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Functional alterations associated with “winter syndrome” in gilthead sea bream (Sparus aurata)
Aquaculture 223 (2003) 15 – 27 www.elsevier.com/locate/aqua-online
Functional alterations associated with ‘‘winter syndrome’’ in gilthead sea bream (Sparus aurata) ´ ngeles Gallardo a,b,*, Mo´nica Sala-Rabanal a,b, M. A Antoni Ibarz a,b, Francesc Padro´s b,c, Josefina Blasco a,b, Jaume Ferna´ndez-Borra`s a,b, Josep Sa´nchez a,b a
Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Avinguda Diagonal, 645, Barcelona E-08028, Spain b Centre de Refere`ncia i Desenvolupament en Aqu¨icultura de la Generalitat de Catalunya, Spain c Servei de Diagno`stic Patolo`gic en Peixos, Facultat de Veterina`ria, Universitat Auto`noma de Barcelona, Bellaterra 08193, Spain Received 22 April 2002; received in revised form 10 February 2003; accepted 10 February 2003
Abstract Gilthead sea bream obtained from a fish farm in the Delta de l’Ebre (Tarragona, Spain) were used to study various functional alterations associated with ‘‘winter syndrome.’’ The fish groups considered were: control fish routinely sampled 3 weeks before an outbreak (which took place in March 1998); winter syndrome symptomatic fish (assessed by eye and corroborated through histopathological analysis); and asymptomatic animals sampled from the same tank where winter syndrome fish were found. Winter syndrome animals weighed less than control and asymptomatic fish. The symptomatic animals suffered a drop in the haematocrit and number of erythrocytes, although red cell volume did not change. Furthermore, the concentration of total plasma proteins in winter syndrome fish was higher than in control animals, due to a rise in h2- and g-globulins; the a2-globulin concentration was lower and albumin and a1- and h1-globulins remained unchanged. A significant increase in the concentration of plasma amino acids, both essential and nonessential, was found in symptomatic fish. This group of animals showed a lower plasma glucose concentration. There were no differences in either the plasma sodium or chloride concentrations between control and symptomatic fish. Instead, the plasma potassium concentration in winter syndrome animals doubled that of control animals. The plasma concentrations of calcium and magnesium were lower in symptomatic animals than in control fish.
* Corresponding author. Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Avinguda Diagonal, 645, Barcelona E-08028, Spain. Tel.: +34-934021557; fax: +34-934110358. E-mail address: [email protected] (M.A. Gallardo). 0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0044-8486(03)00164-9
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Symptomatic fish showed high lipid concentration in liver and elevated plasmatic GOT activity, indicating hepatic damage. No other significant differences in the composition of liver and white muscle were found between control and symptomatic animals. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Winter syndrome; Winter disease; Sparus aurata; Haematology; Plasma determinations; Liver and muscle composition
1. Introduction The gilthead sea bream (Sparus aurata) is a fish species predominantly located in the Mediterranean Sea, but it can also be found around coasts as far north as the UK and as far south as Senegal. In the last 10 –15 years, the commercial farming of gilthead sea bream has become a common practice along the Mediterranean coastline. As with other fish species, one of the main problems associated with sea bream culture is stress due both to the fish being confined in cages, which prevents them from migrating, and handling (Flos et al., 1990). Several studies have shown immunosuppression to be a direct result of stress in this species (Page´s et al., 1995; Tort et al., 1996, 1998a), increasing their susceptibility to opportunistic pathogens and decreasing their ability to fight infections, as clearly demonstrated in other fish species (Pickering and Pottinger, 1985). The effect of temperature on the fish immune system has also been discussed (Bly and Clem, 1992; Zapata et al., 1992). Thus, after exposure to low temperatures, there is a suppression of T-cell mitogenic or antibody responses (Clem et al., 1984; Miller and Clem, 1984; Bly et al., 1986). Wild gilthead sea bream normally live in an environment whose temperature ranges from 11 jC in winter to 23 jC in summer, without any apparent problems related to the temperature changes. There is also evidence that sea bream migrate to greater depths (warmer waters) when surface temperatures start to decline (Davis, 1988). However, the