscerosomatic and splenosomatic indexes were significantly decreased. No significant changes were found in haematological parameters, plasma biochemistry, total antioxidant capacity and respiratory burst. In a second trial, NE was given at 100 ppm alone (D1) or in combination with the prebiotic PREVIDA® (0.5%) (PRE) (D2) for 17 weeks. There were no differences in the growth rates, and FGR was equally improved for D1 and D2. No significant changes in haematology and plasma antioxidant capacity were detected. The histological examination of the liver and the intestine showed no outstanding differences in the liver, but the number of mucosal foldings appeared to be higher in D1 and D2 vs CTRL diet and the density of enterocytes and goblet cells also appeared higher, particularly in the anterior intestine. A 87-gene PCR-array was constructed based on our transcriptomic database (www.nutrigroup-iats.org/seabreamdb) and applied to samples of anterior (AI) and posterior (PI) intestine. It included 54 new gene sequences and other sequences as markers of cell differentiation and proliferation, intestinal architecture and permeability, enterocyte mass and epithelial damage, interleukins and cytokines, pattern recognition receptors (PRR), and mitochondrial function and biogenesis. More than half of the studied genes had significantly different expression between AI and PI segments. The functional significance of this differential tissue expression is discussed. The experimental diets induced significant changes in the expression of 26 genes. The intensity of these changes and the number of genes that were significantly regulated were higher at PI than at AI. At PI, both diets invoked a clear down-regulation of genes involved in cell differentiation and proliferation, some involved in cell to cell communication, cytokines and several PRR. By contrast, up-regulation was mostly found for genes related to enterocyte mass, cell epithelial damage and mitochondrial activity at AI. The changes were of the same order for D1 and D2, except for fatty acid-binding proteins 2 and 6 and the PRR fucolectin, which were higher in D2 and D1 fed fish, respectively. Thus, NE alone or in combination with PRE seems to induce an anti-inflammatory and anti-proliferative transcriptomic profile with probable improvement in the absorptive capacity of the intestine that would explain the improved FGR.

Interleukin gene expression is strongly modulated at the local level in a fish-parasite model.

The goal of this work was to identify interleukin (IL)-related genes in the gilthead sea bream (GSB) (Sparus aurata L.) and how they are modulated by the parasite Enteromyxum leei, a myxozoan that causes severe enteritis with a strong inflammatory response. A Blast-X search of our transcriptomic GSB database (www.nutrigroup-iats.org/seabreamdb) identified 16 new sequences encompassing seven ILs (IL-7, IL-8, IL-10, IL-12ß, IL-15, IL-18, and IL-34), the interleukin enhancer-binding factor 2 (ILF2), and eight IL receptors (IL-R); IL-R1, IL-6RA, IL-6RB, IL-8RA, IL-10RA, IL-10RB, IL-18R1, and IL-22R. Except for ILF2, their expression, plus that of IL-1ß, IL-1R2, IL-6, and TNF-α (from public repositories), were analysed by 96-well PCR array of samples of blood, spleen, head kidney, and intestine of GSB that were anally intubated with E. leei (recipient group, RCPT). Only the expression profile of the intestine of RCPT fish showed significant difference as compared to samples from PBS-inoculated fish. At 17 days post intubation (dpi), the expression of key pro-inflammatory ILs, such as IL-8, IL-8R, IL-12ß, and TNFα was significantly up-regulated, whereas at 64 dpi, anti-inflammatory IL expression (IL-6, IL-6RB, IL-7, IL-10, IL-10RA, and IL-15) was predominant. These results indicate a modification of the IL expression at late times post infection, probably to protect the fish intestine from the parasite and damage inflicted by an excessive inflammatory response. Furthermore, the response is mainly mediated at the local level as no significant changes were detected in blood, spleen and head kidney.

Modulation of leukocytic populations of gilthead sea bream (Sparus aurata) by the intestinal parasite Enteromyxum leei (Myxozoa: Myxosporea).

