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

Fish & Shellfish Immunology 23 (2007) 636e645 www.elsevier.com/locate/fsi

Sharpsnout sea bream (Diplodus puntazzo) humoral immune response against the parasite Enteromyxum leei (Myxozoa) P. Mun˜oz a, A. Cuesta a, F. Athanassopoulou b, H. Golomazou b, S. Crespo c, F. Padro´s c, A. Sitja`-Bobadilla d, G. Albin˜ana c, M.A. Esteban a,*, P. Alvarez-Pellitero d, J. Meseguer a a

Fish Innate Immune System Group, Department of Cell Biology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain b Laboratory of Ichthyology and Fish Pathology, School of Health Sciences, University of Thessaly, Greece c Servei de Diagnostic Patologic en Peixos and Centre de Referencia i Desenvolupament en Aquicultura (Generalitat de Catalunya), Departament de Biologia Animal, de Biologia Vegetal i d’Ecologia, Facultat de Veterinaria, Universitat Autonoma de Barcelona, Bellaterra (Cerdanyola del Valles), Barcelona, Spain d Instituto de Acuicultura Torre de la Sal (CSIC), 12595 Ribera de Cabanes, Castello´n, Spain Received 26 October 2006; revised 11 December 2006; accepted 12 January 2007 Available online 23 January 2007

Abstract 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. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Enteromyxum leei; Myxozoa; Parasite; Humoral innate immunity; Peroxidases; Protease inhibitors; Antitumoral activity; Sharpsnout sea bream (Diplodus puntazzo); Teleostei

1. Introduction The sharpsnout seabream, Diplodus puntazzo [1] (Sparidae) is a marine teleost easily adapted to the on-growing conditions, exhibiting high growth rate and food conversion efficiency [2,3]. Despite the successful rearing of the early * Corresponding author. Tel.: þ34 968363620; fax: þ34 968363963. E-mail address: [email protected] (M.A. Esteban). 1050-4648/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2007.01.014

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life stages of this fish species, high mortality rates (up to 80%) have been reported in on-growing facilities [4] with devastating final consequences. Enteromyxum leei (formerly Myxidium leei) [5] can currently be considered one of the most pathogenic myxosporean in mariculture [6e8]. This parasite causes severe desquamative enteritis, which usually leads to the death of the fish under extreme cachectic conditions. After the first report and description of E. leei in cultured sea bream (Sparus aurata) from southern Cyprus [9], the parasite has been found in association with cultured fish morbidity and mortality in Greece, France, Italy, Spain and Turkey [4,10e13]. Although the life cycle of this myxozoan is not completely known, fish to fish transmission has been achieved by cohabitation, ingestion of infected intestinal mucosa and by waterborne contamination [14]. In spite of the current and potential importance of this enteromyxosis, to date little is known about the mechanisms of infection, development and survival of the parasite outside the fish, and on other aspects of the hosteparasite relationships, including the immune response. The involvement of innate mechanisms in the fish immune response to infectious diseases is well known. Humoral factors like antiproteases, lysines (lysozyme, chitinases, unspecific lysines) or complement, as well as cellular components, mainly phagocytic cells and natural cytotoxic cells, are involved in such responses. However, the information on the immune response of fish to myxosporean infections, both innate and adaptive is very scarce. Only some data on certain aspects of fish humoral or cellular responses are available for Tetracapsula bryosalmonae (PKX organism) [15], Myxobolus cerebralis [16], Myxobolus artus [17] and Sphaerospora dicentrarchi [18,19]. Regarding Enteromyxum spp., the involvement of both innate and adaptive factors in the response to Enteromyxum scophthalmi has been demonstrated in turbot [20]. For E. leei infections, both humoral [21] and cellular [22] innate immune responses have been recently studied in gilthead seabream (S. aurata L.), but only data on the production of reactive nitrogen intermediates are available in the case of D. puntazzo [23]. The aim of this study was to evaluate changes in the innate humoral immune response (serum antiproteases, antitumoral and peroxidase activities) of healthy sharpsnout seabream after exposure to E. leei by cohabitation with infected donor fish. The course of the infection and the histopathological damage were also evaluated in periodical samplings. The involvement of the studied immune factors in the defence against the parasite is discussed. 2. Material and methods 2.1. Fish 2.1.1. Donor fish D. puntazzo used as donor fish (mean weight: 60 g) were obtained from a sea cage of a commercial farm in northwestern Greece, where E. leei infection was diagnosed. Fish of affected stocks showed acute enteritis, followed by anorexia, extensive emaciation and a constant, large, discoloured lesion with scale loss on the dorsal area of the fish and high mortality rate. The infection was confirmed using parasitological examination (10 fish), by the detection of mature parasite spores and sporoblasts in fresh scrapings of the gut mucosa of posterior intestine and in histological sections. High intensity of sporoblasts (>10 sporoblasts/viewing field) and mature spores (>10 mature spores/viewing field) in frequency of 70% and 60%, respectively, were detected in these fish, confirming their suitability as donors. Bacteriological and parasitological examinations for other parasites were also carried out. 2.1.2. Recipient fish Na€ıve fish (mean weight: 2 g) obtained from a commercial hatchery were introduced into 8 m3 raceway tanks receiving a flow through supply of pathogen-free, bore hole water at temperature of 21  1  C. Fish were reared under these controlled conditions (to guarantee the absence of enteromyxosis) for nine months, receiving a commercial dry pellet until a mean weight of 50 g. Bacteriological and parasitological examinations were carried out weekly. 2.2. Cohabitation protocol and samplings The experimental transmission of E. leei was carried out in 8 m3 raceway concrete tanks, under the same rearing conditions as described above. Recipient (n ¼ 150, mean weight: 50 g), and donor (n ¼ 50, mean weight: 60 g) fish were allocated in the same tank, separated by a stable mesh assuring exposure of recipient fish to the parasite stages without contact with donor fish. An additional tank with 50 healthy recipient fish without donor fish at the same

