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.

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

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.

Long-term epidemiological survey of Kudoa thyrsites (Myxozoa) in Atlantic salmon (Salmo salar L.) from commercial aquaculture farms.

Kudoa thyrsites (Myxozoa) encysts within myocytes of a variety of fishes. While infected fish appear unharmed, parasite-derived enzymes degrade the flesh post-mortem. In regions of British Columbia (BC), Canada, up to 4-7% of fillets can be affected, thus having economic consequences and impacting the competitiveness of BC's farms. K. thyrsites was monitored in two farms having high (HP) or low (LP) historical infection prevalence. At each farm, 30 fish were sampled monthly for blood and muscle during the first year followed by nine samplings during year two. Prevalence and intensity were measured by PCR and histology of muscle samples. In parallel, fillet tests were used to quantify myoliquefaction. Infections were detected by PCR after 355 and 509 degree days at LP and HP farms, respectively. Prevalence reached 100% at the HP farm by 2265 degree days and declined during the second year, whereas it plateaued near 50% at the LP farm. Infection intensities decreased after 1 year at both farms. Blood was PCR-positive at both farms between 778 and 1113 degree days and again after 2000 degree days. This is the first monitoring project in a production environment and compares data between farms with different prevalence.

Immunohistochemical characterization of polyclonal antibodies against Enteromyxum leei and Enteromyxum scophthalmi (Myxozoa: Myxosporea), intestinal parasites of fish.

The enteric myxozoan parasites Enteromyxum leei (Diamant, Lom et Dyková) and Enteromyxum scophthalmi Palenzuela, Redondo et Álvarez-Pellitero are responsible for high weight loss in infected fish, which leads to subchronic disease and low mortality rates in gilthead sea bream (GSB), Sparus aurata L., and to high mortality rates in turbot, Psetta maxima (L.). The detection of initial parasite stages in histological sections is particularly difficult, but can be simplified by means of specific antibodies. Rabbit polyclonal antibodies (pAbs) were raised against E. scophthalmi and E. leei, and direct enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry were used to characterize their sensitivity and specificity. Both pAbs were adsorbed (apAb) with non-infected intestines to avoid non-specific labelling of fish tissues and to improve their specificity. The highest titre obtained in ELISA was 1: 32 000 for apAb-Eleei and 1:16 000 for apAb-Escoph. Working dilutions in immunohistochemistry were 1:1000 for apAb-Eleei and 1:8000 for apAb-Escoph. Both apAbs labelled proliferative and sporogonic stages with high specificity. apAb-Escoph was very specific, whereas apAb-Eleei cross-reacted with Sphaerospora dicentrarchi Sitjà-Bobadilla et Álvarez-Pellitero and Sphaerospora testicularis Sitjà-Bobadilla et Álvarez-Pellitero, suggesting the presence of shared antigens. These pAbs stand as new tools for antigenic characterization and the diagnosis of both Enteromyxum species.

Fish immune response to Myxozoan parasites.

Myxozoan parasites are responsible for important economic losses among fisheries and aquaculture industries, and hence the high interest in studying the immune response of fish against them. The most important data available concerning the immune response of fish against myxosporeans are reviewed, with emphasis on the different innate and adaptive immune mechanisms, their relationship with natural and acquired resistance and the strategies to control and prevent myxosporoses. Cellular effectors (lymphocytes, granulocytes, phagocytes, non-specific cytotoxic cells, rodlet cells) and humoral factors (lysozyme, peroxidades, antiproteases, complement, specific antibodies) have been examined for several myxosporoses, and some immune relevant genes have been studied. This information will be crucial for the future development of vaccines and other preventive strategies such as immunomodulation and selection of disease-resistant strains

Living off a fish: a trade-off between parasites and the immune system.

