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

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.

Horizontal transmission of Endolimax piscium, causative agent of systemic amoebiasis in Senegalese sole Solea senegalensis.

Systemic amoebiasis of Senegalese sole Solea senegalensis is caused by Endolimax piscium Constenla, Padrós & Palenzuela, 2014 a cryptic parasitic member of the Archamoebae whose epidemiology is yet unknown. To test whether the parasite can be transmitted horizontally, an experimental trial by cohabitation between non-infected and infected fish was designed. Transmission of the parasite from naturally infected to healthy fish was confirmed in the experiment, with the water as the most likely route of infection. Under the conditions of the study, the infection process was remarkably slow, as parasites could be detected by in situ hybridization within the intestinal mucosa of recipient fish only after 17 wk of cohabitation, and none of the new hosts displayed clinical signs of disease. Long prepatent period and the need for additional triggering factors for the development of the clinical condition are suggested. The intestinal mucosa is proposed as the tissue where the amoeba can survive as endocommensal, but also as an invasion route from which the parasite would disperse to other organs.

Development of different diagnostic techniques for Endolimax piscium (archamoebae) and their applicability in Solea senegalensis clinical samples.

Systemic amoebiasis of sole is caused by Endolimax piscium, a cryptic parasitic archamoeba whose epidemiology and pathogeny are yet unknown. To establish reliable detection methods for this parasite, a battery of molecular diagnostic tools (ISH, PCR and qPCR) were developed and evaluated with a panel of clinical samples from symptomatic diseased fish and from apparently normal animals of different stocks. As there is neither enough background information on the epidemiology of the disease nor a validated reference method, comparison of tests used a composite reference method approach. The ISH technique was the most specific and sensitive in intestine samples and particularly useful as a reference confirmatory method, while the best method in muscle samples was qPCR. Application of the tests to asymptomatic fish demonstrated presence of parasites in a large proportion (>25%) of their intestines, suggesting that this is the point of entry of the amoebae and the initial stage in the development of the disease. The triggering factors that facilitate the breaching of the intestinal barrier by E. piscium, causing granulomatous lesions in other organs and systemic spreading, are not completely understood but our results point to the connective tissue as a preferential target for parasite development and migration.

Endolimax piscium sp. nov. (Amoebozoa), causative agent of systemic granulomatous disease of cultured sole, Solea senegalensis Kaup.

A new amoeba species pathogenic for Senegalese sole is described based on ultrastructural analysis and SSU rDNA phylogenetic inference. The parasite presents round to ovoid trophozoites (<5 µm) with a high degree of intracellular simplification. No mitochondria were observed, but mitosome-like organelles were present. No cysts could be detected. Phylogenetic analysis confirmed the Senegalese sole parasite as an amitochondriate Archamoeba related to Endolimax nana and Iodamoeba spp., and we tentatively describe it as a new species in the genus Endolimax, Endolimax piscium. However, the genetic distance with E. nana is quite large, with only 60% pairwise identity between both SSU rDNA genotypes. Although the overall topology of the Archamoebae cladograms containing E. piscium was consistent, the support for the branching of Endolimax spp. relative to its closest neighbours was variable, being higher with distance or parsimony-based inference methods than with ML or Bayesian trees. The use of stringent alignment sampling masks also caused instability and reduced support for some branches, including the monophyly of Endolimax spp. in the most conservative data sets. The characterization of other Archamoebae parasitizing fish could help to clarify the status of E. piscium and to interpret the large genetic distance observed between Endolimax species.

Detection of carbohydrate terminals in the enteric parasite Enteromyxum scophthalmi (Myxozoa) and possible interactions with its fish host Psetta maxima.

The existence and localisation of carbohydrate terminals in Enteromyxum scophthalmi stages was investigated at light (LM) and transmission electron microscopes (TEM) using lectin histochemistry techniques, with the aim of contributing to elucidate the participation of carbohydrate-lectin interactions in the parasite invasion and relationships with the fish host. The presence of abundant mannose and/or glucose residues was demonstrated by the intense staining by concanavalin A at both LM and TEM. The staining pattern obtained with soybean agglutinin and Bandeiraea simplicifolia I (BSI) indicated the abundance of N-acetyl-galactosamine and D-galactose at a lesser extent. The lectins wheat germ agglutinin, BSI and Ulex europaeus agglutinin produced weaker marks. Most lectins recognised structures present in both pre-sporogonic and sporogonic stages, though the glycosidic pattern and/or staining intensity varied between developmental stages. No staining was obtained with Sambucus nigra agglutinin. The TEM studies demonstrated glucose-mannose, N-acetyl-glucosamine, N-acetyl-galactosamine and alpha-D-galactose as dominant structures at the parasite membrane and host-parasite interface, suggesting a role in host-parasite interactions. All these terminals were also detected in the mitochondria of P cells and were scant in the S cells and nuclei. In turbot intestine, mannose-glucose terminals and N-acetyl-glucosamine were labelled on the epithelial brush border and in the mucous cells and rodlet cells. The relevance of these findings in relation to the host-parasite interaction is discussed.