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stocking density was used as control. All fish were fed daily with commercial pellets. The presence of clinical signs and the mortality were recorded daily along the experiment. Fish were sampled at days 5, 12, 19, 26, 40 and 55 post-exposure (p.e.) (10 recipient and 10 control fish per sampling). Fish were killed by overdose of anaesthetic (300 ppm of 2-phenoxyethanol), weighed and measured. Blood was obtained from caudal vessels, placed in non-heparinised tubes, allowed to clot at 4  C for 4 h. After centrifugation (1300 g), the serum was removed and frozen at 80  C until use. Fragments of middle and posterior intestine, headkidney and spleen from each specimen were excised and processed for histological study. 2.3. Histology Tissue fragments were fixed in 10% neutral buffered formalin and embedded in paraffin for histological processing, following standard histology procedures. Sections were stained with haematoxylineeosin or Giemsa and examined by light microscopy. The infection was evaluated in histological sections, and the prevalence of infection was calculated. Infection intensity was semi-quantitatively evaluated using a scale from 1þ to 3þ, according to the number of stages observed per field at 200 [0: negative; 1þ, mild (scattered stages in some fields); 2þ, moderate (more than 10 stages in most of the fields); 3þ, severe (more than 30 stages in all fields and altered mucosa)]. 2.4. Determination of innate immune factors 2.4.1. Antiprotease activity Total antiproteases activity was determined by the ability of serum to inhibit trypsin activity [24]. Briefly, 20 ml of each serum sample was incubated with the same volume of standard trypsin solution (Sigma, 5 mg ml1) for 10 min at 22  C. After adding 200 ml of 0.1 M phosphate buffer (pH 7.0) and 250 ml of 2% azocasein (Sigma), samples were incubated for 1 h at 22  C, and following the addition of 500 ml of 10% trichloro acetic acid (TCA) a new incubation of 30 min at 22  C was done. The mixture was centrifuged at 6000 g for 5 min. The supernatants (100 ml) were transferred to a 96-well plate containing 100 ml well1 of 1 N NaOH, and the optical density was read at 450 nm using a plate reader (BMG, Fluoro Star Galaxy). For a positive control, buffer replaced the serum and for a negative control, buffer replaced both serum and trypsin. The percentage of inhibition of trypsin activity by each sample was calculated by comparing it to the 100% control sample. All the samples collected were analysed in triplicates. 2.4.2. Antitumoral activity The serum ability to kill tumour cells was determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) viability test [25]. Fish serum (15 ml) was put into each well of a flat-bottomed 96-well microtiter plate, mixed with 50 ml of L-1210 tumour cells (mouse lymphoma, ATCC CCL-219) (5  104 cells well1) and incubated at 37  C for 4 h. After adding 100 ml of MTT in RPMI-1640 culture medium (2 mg ml1), the plates were newly incubated in the same conditions. Following centrifugation, the supernatants were discarded and 100 ml of dimethylsulphoxide was added to each well to solubilise the formazan. Optical density was measured at 570 nm and 690 nm, the latter as reference, using a plate reader. Samples without cells (100% of activity) or without serum (0% of activity) were used to determine the antitumoral activity. 2.4.3. Peroxidase activity The activity of serum peroxidases was measured according to Quade and Roth [26]. Briefly, 5 ml of serum was diluted with 50 ml of Hank’s buffered salt solution (HBSS) without Caþ2 or Mgþ2 in flat-bottomed 96-well plates, and incubated during 1 min with 100 ml of a solution containing 0.1 mM 3,30 ,5,50 -tetramethylbenzidine hydrochloride (TMB) (Sigma) and 2.5 mM H2O2. The reaction was stopped by adding 50 ml of 2 M sulphuric acid, and the optical density was read at 450 nm. The wells without serum were used as blanks. The peroxidase activity was calculated as units/ml, where 1 unit represents the change in absorbance of 1 OD. 2.5. Statistical analysis For each fish group (recipient and control) mean absorbance values were calculated at each sampling time. Graphs show the stimulation index obtained dividing recipient fish values by the mean control fish value at the corresponding