problem of low temperature may be critical in intensive cultured gilthead sea bream because the fish are unable to move to warmer waters. In fact, when the temperature drops to 8– 10 jC, the plasma cortisol of exposed sea bream rises as a stress response (Rotllant et al., 2000) and, below 12 jC, the species stops feeding (Padro´s et al., 1999). ‘‘Winter syndrome’’ or ‘‘winter disease’’ is a pathology exclusively affecting cultured gilthead sea bream in the Mediterranean sea, being more severe in the northern areas. Other fish species, such as sea bass, that are frequently reared in the same facilities seem not to be affected. Winter syndrome causes chronic mortality during the coldest months and acute mortality episodes when the temperature rises (Tort et al., 1998a; Sarusic, 1999). Mortality rates are usually around 7– 10% of the fish stock, although in some very acute cases, they may be as high as 80% (Padro´s et al., 1998). The causes of winter syndrome are not understood and cannot be explained as the result of only one factor (Tort et al., 1998b); although deaths are associated with reduced temperatures, this may not be the only factor affecting mortality as the majority of fish survive through cold periods. Winter syndrome increases plasma cortisol, decreases the complement and lysozyme activities, and reduces circulating lymphocytes (Tort et al., 1998a), therefore reducing the
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ability of fish to resist attacks by opportunistic parasites. Different bacterial and viral agents (Bovo et al., 1995; Doimi, 1996), particularly Pseudomonas anguilliseptica (Berther et al., 1995; Domenech et al., 1997, 1999), have often been isolated from different outbreaks of winter syndrome, although these isolations were not consistent from year to year and no specific pathogen was found (Padro´s et al., 1998). In contrast, infections in other fish species exposed to low temperatures are normally due to specific pathogens (Evensen et al., 1991; Holt et al., 1993; Roberts, 2001). The visible signs of winter syndrome-affected fish are lethargy, sideways or even backwards swimming, and minimal reaction to external stimuli. A variety of significant tissue lesions have been described in symptomatic animals, such as granular degeneration and necrosis in white muscle fibres, atrophy of the exocrine pancreas, pale and friable liver, and a fatty degeneration in hepatocytes (Galeotti et al., 1998; Padro´s et al., 1998; Tort et al., 1998b; Contessi et al., 2000). The digestive tract appears distended and filled with a clear liquid, indicating a potentially reduced nutrient absorption, and many symptomatic fish also have white fibromucous materials in the intestine. Moreover, intestinal mucosa displays a significant hyperplasia of mucous cells and, frequently, the lamina propia shows oedema (Bovo et al., 1995; Padro´s et al., 1998; Tort et al., 1998b; Contessi et al., 2000). Less consistent lesions have also been observed in the brain and kidney (Padro´s et al., 1998). In some cases, blood sampled from symptomatic animals has a clearer appearance due to low red blood cell counts (Padro´s et al., 1999). Most studies related to winter disease are devoted to descriptions of histopathological lesions and immunological variations, but few have shown changes in other aspects of fish physiology. The aim of the present study was to investigate changes in certain physiological parameters related, on one hand, to the homeostasis of animals (proteins, amino acids, glucose, and ions in plasma) and, on the other hand, to haematology and tissue composition. Plasmatic GOT was also analysed as an indicator of hepatic injury. The study of these parameters could help to reveal the aetiology of winter syndrome.
2. Materials and methods 2.1. Animals Gilthead sea bream (S. aurata) were obtained from fish farms located in the Delta de l’Ebre (Tarragona, Spain), where they were maintained in shallow pools with brackish water. Twenty control fish (C) were sampled in February 1998 from a routine seasonal sampling. Twenty-four days later, winter syndrome appeared in the pond and 20 visually symptomatic (WS) fish, along with 10 apparently asymptomatic (A) fish, were sampled. 2.2. Blood collection, morphometric analysis, and tissue sampling Blood was collected by caudal puncture. Lithium EDTA, at a final concentration of 5 mgml 1 blood, was used as an anticoagulant. Fish were killed by severing their spinal cord and were subsequently weighed and measured. Samples of liver and white muscles were taken and immediately frozen in liquid nitrogen for further analysis.