Immune response of turbot, Psetta maxima (L.) (Pisces: Teleostei), to formalin-killed scuticociliates (Ciliophora) and adjuvanted formulations.

The increasing frequency of scuticociliatosis in turbot culture has stressed the need of knowledge on the immune responses to these parasites, for further developing of prevention and control strategies. The immune response of turbot to killed parasites, alone (Ag) or in combination with Montanide ISA 763A (MON), was studied in a laboratory-scale experiment. The variations of several innate immune factors and the antibody response were analysed in immunized vs. non-immunized fish at different times after immunization, and also after a challenge with live ciliates. Amongst innate immune factors, serum lysozyme increased progressively in all inoculated groups. Differences in innate immune factors in Ag and Ag-MON fish with respect to controls were mainly evidenced after challenge, especially for serum complement. Serum antibody levels increased in immunized fish after booster and particularly after challenge. In addition, certain protection was obtained for immunized groups compared to controls or to fish receiving MON alone, and the levels of specific antibodies were also the highest in immunized groups. The obtained information could be useful for further design of immunoprophylactic formulations against scuticociliatosis.

Histopathology of experimental scuticociliatosis in turbot Scophthalmus maximus.

A scuticociliate strain (B-2), originally isolated from an outbreak in a turbot Scophthalmus maximus (= Psetta maxima) farm in Galicia (northwestern Spain) and maintained in axenic culture, was injected intracoelomically (lethal dose 80 equivalent, LD80) in healthy turbot (50 g). Ciliate-injected fish were kept under controlled conditions in a recirculating seawater system and sampled on Days 1 through 8, 10, 12 and 14 postinfection (PI). Necropsies were conducted and included blood collection from the caudal vein and samples of liver, spleen, heart, digestive tract, kidney, gills, abdominal wall and neurocranium taken for routine histology. Mortality occurred from Day 6 until Day 12 PI and reached 66.7% by the end of the experiment. Presence of ciliates in the coelomic fluid was scarce until Day 4 PI. Parasitaemia was only observed from Day 5 until Day 10 PI and its incidence was always low. Presence of scuticociliates in tissue sections followed a progressive pattern of diffusion, with ciliates showing preference for loose connective tissue and also a clear haematophagous activity. The most severely affected organs were the pancreas and digestive tract. No special tropism for nervous tissues was observed in this study. The inflammatory reaction was variable depending on the tissue. After 3 wk, survivors had apparently managed to extinguish the infection.

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

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

An unidentified epi-epithelial myxosporean in the intestine of gilthead sea bream Sparus aurata L.

In the course of experimental infections of gilthead sea bream Sparus aurata with the myxozoan Enteromyxum leei, stages of an unidentified myxozoan were observed attached to the intestinal brush border of some fish. Infection levels of the parasite, which was named "epi-epithelial myxosporean" (EEM) were recorded, and its structure was studied by light microscopy (LM) and electron microscopy (EM). In situ hybridisation (ISH) probes specific for E. leei were developed and used to differentiate between the two parasites. The EEM parasite was observed only in epi-epithelial position on the intestine mucosa and never in any of the other tissues studied (kidney and gall bladder). Prevalence was variable, with values reaching 40.2%. With transmission EM, trophozoites displayed pseudopodia-like projections inserted in between the enterocyte microvilli, producing an intimate interface. No mucosal histopathology that could be attributed to the myxozoan was found. EEM stages did not stain with the E. leei-specific ISH probes. From the results of the LM, EM and ISH studies, we conclude that the EEM parasite found in gilthead sea bream intestine in both Mediterranean and Red Sea sites is a coelozoic myxosporean, distinct from E. leei.

Myxozoan infections in Mediterranean mariculture.