Research in fish immune system and parasite invasion mechanisms has advanced the knowledge of the mechanisms whereby parasites evade or cope with fish immune response. The main mechanisms of immune evasion employed by fish parasites are reviewed and considered under ten headings. 1) Parasite isolation: parasites develop in immuno-privileged host tissues, such as brain, gonads, or eyes, where host barriers prevent or limit the immune response. 2) Host isolation: the host cellular immune response isolates and encapsulates the parasites in a dormant stage without killing them. 3) Intracellular disguise: typical of intracellular microsporidians, coccidians and some myxosporeans. 4) Parasite migration, behavioural and environmental strategies: parasites migrate to host sites the immune response has not yet reached or where it is not strong enough to kill them, or they accommodate their life cycles to the season or the age in which the host immune system is down-regulated. 5) Antigen-based strategies such as mimicry or masking, variation and sharing of parasite antigens. 6) Anti-immune mechanisms: these allow parasites to resist innate humoral factors, to neutralize host antibodies or to scavenge reactive oxygen species within macrophages. 7) Immunodepression: parasites either suppress the fish immune systems by reducing the proliferative capacity of lymphocytes or the phagocytic activity of macrophages, or they induce apoptosis of host leucocytes. 8) Immunomodulation: parasites secrete or excrete substances which modulate the secretion of host immune factors, such as cytokines, to their own benefit. 9) Fast development: parasites proliferate faster than the ability of the host to mount a defence response. 10) Exploitation of the host immune reaction. Knowledge of the evasion strategies adopted by parasites will help us to understand host-parasite interactions and may therefore help in the discovery of novel immunotherapeutic agents or targeted vaccines, and permit the selection of host-resistant strains.

Cardicola aurata sp. n. (Digenea: Sanguinicolidae) from Mediterranean Sparus aurata L. (Teleostei: Sparidae) and its unexpected phylogenetic relationship with Paradeontacylix McIntosh, 1934.

A new sanguinicolid trematode, Cardicola aurata sp. n., is described from gilthead seabream Sparus aurata L., from off the Spanish Mediterranean coast. The morphology of C. aurata sp. n. generally agrees with the diagnosis of the genus, however, in contrast to all other reported Cardicola spp. the male pore is located sub-medially at the posterior end of the body instead of sinistrally before the posterior end of the body. Based on a comparison of the morphology as well as partial 28S and ITS2 rDNA sequence data from the present species with that from closely related species, it was decided to emend the diagnosis of Cardicola rather than create a new genus, as the aberrant position of the male pore is likely to be an autapomorphy. The phylogenetic analyses revealed a close relationship between Cardicola and Paradeontacylix, two genera with considerable morphological differences; C. aurata sp. n. occupies a position intermediate to these genera. Thus, a morphological comparison of Cardicola, Paradeontacylix and Braya, a genus which is morphologically similar to Cardicola but clusters basal to the Cardicola/Paradeontacylix clade, was conducted. The results of this comparison showed that despite large differences with regard to body shape, the organisation of the internal organs is very similar in species of Cardicola and Paradeontacylix. The synopsis of morphological data and molecular phylogeny allows for interpretations regarding the importance of different morphological features for the phylogenetic inference of the Sanguinicolidae.

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.

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.

Sharpsnout sea bream (Diplodus puntazzo) humoral immune response against the parasite Enteromyxum leei (Myxozoa).

The humoral innate immune response of sharpsnout seabream Diplodus puntazzo against the myxozoan Enteromyxum leei was studied. Enteromyxosis was transmitted by cohabitation and a group of uninfected fish served as control. At 5, 12, 19, 26, 40 and 55 days post-exposure (p.e.), control and recipient fish were sampled to determine the prevalence of infection and some humoral innate immune parameters (antiprotease, antitumoral and peroxidase activities). Prevalence of infection was high from day 12 p.e. and reached 100% at days 40 and 55, when intensity of infection was medium to severe. The antiprotease activity was significantly increased in E. leei-exposed fish with respect to control fish at days 12 and 19 p.e. The serum antitumoral activity was slightly lower in recipient than in control fish at all sampling times, except at 40 days p.e., though no statistically significant differences were observed. Serum peroxidases were higher in all recipient fish than in control ones, with the highest stimulation index at 40 days p.e. Within recipient fish, no differences were detected between sampling times in any of the measured activities. The possible implication of these immune factors in the high susceptibility of D. puntazzo to this enteromyxosis 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.

Levamisole activates several innate immune factors in Scophthalmus maximus (L.) Teleostei.