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2.3. Histology Liver, muscle, kidney, digestive tract, and gill samples were fixed in neutral 10% phosphate-buffered formalin for histological studies. Samples were processed in paraffin and stained with hematoxylin – eosin. 2.4. Haematology Haematocrit was measured by using a HaemofugeR microcentrifuge (Heraeus-Christ, Osterode, Germany). Total red blood cell counts and volumes were determined by means of the Coulter principle (Melamed et al., 1990; Shapiro, 1995), using a Multisizer particle counter (Coulter, Hialeah, FL, USA) equipped with a 100-AM orifice. 2.5. Plasmatic determinations The remaining blood was rapidly centrifuged and the obtained plasma was kept at 80 jC until use for analysis. Total protein concentration was determined by the Lowry technique using bovine serum albumin as standard. Protein fractions were obtained by electrophoresis on cellulose acetate bands (CellogelR; Biosystems, Barcelona, Spain), using a Pre´fe´renceR photodensitometer (Sebia, Paris, France) to read the transparent bands. The amino acid content of deproteinized plasma samples was determined by ionic exchange chromatography, following Canals et al. (1992), using an Alpha Plus II autoanalyzer (Pharmacia LKB Biotechnology, Uppsala, Sweden). Plasmatic glucose was determined by means of the glucose oxidase reaction, using a GlucofixR colorimetric kit (Menarini, Florence, Italy). Plasmatic potassium was analysed by flame atomic absorption spectroscopy, using a PU 9200XR spectrometer (Phillips, Eindhoven, the Netherlands), whereas sodium, calcium, and magnesium were determined by inductively coupled plasma spectrometry using a Polyscan 61ER spectrometer (Thermo Jarrell Ash, Franklin, MA, USA). Chloride was quantified by the ClorofixR colorimetric kit (Menarini), based on the mercury thiocyanate reaction. Plasma GOT concentration was determined by a modified version of Beuther (1984) method. In this assay, GOT activity was coupled, through the action of malic dehydrogenase (MDH), to the oxidation of NADH, which was measured spectrophotometrically at 340 nm. 2.6. Liver and white muscle composition The samples of liver and muscle were powdered in liquid nitrogen and separated into three fractions. One fraction was used to determine gravimetrically the tissue water content, drying the sample at 105 jC for 24 h. The protein percentage was determined from another fraction, measuring the elemental nitrogen by burning the sample in a chromatograph (EA 1108 CHNS-O; Carlo Erba Instruments, Milan, Italy). To convert the elemental nitrogen into a protein percentage, the typical correction factor (6.25) was used. From the last fraction, the lipid content was measured after two extractions with chloro-
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Fig. 1. Haematocrit (bars) and number of erythrocytes (dot) in the three experimental groups: (C) control, (A) asymptomatic, and (WS) winter syndrome symptomatic fish. Values are shown as mean F S.E.M. Group differences ( p < 0.05) for each parameter are marked with different letters.
form/methanol (2:1, vol/vol), according to the procedure of Folch et al. (1957), using butylated hydroxy-toluene (BHT; 0.01%) as an antioxidant. The final washed lipid extract was dried on a Buchi rotavap (Bu¨chi Rotavapor R-114, Postfach, Switzerland) and lipid mass was determined gravimetrically. 2.7. Data and statistical analysis Results are given as mean F S.E.M. Differences among groups were tested by one-way ANOVA and the Tukey test using a computerized package (SigmaStat 2.01).
3. Results The diagnosis of fish visually classified as symptomatic for winter syndrome was corroborated though histological analysis, according to the signs described by Padro´s et al. Table 1 Concentrations (gl
Total plasma protein Albumin a1-Globulins a2-Globulins h1-Globulins h2-Globulins Fibrinogen g-Globulins
1
) of total plasma proteins and their fractions in the three experimental groups Control fish
Asymptomatic fish
Symptomatic fish
42.1 F 1.0a 2.4 F 0.7 2.1 F 0.3 10.6 F 0.5a 7.1 F 0.4a 10.0 F 0.4a 1.8 F 0.2 8.1 F 0.5a
48.4 F 0.9b 2.8 F 1.5 1.9 F 0.3 8.2 F 0.7a,b 9.7 F 0.5b 11.9 F 0.7b 2.5 F 0.3 11.4 F 0.6b
46.1 F 1.0b 2.9 F 0.8 2.2 F 0.3 6.7 F 0.3b 8.1 F 0.8a,b 11.9 F 0.5b 1.6 F 0.3 12.7 F 0.9b
Values are shown as mean F S.E.M. Group differences ( p < 0.05) for each parameter are marked with different letters.