SUMMARY The cellular mucosal and systemic effectors of gilthead sea bream (GSB) (Sparus aurata) involved in the acute immune response to the intestinal parasite Enteromyxum leei were studied in fish experimentally infected by the anal route. In the intestinal inflammatory infiltrates and in lymphohaematopoietic organs (head kidney and spleen) of parasitized fish, the number of plasma cells, B cells (IgM immunoreactive) and mast cells (histamine immunoreactive) were significantly higher, whereas the number of acidophilic granulocytes (G7 immunoreactive) decreased, compared with non-parasitized and unexposed fish. These differences were stronger at the posterior intestine, the main target of the parasite, and no differences were found in the thymus. In non-parasitized GSB, the percentage of splenic surface occupied by melanomacrophage centres was significantly higher. These results suggest that the cellular response of GSB to E. leei includes proliferation of leukocytes in lymphohaematopoietic organs and recruitment into intestines via blood circulation involving elements of innate and adaptive immunity. Acidophilic granulocytes and mast cells presented opposite patterns of response to the parasite infection, with an overall depletion of the former and an increased amount of the latter. Some differences between both cell types were also detected in regard to their granule density and cell morphology.

An inside out journey: biogenesis, ultrastructure and proteomic characterisation of the ectoparasitic flatworm Sparicotyle chrysophrii extracellular vesicles.

BACKGROUND: Helminth extracellular vesicles (EVs) are known to have a three-way communication function among parasitic helminths, their host and the host-associated microbiota. They are considered biological containers that may carry virulence factors, being therefore appealing as therapeutic and prophylactic target candidates. This study aims to describe and characterise EVs secreted by Sparicotyle chrysophrii (Polyopisthocotyla: Microcotylidae), a blood-feeding gill parasite of gilthead seabream (Sparus aurata), causing significant economic losses in Mediterranean aquaculture. METHODS: To identify proteins involved in extracellular vesicle biogenesis, genomic datasets from S. chrysophrii were mined in silico using known protein sequences from Clonorchis spp., Echinococcus spp., Fasciola spp., Fasciolopsis spp., Opisthorchis spp., Paragonimus spp. and Schistosoma spp. The location and ultrastructure of EVs were visualised by transmission electron microscopy after fixing adult S. chrysophrii specimens by high-pressure freezing and freeze substitution. EVs were isolated and purified from adult S. chrysophrii (n = 200) using a newly developed ultracentrifugation-size-exclusion chromatography protocol for Polyopisthocotyla, and EVs were characterised via nanoparticle tracking analysis and tandem mass spectrometry. RESULTS: Fifty-nine proteins involved in EV biogenesis were identified in S. chrysophrii, and EVs compatible with ectosomes were observed in the syncytial layer of the haptoral region lining the clamps. The isolated and purified nanoparticles had a mean size of 251.8 nm and yielded 1.71 × 108 particles · mL-1. The protein composition analysis identified proteins related to peptide hydrolases, GTPases, EF-hand domain proteins, aerobic energy metabolism, anticoagulant/lipid-binding, haem detoxification, iron transport, EV biogenesis-related, vesicle-trafficking and other cytoskeletal-related proteins. Several identified proteins, such as leucyl and alanyl aminopeptidases, calpain, ferritin, dynein light chain, 14-3-3, heat shock protein 70, annexin, tubulin, glutathione S-transferase, superoxide dismutase, enolase and fructose-bisphosphate aldolase, have already been proposed as target candidates for therapeutic or prophylactic purposes. CONCLUSIONS: We have unambiguously demonstrated for the first time to our knowledge the secretion of EVs by an ectoparasitic flatworm, inferring their biogenesis machinery at a genomic and transcriptomic level, and by identifying their location and protein composition. The identification of multiple therapeutic targets among EVs' protein repertoire provides opportunities for target-based drug discovery and vaccine development for the first time in Polyopisthocotyla (sensu Monogenea), and in a fish-ectoparasite model.

Mucosal affairs: glycosylation and expression changes of gill goblet cells and mucins in a fish-polyopisthocotylidan interaction.