Fish mariculture has dramatically expanded in recent years in Mediterranean countries. In this scenario, several pathological problems have logically arisen and parasitological etiologies are increasingly being reported, either as primary or secondary pathogens. Myxozoa is the most diverse and economically important group of fish parasites, and several species are known to cause or contribute to losses in mariculture. Species of the genus Enteromyxum currently constitute the most serious parasitological threat. Some unusual biological characters, such as wide host spectrum and direct fish-to-fish transmission, together with high virulence for some host species, combine a dangerous cocktail which is emerging in recent years. Closed-system (recirculation) and heated-water locations are especially sensitive to chronic infections by these parasites, which can cause serious mortality and even discourage culture of some fish species at certain locations (i.e, Diplodus puntazzo). The presentation presents an overview of recent advances in research of marine myxozoans, focusing mainly in the most pathogenic, Enteromyxum spp. The incidence of these and other emerging infections, and the design of potential strategies for control will be introduced.

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

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

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

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

Cryptosporidium scophthalmi n. sp. (Apicomplexa: Cryptosporidiidae) from cultured turbot Scophthalmus maximus. Light and electron microscope description and histopathological study.

Cryptosporidium scophthalmi n. sp. is described from the turbot Scophthalmus maximus L., sampled from different farms on the coast of NW Spain. The parasite was found mainly in the intestinal epithelium and very seldom in the stomach. Oocysts were almost spherical, with 4 naked sporozoites and a residuum, and measured 3.7-5.03 x 3.03-4.69 microm (mean 4.44 x 3.91) (shape index 1.05-1.34, mean 1.14). Sporulation was endogenous, as fully sporulated oocysts were found within the intestinal epithelium, lumen and faeces. Merogonial and gamogonial stages were in the typical extracytoplasmic position, whereas sporogonial stages were deep within the epithelium. Oocysts and other stages of C. scophthalmi comply with most of the diagnostic features of the genus Cryptosporidium, but differ from all hitherto described species. Ultrastructural features, including the characteristic feeding organelle, were mainly comparable with those of other Cryptosporidium species. Mitochondria were frequently observed in sporozoites. Infection prevalence was very variable, and juvenile fish were most frequently and intensively parasitised. External clinical signs were not detected, although some fish showed intestinal distension at necropsy. The marked histopathological damage occurring in severe infection includes distension of epithelial cells by large vacuoles, containing clusters of oocysts, and can lead to sloughing of epithelial cell remnants and oocysts or even detachment of intestinal mucosa. An inflammatory reaction involving leucocyte infiltration was sometimes observed.

Description of Enteromyxum scophthalmi gen. nov., sp. nov. (Myxozoa), an intestinal parasite of turbot (Scophthalmus maximus L.) using morphological and ribosomal RNA sequence data.

A new Myxozoa species causing enteritis and death in cultured turbot, Scophthalmus maximus, is described at light and electron microscope levels. In addition, small subunit ribosomal RNA gene sequences (SSU rDNA) from the new species and from similar myxozoans were obtained and used for phylogenetic inference, as complementary criteria to resolve its taxonomic classification. The new parasite is closely related to Myxidium leei, another enteric histozoic species from marine fish. However, the ascription of M. leei to the genus Myxidium was based on weak morphological evidence and is not supported by our rDNA data analysis. A close relationship with Zschokkella, the other morphologically related myxozoan genus is also not supported. The combined morphological and molecular study results in the establishment of the new genus Enteromyxum to accommodate the new species E. scophthalmi, and the former M. leei, which is transferred to the new genus as Enteromyxum leei (Diamant, Lom & Dyková 1994) n. comb. This genus of marine, histozoic and enteric myxozoans includes significant parasite species for marine finfish culture.

Myxidium leei (Myxozoa) infections in aquarium-reared Mediterranean fish species.

An episode of parasitic enteritis causing trickling mortalities at an exhibition aquarium reproducing Mediterranean ecosystems was found to be caused by the myxozoan parasite Myxidium leei Diamant, Lom & Dykova 1994. The myxozoan was recorded in 25 different fish species belonging to 16 Genera, 10 Families and 4 Orders. It was mainly detected in the intestine of affected fish, and was responsible for severe chronic enteritis. The parasite was probably introduced into the facilities with infected wild fish, and transmitted directly from fish to fish by cohabitation, transfer of infected material and necrophagia. Fish belonging to the Families Labridae and Blenniidae appeared as most susceptible, and the incidence of infections in members of the Sparidae was low. This study significantly widens the host spectrum for this virulent parasite and now includes many ubiquitous coastal Mediterranean species. Wild fish may have a significant role in the transmission of myxidiosis of cultured sparid fish.