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Fig. 2. Changes in the a-amino acid concentration in the three experimental groups. Total a-amino acids (n), essential amino acids ( ), nonessential amino acids ( ), and the essential/nonessential amino acid ratio (.). Values are shown as mean F S.E.M. Group differences ( p < 0.05) for each parameter are marked with different letters.
(1998) and Tort et al. (1998b). All symptomatic fish showed granular degeneration and necrosis in white muscle fibres, different degrees of atrophy (not necrosis) in exocrine pancreas, pale and friable liver, and fatty degeneration in hepatocytes. They also had a distended and liquid-filled digestive tract, hyperplasia of the mucous cells, and oedema in the submucosa. Moreover, white fibrous materials were often found in the intestines. Control and asymptomatic fish did not show any of these signs. Body weights of both control (151 F 2 g) and asymptomatic (153 F 6 g) animals were significantly higher ( p V 0.05) than those of symptomatic fish (125 F 1 g). In contrast, no significant differences were found between the length of asymptomatic and symptomatic fish (192.0 F 5.8 and 188.6 F 2.8 cm, respectively). Some haematological and plasmatic parameters were studied. Fig. 1 shows the haematocrit and the number of erythrocytes (RBC) for the three groups of fish. A significant fall in the haematocrit of symptomatic fish (WS) compared to control (C)
Fig. 3. Concentration of L-alanine (.) and other nonessential amino acids (o) in the three experimental groups. Values are shown as mean F S.E.M. Group differences ( p < 0.05) for each parameter are marked with different letters.
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Table 2 Nonessential a-amino acid and taurine levels (AM) in the plasma of the three experimental groups Control fish Alanine Asparagine Aspartic acid Glycine Glutamic acid Proline Ornitine Serine Tyroxine Taurine
a
222 F 40 46 F 5a 25 F 3 390 F 26 32 F 4a 49 F 12 56 F 7 94 F 7 131 F 14 449 F 37a
Asymptomatic fish a,b
409 F 31 27 F 4a,b 32.0 F 6 392 F 58 53 F 12a,b 35 F 7 25 F 2 98 F 11 164 F 5 777 F 36a,b
Symptomatic fish 803 F 130b 20 F 3b 33 F 3 498 F 77 55 F 7b 91 F 20 54 F 14 116 F 12 182 F 22 818 F 89b
Values are shown as mean F S.E.M. Group differences ( p < 0.05) for each parameter are marked with different letters.
and asymptomatic (A) animals was observed; this seems to be related to a significant drop in RBC number because no changes in RBC volume among C, A, and WS fish were found (around 85 Am3). The concentration of total plasma proteins and their fractions were also measured (Table 1). There was a significant increase in the total protein concentration of winter symptomatic (WS) and asymptomatic (A) fish compared to controls (C), the globulin fraction being responsible for most of the change, whereas the albumin fraction remained constant. The different globulin fractions did not change uniformly. h2- and g-globulins increased significantly in WS and A fish, while the concentration of a2-globulins was lower in WS animals than in both controls. Additionally, the levels of h1-globulins in A animals were higher than in C and WS fish. Finally, the levels of a1-globulins and fibrinogen did not change. A significant increase in plasma amino acid concentration was also observed in the WS group (Fig. 2). Although this rise affects both essential and nonessential amino acids and the essential/nonessential ratio did not differ significantly between the C and WS groups (Fig. 2), the nonessential amino acids increased more than essential amino acids (82.5% Table 3 Essential amino acid levels (AM) in the plasma of the three experimental groups
Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine
Control fish
Asymptomatic fish
Symptomatic fish
58 F 10 61 F 5a 83 F 6a 189 F 10 148 F 12 24 F 3 71 F 9 75 F 5 209 F 14
85 F 20 106 F 25a,b 95 F 11a,b 174 F 11 170 F 23 23 F 12 82 F 11 81 F 18 218 F 21
152 F 61 117 F 8b 154 F 17b 244 F 27 202 F 26 41 F 10 72 F 8 96 F 15 285 F 34
Values are shown as mean F S.E.M. Group differences ( p < 0.05) for each parameter are marked with different letters.
