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.

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

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

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

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