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sampling time. Thus, values higher than 1 indicate activation while lower values reflect inhibition. Data are represented by means þ SE. Data were analysed by one-way analysis of variance (ANOVA) followed by a Tukey’s test to determine differences between recipient and control fish and between sampling points within each group (p  0.1 or p  0.05). Comparison of parasitized vs non-parasitized recipient fish could not be carried out due to the scarce number of non-parasitized fish. In order to measure the strength of a possible association between the three immunological variables within individual fish, Pearson correlation coefficients were calculated. Control and recipient fish were analysed separately, and data from each sampling were analysed to detect some time-related particular associations. In addition, data from all the samplings were merged to analyse a possible global pattern. 3. Results 3.1. Course of Enteromyxum leei infections The histological examination of recipient fish allowed the identification of different parasitic stages in the gut mucosa. Some histological samples could not be accurately evaluated due to the high level of autolysis, mainly those with low or very low infection level. Thus, prevalence was calculated considering only fish in which autolysis did not interfere the evaluation of infectious status (Table 1). Prevalence of infection was already high (66.6%) after 12 days of cohabitation and reached 100% at 19 days p.e. and onwards, when infection intensity was moderate to severe in all the infected fish. During the experiment only one fish died (day 54 p.e.), showing an extensive emaciation and a constant, large, discoloured lesion with scale loss on the dorsal area. Furthermore, extensive haemorrhage in the intestinal mucosa was observed. Parasite developmental stages were found in both the middle and posterior intestinal epithelium, although the intensity of infection was slightly higher in the middle gut. Most initial stages consisted of trophozoites with several inner cells. Many sporogonic stages were detected, with one or two mature spores and several accompanying secondary cells. Extensive infections involving the entire gut were frequently observed. Increased infiltration of eosinophilic granular cells, lymphocytes and macrophages in the intestinal submucosa was noticed in infected fish (Fig. 1). No parasitic stages were found in head-kidney and spleen. All control fish resulted negative for E. leei, and none of them died during the experiment nor showed signs of disease. Bacteriological and histological examinations failed to detect any other pathogens. 3.2. Humoral immune factors The antiproteases activity in recipient fish (stimulation index 0.91e1.32) fluctuated around the mean control value (stimulation index 1), though the values registered at 12 and 19 days of exposure were statistically significantly higher than in controls (Fig. 2A).