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Fig. 4. Changes in the plasmatic concentration of minority cations in the three experimental groups: potassium (bars), calcium (.), and magnesium (o). Values are shown as mean F S.E.M. Group differences ( p < 0.05) for each parameter are marked with different letters.
versus 46.3%). Among the nonessential amino acids, the rise in L-alanine concentration (260%) was notable (Fig. 3 and Table 2). However, there was not a single entity responsible for the change in essential amino acids (Table 3). Finally, plasma taurine rose significantly in WS fish compared with control animals (Table 2). Another change found in the plasma of symptomatic fish was a significant ( p < 0.05) fall in the glucose concentration, in winter syndrome animals (3.7 F 0.2 mM) being 21% and 33% lower with respect to control (4.7 F 0.2 mM) and asymptomatic fish (5.5 F 0.3 mM), respectively. The ionic composition of plasma was also analysed. Main ions such as sodium (172.1 F 3.0, 178.0 F 4.0, 166.8 F 2.2 mM in C, A, and WS, respectively) and chloride (116.1 F 3.1, 113.6 F 2.9, 114.9 F 2.2 mM in C, A, and WS, respectively) did not change significantly. In contrast, the potassium concentration increased by 100% in A and WS fish
Fig. 5. Lipid percentage in liver (bars) and plasmatic GOT activity (dot) in the three experimental groups. Values are shown as mean F S.E.M. Group differences ( p < 0.05) for each parameter are marked with different letters.
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Table 4 Liver and muscle composition (%) of the three experimental groups
Liver
Muscle
Water Lipid Protein Water Lipid Protein
Control fish
Asymptomatic fish
Symptomatic fish
71.0 F 0.5 8.4 F 0.6a 14.6 F 0.3 73.6 F 0.4 3.8 F 0.2 19.8 F 0.3
70.7 F 1.6 10.4 F 2.0a,b 14.9 F 0.6 76.2 F 0.5 3.3 F 0.2 19.8 F 0.6
70.5 F 0.9 12.2 F 0.7b 14.1 F 0.2 75.5 F 0.4 3.9 F 0.2 20.7 F 0.3
Values are shown as mean F S.E.M. Group differences ( p < 0.05) for each parameter are marked with different letters.
compared with C animals; the concentrations of calcium and magnesium were the same in C and A animals, but decreased by 10% and 34%, respectively, in WS animals (Fig. 4). The greatest change in the liver morphology of symptomatic fish was the increase of friability of the organ, probably due to a rise in lipid content (Fig. 5 and Table 4). As a result of apparent alterations in liver morphology, the GOT plasmatic concentration was measured as an indicator of hepatic damage. Plasmatic GOT levels in WS fish showed a dramatic increase (Fig. 5), confirming the existence of liver damage in these animals. On the other hand, no significant differences among groups were found in liver water and protein contents (Table 4). Neither were significant differences found among C, A, and WS fish with respect to water, protein, and lipid percentages in white muscle (Table 4).
4. Discussion As stated above, most studies related to winter syndrome in gilthead sea bream describe histopathological changes (Galeotti et al., 1998; Padro´s et al., 1998; Tort et al., 1998b; Contessi et al., 2000) and immunological variations (Tort et al., 1998a,b; Contessi et al., 2000), but few provide other functional information (Galeotti et al., 1999; Padro´s et al., 1999). Identifying modifications in certain physiological parameters would offer a more complete understanding of the pathogenesis of this syndrome. From an epidemiological point of view, a fall in water temperature seems to be the single common factor associated with the development of the disease, although there are obviously other factors involved as the majority of fish survive through cold periods (Tort et al., 1998b). One of most noticeable changes in affected fish is lipid accumulation in liver. It has been argued that for some fish, this change represents an adaptive mechanism in response to a drop in water temperature (Giardina et al., 1998), as it helps oxygen to diffuse into the mitochondria. However, a potential intoxication related to bacterial or other toxins should not be ruled out (Pascale et al., 1989; Sornaraj et al., 1995). In gilthead sea bream, feeding stops at low temperatures ( < 12 jC) (Padro´s et al., 1999) and, therefore, the liver lipid accumulation cannot be due to lipid absorption from the gut. Symptomatic fish may present an imbalance between the entry and exit of liver nutrients, possibly as a result of a hepatic dysfunction. However, in spite of the raised liver lipid percentage and the