Levamisole, originally synthesized as an anti-helminthic, has been widely used in human and veterinary medicine as an immunomodulator or adjuvant. However, data on its use in fish are scarce, and no information is available for turbot. To study the effects of levamisole treatment on the innate immune system of juvenile turbot, two different doses (D1=500 mg/kg; D2=250 mg/kg dry food) were orally administered for 2 weeks and samplings were performed at -10, 14, 28, 49 and 77 days post treatment (p.t.). Biometrical, haematological, histological and immunological data were obtained. Specific growth rate was higher in the medicated groups than in the control (C), but the difference was statistically significant only for D1 fish at day 49 p.t. The leucocytes/thrombocytes ratio was significantly higher in D1 than in C fish at 14 days p.t., but decreased subsequently. At most samplings, the percentage of the circulating lymphocytes was lower and that of the progranulocytes was higher in the medicated fish than in the C ones. The percentage of fish with high haemotopoietic activity in the kidney was clearly higher in D1 and D2 fish than in C ones at some sampling points. The respiratory burst activity of blood leucocytes was significantly higher in D1 fish than in C ones in all samplings, except at day 77 p.t. when control fish experienced a rebound effect. In all medicated fish, an initial increase of such activity was observed, followed by a further decrease. Their serum peroxidases followed a contrary pattern, with a decrease in the second sampling and a subsequent and non-significant recovery, a situation also observed for serum lysozyme and complement. Therefore, oral levamisole treatment actually affects some turbot immune factors, although stimulation or depression can occur depending on the considered factor and the administered dose. These results point out the interest of further studies on the mechanisms involved in the levamisole action for its adequate use as immunomodulator.

Cell-mediated cytotoxicity is the main innate immune mechanism involved in the cellular defence of gilthead seabream (Teleostei: Sparidae) against Enteromyxum leei (Myxozoa).

The cellular innate immune response of gilthead seabream (Sparus aurata L.) against the myxozoan Enteromyxum leei was studied. Enteromyxosis was transmitted by maintaining uninfected fish (recipients) together with infected animals. A group of fish not exposed to the infection served as controls. After 10, 22, 38, 52 and 108 days, control and recipient fish were sampled and leucocyte subpopulations and cellular immune responses (leucocyte peroxidases, phagocytosis, respiratory burst and cytotoxicity) of the head-kidney leucocytes were determined. The percentage of acidophilic granulocytes was significantly lower in non-parasitized and parasitized recipient fish than in control fish after 22 days but no significant differences were seen between non-parasitized and parasitized recipient animals. The leucocyte peroxidase content, phagocytosis and respiratory burst activity were seen to have decreased significantly at different sampling times in both non-parasitized and parasitized recipient fish with respect to the controls, whereas cytotoxic activity was up to 2.3 times higher than in control fish. Within the recipient group, little difference was observed in the studied parameters between non-parasitized and parasitized fish. These data demonstrate that cytotoxic activity may have an important role in the defence of gilthead seabream against the myxosporean E. leei. Immunological implications of E. leei infections are discussed.

Interactions between bacteria and Cryptosporidium molnari in gilthead sea bream (Sparus aurata) under farm and laboratory conditions.

The possible interaction of Cryptosporidium molnari and bacteria in gilthead sea bream (Sparus aurata) was studied. Epidemiological data from a pathological survey under farm conditions were analyzed. In addition, parasite and bacteria burdens were studied in experimental models in which naturally and experimentally parasitized fish were challenged with a particular strain of Vibrio harveyi (H57). All the bacteria species present were studied. Under farm conditions, the parasite was more prevalent when mortality or morbidity cases (study C) occurred than in randomly sampled fish (study B). In study C, parasite abundance was significantly higher in bacteria-negative fish, and total bacteria abundance was significantly higher within non-parasitized fish. V. harveyi and V. splendidus were the most prevalent among bacteria carriers in studies B and C, respectively. In study C, among bacteria carriers, most isolates were slightly more prevalent in parasitized than in non-parasitized fish. Two groups (G1, G2) of naturally parasitized fish were inoculated with H57 by intracoelomic injection (ICI) and by oral intubation (OI). H57 was recovered only in G1 inoculated fish, which had a significantly higher basal abundance of total bacteria, and where the only ones with mortalities. In G1, the mortality rate and the prevalence of other V. harveyi strains different from the H57 molecular type were higher in ICI than in OI fish, and the total bacteria abundance was also significantly higher in ICI fish. C. molnari abundance was significantly higher in G1 than in G2, and also in OI than in ICI fish within G1. When H57 was IC inoculated to fish (G3, from the same farm as G2) experimentally infected with C. molnari, H57 was not recovered from any fish. A low mortality was recorded, and only in those fish inoculated with both pathogens. Also in these fish, the prevalence of infection of C. molnari was higher and histopathological damage to the stomach was greater than in fish inoculated only with the parasite. Therefore, the impact of the parasite would be reduced notably when the bacterial burden or the intensity of parasite infection are low (G2, G3).