Table 1 Progression of the infection and mortality of recipient sharpsnout seabream after cohabitation with Enteromyxum leei-donor fish Days of cohabitation

No. Sampled fish

Prevalence (%)a

Mortality (n)

Mean intensityb

5 12 19 26 40 54c 55

10 10 10 10 10 e 10

0 (4) 66.6 (6) 100 (8) 100 (9) 100 (10) e 100 (10)

e e e e e 1 e

0 1.5þ 1.5þ 2.7þ 2.8þ 3þ 2.7þ

Total

60

87.2

1

2.4þ

a

Prevalence recorded in posterior gut. Number of fish used to evaluate infection in histological sections in parenthesis. Infection intensity was semi-quantitatively evaluated using a scale of 1þ to 3þ. 0, no infection; 1þ, scattered stages in some fields; 2þ, more than 10 stages in most of the fields; 3þ, more than 30 stages in all the fields and altered mucosa. c Days of cohabitation at which the indicated number of mortalities (n) caused by enteromyxosis was registered. b

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Fig. 1. Histological sections of sharpsnout seabream intestine infected by Enteromyxum leei. (A) Severe infection. Different development stages observed in the gut epithelium. Bar 50 mm. (B) Moderate infection. Few development stages of E. leei observed in the gut epithelium, including trophozoites (arrow) and sporogonic stages (arrowhead). Bar 20 mm.

The serum ability of recipient fish to kill tumour cells was slightly decreased at all the sampling times, except at 40 days p.e., but no significant differences were observed with respect to the controls (Fig. 2B). Serum peroxidases, which are released by activated circulating leucocytes, were higher in all recipient fish than in control fish, and statistically significant differences were observed (p < 0.1 at 5, 12 and 19 days p.e.; p < 0.05 at 26, 40 and 55 days p.e.), with the highest activity at 40 days p.e. (Fig. 2C). Within recipient fish, no differences were detected between sampling times in any of the measured factors. Some statistically significant correlations were detected between the studied immune factors within individual fish. A negative correlation was found between antitumoral activity and antiproteases at 19 days p.e. (r ¼ 0.66, p ¼ 0.0379) in control fish, and also in recipient fish when analysed globally (r ¼ 0.367, p ¼ 0.00536). The remaining significant correlations were positive and were observed exclusively in control fish. They involved antitumoral activity vs peroxidases at 5 (r ¼ 0.789, p ¼ 0.00668) and 19 (r ¼ 0.69, p ¼ 0.0274) days p.e., and antiproteases vs peroxidases in control fish at 55 days p.e. (r ¼ 0.762, p ¼ 0.0171).

4. Discussion Sharpsnout sea bream is a highly valued fish species for human consumption in the Mediterranean area but significant losses due to the marine myxosporean parasite E. leei have questioned the viability of its farming [27]. For most Myxozoans, experimental infection models are hampered by the lack of continuous in vitro culture and the scarce knowledge of their life cycle. However, E. leei is an excellent model for myxosporean study since the direct fish to fish infection, previously demonstrated by Diamant [14], is effective, as in the case of the related species E. scophthalmi from turbot [28]. E. leei displays a low degree of host specificity [13], but differential susceptibility to this myxozoan has been reported among sparids cultured in netpens at the Eastern Mediterranean [4, 11], showing D. puntazzo the highest mortalities rates. In the present work, the parasitosis caused by E. leei in sharpsnout sea bream was effectively transmitted by cohabitation of infected and recipient fish, as it was previously demonstrated in gilthead sea bream [14]. However, the particularly faster development and the higher severity of the infection in this species in comparison with gilthead sea bream are remarkable. Parasite developmental stages were found in both middle and posterior intestinal epithelium. In gilthead seabream, the hind gut is the most parasitized intestinal part, and the predominant pattern of dispersion (as deduced from both natural and cohabitation infections) is anteriorly from the rectum, with progressive invasion of the medium and anterior parts of intestine (authors’ unpublished data). Although the course of infection in D. puntazzo has not been elucidated yet, it could occur as in gilthead sea bream. In this study, the inflammatory response associated to the infection (involving mainly eosinophilic granular cells but also lymphocytes and