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pathological levels of GOT in plasma (Galeotti et al., 1999 and present data), it is not clear if this situation induces a general failure in this organ functioning. This is because some hepatic-dependent systems remain active; for instance, symptomatic animals show an increase in h2-globulin concentration, although other plasmatic protein fractions, such as a2-globulins, are reduced in these fish. No significant differences were found in the albumin fraction concentration among the three groups studied. This is not consistent with the fall described by Galeotti et al. (1999) in symptomatic animals. Observation of a seasonal cycle showed that albumin normally reaches its minimum value in winter (data not published), suggesting that this difference may be the reason for this discrepancy. In February – March, the analysed fish (C, A, and WS) showed a particularly low albumin concentration. The increase in the g-globulin fraction in WS and A animals suggests that fish are responding to an infectious process, although this increase was not found by Galeotti et al. (1999) in symptomatic winter syndrome animals. Additionally, a2-globulin concentration was the only plasma protein fraction that varied in symptomatic fish but not in asymptomatic animals. This latter group showed a h1-globulin concentration significantly higher that did the C and WS groups; no explanation for this change can be found, unless the h1-globulin fraction contains a protective protein. Neither the a2- or h1-globulins were homogeneous fractions and no further analysis was carried out to assess which entity(ies) was actually responsible for the observed changes. Due to the heterogeneity of plasma proteins, the total protein concentration cannot be considered as a good indicator of this disease. In spite of lipid accumulation in the liver and the elevated gluconeogenic substrate in plasma (L-alanine), the symptomatic gilthead sea bream could not maintain the glucose level; this fall in glucose concentration in affected animals is in agreement with the findings of Galeotti et al. (1999). These data confirm the idea that there is a partial liver failure in fish affected by winter syndrome. Although winter disease is associated with a granular degeneration of white muscle (Padro´s et al., 1998; Tort et al., 1998a,b), nonsignificant changes were found in tissue percentages of water, lipids, and proteins. However, muscle destruction may be reflected in the observed rise in the concentration of plasmatic amino acids. Proteolysis may be detected earlier in plasma than in muscle, due to the differences in size of the two compartments. Fasting, as a simple cause of this increase, can be ruled out because control and asymptomatic animals were subjected to the same water temperature ( < 12 jC) and did not eat either. In addition, although starvation can induce changes in muscle related to catabolic reactions, granular degeneration was not observed in C and A animals. The dramatic increase in L-alanine, the only nonessential amino acid that increased significantly, could be related to the rise in branched-chain amino acid oxidation, which would supply amino acid groups for L-alanine synthesis, as has been observed in fasting fish (Blasco et al., 1991, 1992). Apart from the observed fall in the number of erythrocytes in symptomatic fish, the drop in haematocrit could be partially due to haemodilution, but this can be ruled out because of the increase in the total concentration of plasma proteins. In some cases, sea bream affected by winter syndrome also showed a clear hypoplasia of the haematopoietic tissue (Padro´s, personal communication) and this could also be associated with the fall in erythrocytes; however, this hypoplasia was not found in the WS group.
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The rise in potassium plasma levels detected in WS fish may be related to the cellular destruction in muscles and liver, and haemolysis can be ruled out as cause because no free haemoglobin was detected in plasma. WS fish also presented an imbalance in the concentrations of magnesium and calcium. The fall in these ions could have important consequences for the metabolism of symptomatic animals because these cations are involved in metabolic processes, such as muscular activity or coenzymes. Asymptomatic fish were a heterogeneous group for some parameters. These animals also showed intermediate values, between control and winter syndrome fish, on some of the analysed parameters, such as number of red blood cells, amino acids, or a2-globulins. This could be due to the fish living under compromising conditions and some of them developing winter syndrome subsequently. Moreover, some variations between C and A animals may be due to seasonal changes, such as the variation in the concentration of h2globulins or the number of erythrocytes. The number of erythrocytes of gilthead sea bream is subject to a seasonal cycle that reaches its minimum in winter (data not published). This evolution has already been described for some species of fish, such as trout (Lane, 1979), as being due to a fall of erythropoiesis in winter (Cossins and Kilbey, 1989). However, if the described changes in h1-globulins between C and A fish are due to seasonal variation, then WS animals must be unable to maintain the levels of this protein fraction. Winter syndrome appears to affect only the weakest farmed gilthead sea bream, either the smallest fish or those most sensible to stress factors or toxins. Symptomatic animals show partial liver failure, either as cause or effect.