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.

Response of Ig-positive cells to Enteromyxum scophthalmi (Myxozoa) experimental infection in turbot, Scophthalmus maximus (L.): A histopathological and immunohistochemical study.

In recent years, a new parasite that causes severe losses has been detected in farmed turbot, Scophthalmus maximus (L.), in Northwestern Spain. Dead fish showed emaciation and cachexia caused by severe necrotizing enteritis that affected all portions of the digestive tract. The parasite was classified as a myxosporean and named Enteromyxum scophthalmi. This study was set up to gain insights into the immune response of fish against this parasitic infection. The kinetics of immunoglobulin positive (Ig+) cells in spleen, kidney and intestine from turbot experimentally infected with E. scophthalmi was studied. For evaluating both the progress of infection and the lesions induced by the parasite, we performed histopathological studies and for detecting Ig+ cells in situ we used an indirect immunohistochemical method. In fish exposed to the parasite, the number of Ig+ cells significantly increased in the intestine, the target organ of the parasite, whereas in spleen and kidney, haematopoietic organs where the parasite was not detected, the number of Ig+ cells decreased. Furthermore, the pattern of distribution of Ig+ cells changed in all three organs examined in recipient/infected fish compared with control fish (not exposed to the parasite). The results obtained in this study indicate that the infection by E. scophthalmi in turbot induced an immune response that involved changes in the number and distribution of Ig+ cells.

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.

Gilthead seabream ( Sparus aurata L.) innate defence against the parasite Enteromyxum leei (Myxozoa).

The humoral innate immune response of gilthead seabream (Sparus aurata L.) against the myxozoan Enteromyxum leei has been studied. At 10, 22, 38, 52 and 108 days of cohabitation fish were sampled to examine gut histology and to determine serum innate immune parameters and the mRNA expression of pro-inflammatory cytokines (IL-1beta and TNFalpha) in head-kidney. The parasite was successfully transmitted to 45% of the recipient fish and prevalence reached a maximum (62.5%) at the last sampling time (108 days). Recipient fish started to die after 74 days of cohabitation. In general, alternative complement activity was higher whereas the peroxidase level was lower in recipient fish than in controls. Moreover, IL-1beta mRNA expression increased while the TNFalpha gene expression decreased in recipient fish. These data demonstrate the involvement of complement activity in the defence mechanisms of the gilthead seabream against the myxosporean E. leei. Within the recipient fish group, few differences were observed in the studied immune parameters between E. leei-parasitized and non-parasitized recipient fish. Parasitological and immunological implications of E. leei infections in Mediterranean fish farms are discussed.

Epidemiology of Cryptosporidium molnari in Spanish gilthead sea bream (Sparus aurata L.) and European sea bass (Dicentrarchus labrax L.) cultures: from hatchery to market size.

A long-term epidemiological study of Cryptosporidium molnari in aquacultured European sea bass (ESB) and gilthead sea bream (GSB) was performed in different types of facilities on the Atlantic, Cantabric, and Mediterranean coasts. Four types of studies were carried out. In study A, fish raised from juveniles to marketable size (ongrowing stage) were periodically sampled in three different types of cultures. Studies B and C focused on hatchery and nursery facilities. In study D, occasional samplings were performed during mortality or morbidity outbreaks. As a general trend, C. molnari was more prevalent in GSB than in ESB. Data on the distribution pattern of C. molnari in total sampled GSB (studies A, B, and D) had a variance higher than the mean (overdispersion). In GSB (study A), the type of ongrowing system (sea cages, earth ponds, or indoor tanks) was found to have no significant effect. There was a significant relationship between the presence of the parasite and both fish weight and season. The highest infection values were recorded in spring. Prevalence and intensity had convex weight profiles, with a peak in 30- to 100-g fish. In study D, the prevalence of infection was higher in fish recently introduced in sea cages and in preongrowing systems. In studies B and C, fish were almost never infected before entering the postlarval and nursery facilities. The parasite seems to enter the host mainly through the water in production steps with less stringent water treatment. Recirculation systems and fish cannibalism could contribute to oocyst concentration and dispersion in aquaculture facilities.