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Antiprotease activity (SI)

A

641

2

*

*

1

B

2

Anti-tumoral activity (SI)

0

1

5

12

19

26

40

55

5

12

19

26

40

55

0

Serum peroxidases (SI)

C

4

*

*

3

+ 2

+

+

5

12

+

1

0 19

26

40

55

Days of cohabitation Fig. 2. Humoral innate immune parameters in sharpsnout sea bream specimens exposed to Enteromyxum leei by cohabitation. (A) Serum antiprotease activity. (B) Antitumoral activity. (C) Antiprotease activity. Data are presented as the stimulation index (SI) (mean þ SE; n ¼ 10) obtained by dividing each recipient fish value by the mean value of the control group. Thus, values > or <1 mean an increase or decrease with respect to controls, respectively. Symbols * and þ denote statistically significant differences (p  0.05 and p  0.1, respectively) between recipient and control fish.

macrophages) is particularly important, though, as deduced from our results, it does not seem to be able to prevent the progression of the infection. Data on the non-specific immune reaction against myxosporeans are scarce [18, 29e32], and most of the knowledge is based on histopathological observations. For D. puntazzo, only some preliminary data on innate immune response and its relationship with treatments [33], and on variations in the nitric oxide production in response to E. leei infection [23] have been recently published. Proteases are known to contribute to the pathogeniticy of parasites. Although the knowledge of myxosporean proteases is still fragmentary and no information is available on their production by Enteromyxum spp., some Myxosporea have been demonstrated to produce proteases such us Henneguya salminicola [34], Kudoa spp. [35], Sphaerospora