Introduction: Secreted mucins are highly O-glycosylated glycoproteins produced by goblet cells in mucosal epithelia. They constitute the protective viscous gel layer overlying the epithelia and are involved in pathogen recognition, adhesion and expulsion. The gill polyopisthocotylidan ectoparasite Sparicotyle chrysophrii, feeds on gilthead seabream (Sparus aurata) blood eliciting severe anemia. Methods: Control unexposed and recipient (R) gill samples of gilthead seabream experimentally infected with S. chrysophrii were obtained at six consecutive times (0, 11, 20, 32, 41, and 61 days post-exposure (dpe)). In histological samples, goblet cell numbers and their intensity of lectin labelling was registered. Expression of nine mucin genes (muc2, muc2a, muc2b, muc5a/c, muc4, muc13, muc18, muc19, imuc) and three regulatory factors involved in goblet cell differentiation (hes1, elf3, agr2) was studied by qPCR. In addition, differential expression of glycosyltransferases and glycosidases was analyzed in silico from previously obtained RNAseq datasets of S. chrysophrii-infected gilthead seabream gills with two different infection intensities. Results and Discussion: Increased goblet cell differentiation (up-regulated elf3 and agr2) leading to neutral goblet cell hyperplasia on gill lamellae of R fish gills was found from 32 dpe on, when adult parasite stages were first detected. At this time point, acute increased expression of both secreted (muc2a, muc2b, muc5a/c) and membrane-bound mucins (imuc, muc4, muc18) occurred in R gills. Mucins did not acidify during the course of infection, but their glycosylation pattern varied towards more complex glycoconjugates with sialylated, fucosylated and branched structures, according to lectin labelling and the shift of glycosyltransferase expression patterns. Gilthead seabream gill mucosal response against S. chrysophrii involved neutral mucus hypersecretion, which could contribute to worm expulsion and facilitate gas exchange to counterbalance parasite-induced hypoxia. Stress induced by the sparicotylosis condition seems to lead to changes in glycosylation characteristic of more structurally complex mucins.

Deep sequencing for de novo construction of a marine fish (Sparus aurata) transcriptome database with a large coverage of protein-coding transcripts.

BACKGROUND: The gilthead sea bream (Sparus aurata) is the main fish species cultured in the Mediterranean area and constitutes an interesting model of research. Nevertheless, transcriptomic and genomic data are still scarce for this highly valuable species. A transcriptome database was constructed by de novo assembly of gilthead sea bream sequences derived from public repositories of mRNA and collections of expressed sequence tags together with new high-quality reads from five cDNA 454 normalized libraries of skeletal muscle (1), intestine (1), head kidney (2) and blood (1). RESULTS: Sequencing of the new 454 normalized libraries produced 2,945,914 high-quality reads and the de novo global assembly yielded 125,263 unique sequences with an average length of 727 nt. Blast analysis directed to protein and nucleotide databases annotated 63,880 sequences encoding for 21,384 gene descriptions, that were curated for redundancies and frameshifting at the homopolymer regions of open reading frames, and hosted at http://www.nutrigroup-iats.org/seabreamdb. Among the annotated gene descriptions, 16,177 were mapped in the Ingenuity Pathway Analysis (IPA) database, and 10,899 were eligible for functional analysis with a representation in 341 out of 372 IPA canonical pathways. The high representation of randomly selected stickleback transcripts by Blast search in the nucleotide gilthead sea bream database evidenced its high coverage of protein-coding transcripts. CONCLUSIONS: The newly assembled gilthead sea bream transcriptome represents a progress in genomic resources for this species, as it probably contains more than 75% of actively transcribed genes, constituting a valuable tool to assist studies on functional genomics and future genome projects.

Antigenic characterization of Enteromyxum leei (Myxozoa: Myxosporea).

Enteromyxum leei, an intestinal myxozoan parasite affecting a wide range of fish, was partially purified, and the immunogenic composition of its glycoproteins as well as the proteolytic activity were studied. Parasite extracts, mainly containing spores, were separated by SDS-PAGE, and thereafter, immunoblots were carried out with a polyclonal antiserum (Pab) raised against E. leei. Periodic acid/Schiff staining of gels, periodate- and Proteinase K-treated Western blots and Lectin blots were performed to analyse the terminal carbohydrate composition of the parasite's antigens. Additionally, the cross-reaction of the parasite extracts with a Pab raised against the polar filament of the myxozoan Myxobolus pendula was studied. Both Pabs detected proteic epitopes on antigenic proteins and glycoproteins of E. leei, ranging between 15 and 280 kDa. In particular, 2 glycoproteic bands (15 and 165 kDa), immunoreactive to both Pabs and with glucose and mannose moieties, could correspond to common antigens shared among myxozoans. The 165 kDa band also presented galactose, N-acetyl-galactosamine and N-acetyl-glucosamine, pointing to its possible origin on chitin-built spore valves and to its possible involvement in host-parasite interactions. The molecular weight of the 15 kDa glycoproteic antigen matches that of minicollagen, a cnidarian-specific protein of nematocysts with a myxozoan homologue. Several proteases with apparent molecular weights ranging between 43 and 245 kDa were found in zymographies of E. leei extracts, and these may have a potential role in the parasite's pathogenesis. This is a useful approach for further studies to detect targets for antiparasitic therapy.