Development of a polymerase chain reaction diagnostic assay for Ceratomyxa shasta, a myxosporean parasite of salmonid fish.

A diagnostic procedure based on the polymerase chain reaction (PCR) was developed for the myxosporean parasite Ceratomyxa shasta. Three sets of oligonucleotide primers were designed to specifically amplify C. shasta ribosomal RNA genes and several parameters of the assay were tested and optimised. A simple protocol for the processing of fish tissue samples was also developed. In a single round, 20 microliters volume reaction the optimised procedure allows the detection of 50 fg of purified C. shasta genomic DNA, or 0.01 spore from a seeded fish intestine sample. This protocol is considerably faster, cheaper and more reliable than any previous diagnostic procedure for a myxosporean parasite, and can be an invaluable tool for the monitoring of early and/or subclinical C. shasta infections in wild and cultured salmon populations.

Immunohistochemical reactivity of polyclonal antibodies against Sphaerospora testicularis and Ceratomyxa labracis (Myxosporea: Bivalvulida), with other myxosporean parasites.

Immunological staining with rabbit antibodies raised against Sphaerospora testicularis and Ceratomyxa labracis was used to characterise their specificity and their reactivity towards other fish parasites. Polar capsules and valves of S. testicularis and C. labracis were labelled with their homologous antibody and cross reaction was observed with all the myxosporean parasites assayed from marine and freshwater fish hosts. All polar capsules were stained with both antibodies, except those of Zschokkella mugilis, which were not labelled with anti-S. testicularis serum. These observations suggest that polar capsules may be very conserved structures in myxosporean parasites from different hosts.

Glomerular disease associated with Polysporoplasma sparis (Myxozoa) infections in cultured gilthead sea bream, Sparus aurata L. (Pisces: Teleostei).

Polysporoplasma sparis infection was studied in gilthead sea bream from different mariculture systems of the Spanish coasts. Culture conditions influenced the infection dynamics, as the parasite appeared only in semi-intensive cultures and was not found in intensive closed systems nor in open ones. No clear seasonal pattern was observed. No fish weighing less than 51 g was found parasitized in any group. A statistically significant dependence between infection prevalence and host weight was observed in some growing stocks. Light and transmission electron microscope observations revealed serious damage in the trunk kidney. Glomerular disease was provoked by the progressive occupation of the glomerular capillaries by P. sparis spores. Tubular epithelial cells were also affected. Inflammatory responses appeared towards the end of the infection, and consisted mainly of melanomacrophages and eosinophils. Rodlet cells were common close to infected capillaries and debris of rodlet sacs formed a belt encircling capillary vessels. Cytochemistry demonstrated the lipidic nature of these sacs and the glycogen and glycoprotein composition of the cytoplasmic granules of rodlet cells.

Comparative studies on carbohydrates of several myxosporean parasites of fish using lectin histochemical methods.

A histochemical study using lectin methods was performed on myxosporean parasites from vastly different fish hosts from marine and fresh waters. Six biotinylated lectins were used (WGA, SBA, BS-I, Con-A, UEA-I and SNA). The binding pattern of Con-A and WGA revealed the presence of mannose and/or glucose, and N-acetyl-D-glucosamine respectively, in polar capsules and valves of most of the myxosporea assayed. Thus, chitin may be present in polar capsules and/or valves of myxosporean spores. The BS-I binding pattern showed the presence of alpha-D-galactose and/or N-acetyl-D-galactosamine residues in polar capsules of Kudoa sp., Zschokkella mugilis Sitjà-Bobadilla et Alvarez-Pellitero, 1993 and Leptotheca sp., and in the valves of the latter. Scarce amounts of N-acetyl-D-galactosamine and/or alpha-D-galactose were demonstrated by SBA binding in Sphaerospora dicentrarchi Sitjà-Bobadilla et Alvarez-Pellitero 1992, Leptotheca sp. and Kudoa sp. valves, and in Leptotheca sp. polar capsules. The UEA-I staining indicated the absence of alpha-L-fucose in all the myxosporea assayed except in Leptotheca sp. N-acetylneuraminic acid was detected with SNA in the polar capsules and sporoplasms of Polysporoplasma sparis Sitjà-Bobadilla et Alvarez-Pellitero, 1995 and in the polar capsules and valves of Kudoa sp. These results indicate that, although Myxosporea may have conserved carbohydrate structures, some of them can show significantly different binding patterns, which may be useful in diagnostic and functional studies.