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dicentrarchi [36], and Myxobolus cerebralis [37,38]. Host protease inhibitors modulate protease activities and control a variety of the critical protease-mediated processes, including the resistance to invasion by infectious agents. Fish plasma contains a number of protease inhibitors, mainly a-1-antiprotease, a-2-anti-plasmin and of a-2-macroglobulin, which play a role in restricting the ability of bacteria to invade and grow in vivo [39]. Up-regulated expression of a-2-macroglobulin has been reported in grass carp Ctenopharyngodon idella in response to infection of the parasitic copepod Sinergasilus major [40] and in carp intraperitoneally injected with Trypanoplasma borreli [41]. Moreover, in rainbow trout infected with Cryptobia salmositica, resistance was associated with a-2-macroglobulin concentrations in plasma and it was demonstrated that this antiprotease was able to neutralize a secreted haemolytic metalloprotease [42]. In the current work, E. leei was able to enhance the total antiproteases activity in the serum of D. puntazzo soon after exposure. In contrast, in turbot experimentally infected with the related myxosporean E. scophthalmi, total serum antiproteases oscillated in a similar manner in recipient and control fish, while a-2-macroglobulin followed a more similar pattern to that of total antiproteases in D. puntazzo, with an increase soon after exposure in recipient fish [20]. The rise in serum antiproteases observed in recipient D. puntazzo could be partly due to a feedback mechanism to prevent proteolysis in skeletal muscle under the extensive emaciation occurring in E. leei-infected fish, as it was suggested for E. scophthalmi-infected turbot. Cytotoxicity is one of the most important mechanisms of innate immunity, particularly in vertebrate response against parasites. This response may be mediated by a heterogeneous population of cells [43], but it can also be attributable to the release of soluble factors with lytic activity. In the present work, although no statistically significant differences were observed in antitumoral activity with respect to the controls, E. leei-exposed fish showed slightly decreased lytic activity against tumoral cells at most sampling times, except at 40 days p.e. On the contrary, cytotoxic activity of head-kidney leucocytes was greatly and significantly enhanced in E. leei-exposed gilthead sea bream, and it showed an opposite pattern respect to sharpsnout, as it was significantly higher than in control fish at all sampling times except at 38 days p.e. [22]. The exact mechanisms implicated in the cytotoxic response to E. leei infections are unknown. Complement could be involved as it is included amongst the potential cytotoxic effector mechanisms in mammals [44]. In fish, complement-fixing antibodies have been considered an important part of the protective mechanism against parasites such as Cryptobia salmositica [45]. Complement activity is regulated by the serum levels of its different components, natural decay of the activated fragments, specific complement inhibitors and serum protease inhibitors. Thus, the negative correlation observed in the present study between antitumoral activity and antiproteases at 19 days p.e. in control fish, and also in recipient fish when analysed globally, could be a consequence of the effect of increased serum protease inhibitors through complement depletion. Phagocytes release two distinct peroxidases from their cytoplasmatic granules. Monocytes and neutrophils contain myeloperoxidase (MPO), whereas eosinophils produce eosinophil-peroxidase [46]. The interaction of peroxidases with H2O2 forms hypohalous acids, which are potent oxidants known to have several cytotoxic effects on mammalian and bacterial cells. Cell membrane integrity may be violated by membrane peroxidation and the oxidation and/or decarboxylation of membrane proteins [47,48]. In the current study, serum peroxidases significantly increased in D. puntazzo exposed to E. leei at all sampling points with the highest stimulation at 40 days p.e.(three fold). Previous studies in gilthead seabream (S. aurata) exposed to E. leei showed an increment in serum peroxidase levels after 10 days of cohabitation, but a decrease occurred in subsequent samplings [21]. Such differences in response to E. leei infection between sharpsnout seabream and gilthead seabream regarding peroxidase levels could explain the highest mortalities reported in D. puntazzo. The ability of S. aurata to recover from E. leei infection could be partly related to the capacity of this fish to return peroxidases to preinfection levels. In fact, excessive or misplaced generation of this oxidant is known to cause damage to tissues [49]. Sea bass (Dicentrarchus labrax L.) with sudden and unexplained mortalities presented high levels of peroxidase in serum, 50 times higher than basal levels (unpublished data). Also, common dentex, an extremely stress-sensitive sparid, has significantly higher peroxidase levels than gilthead sea bream, but with the same plasma antioxidant capacity [50]. Peroxidases, and more specifically MPO, are pivotal enzymes involved in leucocyte-mediated host defences and in the regulation of inflammation. MPO, as a major nitric oxide (NO) scavenger, up-regulates the catalytic activity of inducible NO synthase by preventing NO feedback inhibition [51]. In fact, MPO can oxidize nitrite to nitrogen oxide [52], which increases nitric oxide accumulation that mediates toxicity in cells. In E. leei-infected D. puntazzo, an increment in serum peroxidase activity was observed in the current experiment, and an increase in serum nitrite level has been reported [23]. Data on leucocyte peroxidase content are not available, but, considering the observed cellular response, we can speculate that an increase could also occur, which could scavenge NO from the cells and increase the serum nitrite level.