Effects of Enteromyxum leei (Myxozoa) infection on gilthead sea bream (Sparus aurata) (Teleostei) intestinal mucus: glycoprotein profile and bacterial adhesion.

The intestinal myxosporean parasite Enteromyxum leei causes severe desquamative enteritis in gilthead sea bream (Sparus aurata) (Teleostei) that impairs nutrient absorption causing anorexia and cachexia. In fish, as in terrestrial vertebrates, intestinal goblet cells are responsible for the adherent mucus secretion overlying epithelial cells, which constitutes a first line of innate immune defense against offending microorganisms but serves also as substrate and nutrient source for the commensal microflora. The secreted intestinal mucus of parasitized (n = 6) and unexposed (n = 8) gilthead sea bream was isolated, concentrated, and subjected to downward gel chromatography. Carbohydrate and protein contents (via PAS and Bradford stainings), terminal glycosylation (via lectin ELISA), and Aeromonas hydrophila and Vibrio alginolyticus adhesion were analyzed for the isolated intestinal mucins. Parasitized fish, compared with unexposed fish, presented intestinal mucus mucins with a lower glycoprotein content and glycosylation degree at the anterior and middle intestine, whereas both glycoprotein content and glycosylation degree increased at the posterior intestine section, though only significantly for the total carbohydrate content. Additionally, a slight molecular size increase was detected in the mucin glycoproteins of parasitized fish. Terminal glycosylation of the mucus glycoproteins in parasitized fish pointed to an immature mucin secretion (N-acetyl-α-D-galactosamine increase, α-L-fucose, and neuraminic-acid-α-2-6-galactose reduction). Bacterial adhesion to large-sized mucus glycoproteins (>2,000 kDa) of parasitized fish was significantly lower than in unexposed fish.

Modulation of the IgM gene expression and IgM immunoreactive cell distribution by the nutritional background in gilthead sea bream (Sparus aurata) challenged with Enteromyxum leei (Myxozoa).

The aim of the present work was to determine if a plant protein-based diet containing vegetable oils (VO) as the major lipid source could alter the distribution of IgM immunoreactive cells (IRCs) and the IgM expression pattern in the intestine and haematopoietic tissues of gilthead sea bream (GSB) (Sparus aurata) challenged with the myxosporean Enteromyxum leei. In a first trial (T1), GSB fed for 9 months either a fish oil (FO) diet or a blend of VO at 66% of replacement (66VO diet) was challenged by exposure to parasite-contaminated water effluent. All fish were periodically and non-lethally sampled to know their infection status. After 102 days of exposure, samples of intestine and head kidney were obtained for IgM expression and immunohistochemical detection (IHC). Additional samples of spleen were taken for IHC. Fish were categorized as control (C, not exposed), and early (E), or late (L) infected. The 66VO diet had no effect on the number of IgM-IRCs in any of the tissues or on IgM expression in C fish, whereas the infection with E. leei had a strong effect on the intestine. A combined time-diet effect was also observed, since the highest expression and IRCs values were registered in the posterior intestine (Pi) of E-66VO fish. A positive correlation was found between IgM expression and the presence of IgM-IRCs in the Pi. The effect of the time of infection was studied more in detail in a second trial (T2) in which samples of Pi were taken at 0, 24, 51, 91 and 133 days after exposure to the parasite. A significant increase of the IgM expression was detected only in parasitized fish, and very late after exposure. These results show that the duration of the exposure to the parasite is the most determinant factor for the observed intestinal IgM increased phenotype which gets magnified by the feeding of a high VO-based diet.

