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

Enterospora nucleophila is a microsporidian responsible for an emaciative disease in gilthead sea bream (Sparus aurata). Its intranuclear development and the lack of in vitro and in vivo 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 E. nucleophila 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 in situ hybridization in the submucosa when intracytoplasmic stages were present. This study describes the lesions of E. nucleophila in gilthead sea bream, an important aquaculture species.

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.

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).

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.

Effects of Different Co-Feeding Protocols on the Early Weaning of Flathead Grey Mullet (Mugil cephalus) Larvae.

The sustainable expansion of aquaculture relies on a sufficient supply of eggs and larvae, which are the first step of life cycle management. However, marine fish larval rearing generally depends on live feed production, which needs additional facilities and labor. The flathead grey mullet (Mugil cephalus), a promising species for aquaculture diversification, has a precocious digestive system development, supporting the feasibility of early weaning strategies. For these reasons, this study evaluated survival, growth, proximate and fatty acid composition, and gene expression of Mugil cephalus larvae reared under three different weaning protocols. Three co-feeding treatments, two with different Artemia sp. concentrations (A100 and A50, 2 and 1 Artemia sp. mL-1 day-1, respectively) and one with only rotifers administered as live feed along the feeding trial (A0), were assessed from 22 to 36 days post-hatching (dph). The A0 treatment performed better in survival (64.79 ± 7.40%) than the A100 protocol (32.46 ± 12.82%). In contrast, the larvae of the A100 treatment presented significantly higher final length (15.51 ± 0.86 mm) than those of the A0 treatment (12.19 ± 1.45 mm) and higher final weight (41.28 ± 1.48 mg) than those of the A50 and A0 treatments (31.23 ± 3.65 mg and 24.03 ± 7.99 mg, respectively). On the other hand, the expression of digestive enzyme- and somatotropic factor-related genes did not show differences between treatments. The present results support the convenience of treatment A0 in maximizing survival, as rotifers should be maintained until 30-32 dph (until a total larval length of at least 10 mm). However, to improve growth and minimize size dispersion, Artemia sp. addition is recommended from day 26 to day 29 post-hatching (total larval length of 8 to 9 mm).

Evaluation of the Effects of Two Different Feeding Frequencies on the Digestive Biochemistry of Two Mullet Species (Chelon labrosus and Liza aurata).

Mullets (Mugilidae) present significant potential for sustainable aquaculture diversification due to their eurythermal, euryhaline, and low-trophic nature. However, the physiological differences and optimal cultured conditions among the diverse mullet species are quite unknown. For these reasons, the present study aimed to address two main objectives: (1) to characterize the differences in digestive biochemistry, somatic indexes, and body composition between two mullet species (Liza aurata and Chelon labrosus); and (2) to evaluate the interactions of two different feeding frequencies (one against three meals per day) on the above-mentioned parameters, and also on the potential bioavailability of nutrients determined using in vitro assays. The results evidenced higher protease and amylase activities for Chelon labrosus than for Liza aurata, while the latter species presented a higher percentage of eviscerated weight and muscle lipids. Furthermore, the results from in vitro assays supported the higher enzyme activity of Chelon labrosus by an observed increase in the release of amino acids and reducing sugars measured for this species. Regarding feeding patterns, the results of the in vitro assays simulating enzyme: substrate (E:S) ratios corresponding to one or three meals per day point to a clear increase of nutrient bioavailability when the daily ration is split into several meals. The present results improve the physiological knowledge of mullet species and define criteria to develop better management protocols by producers.

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.

Determination of heavy metals from Aloe vera by- product in golden mullet (Liza aurata); A consumer health risk assessment.

Heavy metals have become subject of concern in the recent years because of its potency to cause cardiovascular diseases and other toxic health effects. Therefore, this research was assumed to investigate the level of toxicity in terms of heavy metals accumulation in the fish samples and its benefits and risk for human consumers health and also evaluate the partial replacement of plant sources by canarian Aloe vera diets as a pure product or like a by-product on toxicological effects on the golden mullet (Liza aurata) fillet and whole body. In this study risks arising from fish metal content has been measured using various parameters as Estimated Daily Intake (EDI), Maximum Safe Consumption (MSCA), Target Hazard quotient (THQ), Hazard Index (HI) Carcinogenic risk of As (As- CR), the Value Selenium Health Benefit (Se HBV) and also the Nutritional Values has been evaluated. The results showed that all heavy metal levels in the fish tissue and diets were below the confirmed safe limits for consumption. In case of diets, it is obvious that with the exception of As, Hg, and Se, the presence of heavy or essential metals in both whole fish and raw fillet in golden grey mullet given experimental diets revealed that the whole fish had the highest concentration. Thus, it can be concluded that Aloe vera product and byproduct were in safety limits for fish and also for humans through food chain. Various risk and benefit assessment measures established by national and international authorities concluded that Liza aurata use was mostly safe.

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.

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.

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.

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 TH1, CTL and TH17 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.

Anestesia para cesárea en unapaciente con siringomielia. Reporte de un caso / Anesthesia for cesarean section in a patient with syringomiella. a case report

Paciente femenina de 39 años de edad con diagnóstico de Siringomielia a la que se le realiza cesárea. Se presenta el manejo anestésico y se discute el caso