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The analysis of correlations between immune factors within individual fish can aid to a better knowledge of their relationships and significance in relation to infection. However, scarce statistically significant relationships were detected in the current experiment. Control fish with high peroxidase levels also showed high antitumoral activity at days 15 and 19 p.e., and high antiprotease level at day 55 p.e., but these relationships were not observed in recipient fish. This could be related to the absence of variation of antitumoral activity and antiproteases at those sampling points in recipient fish, which showed, however, increased peroxidase levels from day 5 after exposure. In conclusion, some innate immune factors are affected in D. puntazzo by exposure to E. leei, mainly the serum peroxidase levels and the total antiprotease activity. The increase in peroxidases could be related to the cellular response observed in the intestinal submucosa. However, the immune response to E. leei seems to be lower in sharpsnout than in gilthead sea bream, which could explain the clearly higher severity of infections produced in the former species. Moreover, the main mechanisms involved in the innate response to E. leei infection seem to be different in both hosts. Cytotoxic activity is considered the main innate mechanism of response in S. aurata and is clearly enhanced, not only in E. leei-exposed fish, but also in parasitized vs non-parasitized fish [22]. By contrast, cytotoxic activity was scarcely affected by E. leei infection in D. puntazzo, whereas the significant and sustained increase in serum peroxidases could contribute to the severity of damage in this fish through immunopathological effects. More studies are needed to elucidate the mechanisms involving these and other immune factors, innate or adaptive in the fish response to the parasite. Such information could be useful to understand the higher susceptibility of sharpsnout sea bream to this enteromyxosis and it would contribute to the development of control measures for this important disease. Acknowledgements This work has been funded by the EU Project MyxFishControl (QLRT-2001-00722). A. Cuesta is recipient of a fellowship from Fundacio´n CajaMurcia. References [1] Cetti F. Anfibi e pesci di Sardegna. Storia naturale di Sardegna. Sassari Stor natur Sardegna 1777;3:1e208. [2] Favaloro E, Lopiano L, Mazzola A. Rearing of sharpsnout seabream (Diplodus puntazzo, Cetti 1777) in a Mediterranean fish farm: monoculture versus polyculture. Aquac Res 2002;33:137e40. [3] Herna´ndez MD, Egea MA, Rueda FM, Martı´nez FJ, Garcia Garcı´a B. Seasonal condition and body composition changes in sharpsnout seabream (Diplodus puntazzo) raised in captivity. Aquaculture 2003;220:569e80. [4] Athanassopoulou F, Prapas T, Rodger H. Diseases of Puntazzo puntazzo Cuvier in marine aquaculture systems in Greece. J Fish Dis 1999;22:215e8. [5] Diamant A, Lom J, Dykova´ I. Myxidium leei n. sp., a pathogenic myxosporean of cultured sea bream Sparus aurata. Dis Aquat Organ 1994;20:137e41. [6] Kent ML, Andree KB, Bartholomew JL, El-Matbouli M, Desser SS, Devlin RH, et al. Recent advances in our knowledge of the Myxozoa. J Eukaryot Microbiol 2001;48:395e413. [7] Ghittino C, Latini M, Agnetti F, Panzieri C, Lauro L, Ciappelloni R, et al. Emerging pathologies in aquaculture: effects on production and food safety. Vet Res Commun 2003;27:471e9. [8] Golomazou E, Karagouni E, Athanassopoulou F. The most important myxosporean parasite species affecting cultured Mediterranean fish. J Hellenic Vet Med Soc 2004;55:42e352. [9] Diamant A. A new pathogenic histozoic Myxidium (Myxosporea) in cultured gilt-head seabream Sparus aurata L. Bull Eur Assoc Fish Pathol 1992;12:64e6. [10] Le Breton A, Marques A. Occurrence of an histozoic Myxidium infection in two marine cultured species. Puntazzo puntazzo C. and Pagrus major. Bull Eur Assoc Fish Pathol 1995;15:210e2. [11] Rigos G, Christophilogannis P, Yiagnisi M, Andriopolulou A, Koutsodimou M, Nengas I, et al. Myxosporean infection in Greek mariculture. Aquac Int 1999;7:361e4. [12] Sakiti PN, Tarer V, Jacquemin D, Marques A. Pre`sence en Me´diterrane´ occidentale d’une Myxosporidie histozo€ıque pathoge`ne dans les e´levages de daurade, Sparus aurata L. Ann Sci Nat 1996;17:123e7. [13] Padro´s F, Palenzuela O, Hispano C, Tosas O, Crespo S, Alvarez-Pellitero P. Myxidium leei (Myxozoa) infections in aquarium-reared Mediterranean fish species. Dis Aquat Organ 2001;47:57e62. [14] Diamant A. Fish-to-fish transmission of a marine myxosporean. Dis Aquat Organ 1997;30:99e105. [15] Saulnier D, Kinkenlin P. Antigenic and biochemical study of PKX, the myxosporean causative agent of proliferative kidney disease of salminid fish. Dis Aquat Organ 1996;27:103e14. [16] Griffin BR, Davis EM. Myxosoma cerebralis: detection of circulating antibodies in infected rainbow trout (Salmo gairdneri). J Fish Res Board Can 1978;35:1186e90.

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