Acknowledgements This work has been supported by a grant from the CICYT (MAR97-0408-00-02) of the Spanish Government, and was partially funded by the Comisio´ Interdepartamental de Recerca i Innovacio´ Tecnolo`gica (CIRIT) of the Generalitat de Catalunya. M. SalaRabanal and A. Ibarz received fellowships from the Generalitat de Catalunya and the Spanish Government, respectively. We wish to thank Joana Valentı´n for technical help.
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Canals, P., Gallardo, M.A., Blasco, J., Sa´nchez, J., 1992. Uptake and metabolism of L-alanine by freshly isolated trout (Salmo trutta) hepatocytes: the effect of fasting. J. Exp. Biol. 169, 37 – 52. Clem, L.W., Faulmann, E., Miller, N.W., Ellsaesser, C.F., Lobb, C.J., Cuchens, M.A., 1984. Temperaturemediated processes in teleost immunity. Differential effects of temperature on mitogenic response of channel catfish lymphocytes. Dev. Comp. Immunol. 8, 313 – 322. Contessi, B., Votpatti, D., Gusmani, L., Bovo, G., Maltese, C., Mutinelli, F., Borghesan, F., Gennari, L., Sarli, G., Galeotti, M., 2000. ‘‘Winter disease’’ in farmed gilthead sea bream (S. aurata): immunological aspects. Boll. Soc. Ital. Patol. Ittica 12, 2 – 16. Cossins, A.R., Kilbey, R.V., 1989. The seasonal modulation of Na+/H+ exchanger activity in trout erythrocytes. J. Exp. Biol. 144, 463 – 478. Davis, P.S., 1988. Two occurrences of the gilthead, Sparus aurata Linnaeus 1758, on the coast of Northumberland, England. J. Fish Biol. 33, 951 – 952. Doimi, M., 1996. A new winter disease in sea bream (Sparus aurata): a preliminary report. Bull. Eur. Assoc. Fish Pathol. 16, 17 – 18. Domenech, A., Ferna´ndez-Garayzabal, J.F., Lawson, P., Garcı´a, J.A., Cutili, M.T., Blanco, M., Gibello, A., Moreno, M., Collins, M.D., Domı´nguez, L., 1997. Winter disease outbreak in sea bream (Sparus aurata) associated with Pseudomonas anguillieptica infection. Aquaculture 156, 317 – 326. Domenech, A., Ferna´ndez-Garayzabal, J.F., Garcı´a, J.A., Cutili, M.T., Moreno, M., Domı´nguez, L., 1999. Association of Pseudomonas anguilliseptica infection with ‘‘winter disease’’ in sea bream, Sparus aurata L. J. Fish Dis. 22, 69 – 71. Evensen, O., Espelid, S., Haastein, T., 1991. Immunohistochemical identification of Vibrio salmonicida in stored tissues of Atlantic Salmo salar from the first known outbreak of coldwater vibriosis. Dis. Aquat. Org. 10, 189 – 195. Flos, R., Tort, L., Torres, P., 1990. The development of better conditions and handling procedures for intensive cultures: the incidence of stress. In: Flos, R., Tort, L., Torres, P. (Eds.), Mediterranean Aquaculture. Ellis Horwood, New York. 213 pp. Folch, J., Lees, M., Sloane Stanley, G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497 – 509. Galeotti, M., Beraldo, P., Contessi, B., Manetti, M.F., Votpatti, D., 1998. ‘‘Winter disease’’ in farmed gilthead sea bream: immunological aspects. Biol. Mar. Mediterr. 5, 1600 – 1608. Galeotti, M., Contessi, B., Volpatti, L., Fusmani, L., Maltese, C., Mutinetti, F., Borghesan, F., Gennari, L., Bovo, G., 1999. ‘‘Winter disease’’ in farmed gilthead sea bream (Sparus aurata): etiopathologenetical and immunological aspects. European Association of Fish Pathologists. 