Effect of nutrition and Enteromyxum leei infection on gilthead sea bream Sparus aurata intestinal carbohydrate distribution.

The effect of a practical plant protein-based diet containing vegetable oils (VO) as the major lipid source on the mucosal carbohydrate pattern of the intestine was studied in gilthead sea bream Sparus aurata challenged with the myxosporean parasite Enteromyxum leei. Fish fed for 9 mo either a fish oil (FO) diet or a blend of VO at 66% of replacement (66VO diet) were exposed to parasite-contaminated water effluent. Samples of the anterior, middle and posterior intestine (AI, MI and PI, respectively) were obtained for parasite diagnosis and histochemistry. Fish were categorised as control (C, not exposed), early (E) or late (L) infected. Mucin and lectin histochemistry was applied to detect the different types of mucins and sialic acid in goblet cells (GC), the brush border and enterocytes. The number of GC stained with periodic acid Schiff (PAS), alcian blue (AB), aldehyde fuchsin-alcian blue (AF-AB), for the detection of neutral, acidic, sulphated and carboxylic mucins, and with the lectin Sambucus nigra agglutinin (SNA), were counted in digital images. The 66VO diet produced a significant decrease of GC with neutral and acidic mucins in the AI and MI, and also of those with carboxylic mucins and sialic acid in the MI. Sulphated mucins and sialic acid were less abundant in the AI than in the MI and PI in the C-66VO treatment. E. leei infection had a strong effect on the number of GC, as E and L infected fish had a significant decrease of GC positive for all the stains versus C fish in PI. Time and diet effects were also observed, since the lowest values were mostly registered in E-66VO fish in PI. In conclusion, though GC depletion was mainly induced by enteromyxosis, an effect of the diet was also observed. Thus, the diet can be a predisposing factor that worsens the disease course.

A bloody interaction: plasma proteomics reveals gilthead sea bream (Sparus aurata) impairment caused by Sparicotyle chrysophrii.

BACKGROUND: Sparicotylosis is an enzootic parasitic disease that is well established across the Mediterranean Sea. It is caused by the polyopisthocotylean monogenean Sparicotyle chrysophrii and affects the gills of gilthead sea bream (GSB; Sparus aurata). Current disease management, mitigation and treatment strategies are limited against sparicotylosis. To successfully develop more efficient therapeutic strategies against this disease, understanding which molecular mechanisms and metabolic pathways are altered in the host is critical. This study aims to elucidate how S. chrysophrii infection modulates the plasma proteome of GSB and to identify the main altered biological processes involved. METHODS: Experimental infections were conducted in a recirculating aquaculture system (RAS) in which naïve recipient GSB ([R]; 70 g; n = 50) were exposed to effluent water from S. chrysophrii-infected GSB (98 g; n = 50). An additional tank containing unexposed naïve fish (control [C]; 70 g; n = 50) was maintained in parallel, but with the open water flow disconnected from the RAS. Haematological and infection parameters from sampled C and R fish were recorded for 10 weeks. Plasma samples from R fish were categorised into three different groups according to their infection intensity, which was based on the number of worms fish-1: low (L: 1-50), medium (51-100) and high (H: > 100). Five plasma samples from each category and five C samples were selected and subjected to a SWATH-MS proteome analysis. Additional assays on haemoglobin, cholesterol and the lytic activity of the alternative complement pathway were performed to validate the proteome analysis findings. RESULTS: The discriminant analysis of plasma protein abundance revealed a clear separation into three groups (H, M/L and C). A pathway analysis was performed with the differentially quantified proteins, indicating that the parasitic infection mainly affected pathways related to haemostasis, the immune system and lipid metabolism and transport. Twenty-two proteins were significantly correlated with infection intensity, highlighting the importance of apolipoproteins, globins and complement component 3. Validation assays of blood and plasma (haemoglobin, cholesterol and lytic activity of alternative complement pathway) confirmed these correlations. CONCLUSIONS: Sparicotylosis profoundly alters the haemostasis, the innate immune system and the lipid metabolism and transport in GSB. This study gives a crucial global overview of the pathogenesis of sparicotylosis and highlights new targets for further research.

Intestinal Transcriptomic and Histologic Profiling Reveals Tissue Repair Mechanisms Underlying Resistance to the Parasite Ceratonova shasta.

BACKGROUND: Myxozoan parasites infect fish worldwide causing significant disease or death in many economically important fish species, including rainbow trout and steelhead trout (Oncorhynchus mykiss). The myxozoan Ceratonova shasta is a parasite of salmon and trout that causes ceratomyxosis, a disease characterized by severe inflammation in the intestine resulting in hemorrhaging and necrosis. Populations of O. mykiss that are genetically fixed for resistance or susceptibility to ceratomyxosis exist naturally, offering a tractable system for studying the immune response to myxozoans. The aim of this study was to understand how steelhead trout that are resistant to the disease respond to C. shasta once it has become established in the intestine and identify potential mechanisms of resistance. RESULTS: Sequencing of intestinal mRNA from resistant steelhead trout with severe C. shasta infections identified 417 genes differentially expressed during the initial stage of the infection compared to uninfected control fish. A strong induction of interferon-gamma and interferon-stimulated genes was evident, along with genes involved in cell adhesion and migration. A total of 11,984 genes were differentially expressed during the late stage of the infection, most notably interferon-gamma, interleukin-6, and immunoglobulin transcripts. A distinct hardening of the intestinal tissue and a strong inflammatory reaction in the intestinal submucosa including severe hyperplasia and inflammatory cell infiltrates were observed in response to the infection. The massive upregulation of caspase-14 early in the infection, a protein involved in keratinocyte differentiation might reflect the rapid onset of epithelial repair mechanisms, and the collagenous stratum compactum seemed to limit the spread of C. shasta within the intestinal layers. These observations could explain the ability of resistant fish to eventually recover from the infection. CONCLUSIONS: Our results suggest that resistance to ceratomyxosis involves both a rapid induction of key immune factors and a tissue response that limits the spread of the parasite and the subsequent tissue damage. These results improve our understanding of the myxozoan-host dialogue and provide a framework for future studies investigating the infection dynamics of C. shasta and other myxozoans.

Experimental Horizontal Transmission of Enterospora nucleophila (Microsporea: Enterocytozoonidae) in Gilthead Sea Bream (Sparus aurata).

Enterospora nucleophila is a microsporidian enteroparasite that infects mainly the intestine of gilthead sea bream (Sparus aurata), 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 E. nucleophila 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 E. nucleophila 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.

Novel horizontal transmission route for Enteromyxum leei (Myxozoa) by anal intubation of gilthead sea bream Sparus aurata.

The aim of the present study was to determine whether Enteromyxum leei, one of the most threatening parasitic diseases in Mediterranean fish culture, could be transmitted by peranal intubation in gilthead sea bream Sparus aurata L. Fish were inoculated either orally or anally with intestinal scrapings of infected fish in 3 trials. Oral transmission failed, but the parasite was efficiently and quickly transmitted peranally. Prevalence of infection was 100% at 60 d post inoculation (p.i.) in Trial 1 under high summer temperature (22 to 25 degrees C; fish weight = 187.1 g), and 85.7% in just 15 d p.i. in Trial 3 using smaller fish (127.5 g) at autumn temperature (19 to 22 degrees C). In Trial 2, prevalence reached 60% at 60 d p.i. in the group reared at constant temperature (18 degrees C), whereas no fish was infected in the group that was kept at low winter temperature (11 to 12 degrees C), although infection appeared (46.1% at 216 d p.i.) when temperature increased in spring. The arrested development at low temperature has important epidemiological consequences, as fish giving false negative results in winter can act as reservoirs of the parasite. Histopathological examination showed a posterior-anterior intestinal gradient in the progression of the infection, in terms of both intensity and parasite maturation. Thus, per-anal intubation provides a very uniform, reliable and faster mode of transmission of E. leei than the commonly used transmission methods (cohabitation, exposure to infected effluent and oral inoculation), which require long exposure times or give variable and unpredictable results.

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

Passive immunization constitutes an emerging field of interest in aquaculture, particularly with the restrictions for antibiotic use. Enteromyxum leei 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, Sparus aurata) that survive E. leei 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 E. leei-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 E. leei 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 E. leei and set the basis for the development of effective treatment or prophylactic methods for aquaculture.