9th International Conference on ‘‘Diseases of Fish and Shellfish’’. Giardina, B., Mordente, A., Zappacosta, B., Calla, C., Colacicco, L., Gozzo, M.L., Lippa, S., 1998. The oxidative metabolism of Antarctic fish: some peculiar aspects of cold adaptation. In: Prisco, G., Pisano, E., Clarke, A. (Eds.), Fishes of Antarctica. A Biological Overview. Di Milano Italy Springer-Verlag, Italy, pp. 129 – 138. Holt, R.A., Rohovec, J.S., Freyer, J.L., 1993. Bacterial cold-water disease. In: Inglis, V., Roberts, R.J., Bromage, N.R. (Eds.), Bacterial Diseases of Fish. Blackwell, Oxford, pp. 3 – 22. Lane, H.C., 1979. Progressive changes in haematology and tissue water of sexually mature trout, Salmo gairdneri Richardson, during the autumn and winter. J. Fish Biol. 15, 425 – 436. Melamed, M.R., Lindmo, T., Mendelsohn, M.L., 1990. Flow Cytometry and Sorting: Chapter 4. Electrical Resistance Pulse Sizing: Coulter Sizing. Wiley-Liss, New York, USA. Miller, N.W., Clem, L.W., 1984. Temperature-mediated processes in teleost immunity. Differential effects of temperature on catfish in vitro antibody responses to thymus-dependent and thymus-independent antigens. J. Immunol. 133, 2356 – 2359. Padro´s, F., Crespo, S., Sala, S., Sa´nchez, J., Blasco, J., Ferna´ndez, J., Rotllant, J., Tort, L., 1998. Winter syndrome in gilthead seabream (Sparus aurata). Structural and functional alterations and the influence of stress. International Congress on the Biology of Fish, Baltimore, MD. Padro´s, F., Herna´ndez, A., Rotllant, J., Puigcerver, M., Sala, R., Crespo, S., Tort, L., Ibarz, A., Sala, M., Gallardo, M.A., Blasco, J., Ferna´ndez, J., Sa´nchez, J., 1999. La enfermedad de invierno en la dorada (Sparus aurata L.). Caracterı´sticas del sı´ndrome, disfunciones observadas y metodologı´a de ana´lisis y prevencio´n. VII Congreso Nacional de Acuicultura, Las Palmas de Gran Canaria, Spain.
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Page´s, T., Gomez, E., Suner, O., Viscor, G., Tort, L., 1995. Effects of daily management stress on haematology and blood rheology of the gilthead seabream. J. Fish Biol. 46, 775 – 786. Pascale, R., Daino, L., Garcea, R., Frassetto, S., Ruggiu, M.E., Vannini, M.G., Cozzolino, P., Feo, F., 1989. Inhibition by ethanol of rat liver plasma membrane sodium potassium ATPase protective effect of S-adenosylL-methionine, L-methionine and N-acetylcysteine. Toxicol. Appl. Pharmacol. 97, 216 – 229. Pickering, A.D., Pottinger, T.G., 1985. Cortisol can increase the susceptibility of brown trout, Salmo trutta L., to disease without reducing the white blood cell count. J. Fish Biol. 27, 611 – 619. Roberts, R.J., 2001. Fish Pathology, 3rd ed. Baillie`re Tindall, London. 492 pp. Rotllant, J., Balm, P.H.M., Wendelaar, B.S.E., Perez-Sa´nchez, J., Tort, L., 2000. A drop in ambient temperature results in a transient reduction of interrenal ACTH responsiveness in the gilthead sea bream (Sparus aurata, L.). Fish Physiol. Biochem. 23, 265 – 273. Sarusic, G., 1999. Clinical signs of the winter disease phenomenon in sea bream (Sparus aurata, L.). Bull. Eur. Assoc. Fish Pathol. 19, 113. Shapiro, H.M., 1995. Practical Flow Cytometry: Chapter 4. The Coulter Principle: Electronic Cell Sizing. WileyLiss, New York. Sornaraj, R., Thanalasmi, S., Baskaran, P., 1995. Influence of heavy metals on biochemical responses of the freshwater air-breathing fish Channa punctatus. J. Ecotoxicol. Environ. Monit. 5, 19 – 27. Tort, L., Gomez, E., Montero, D., Sunyer, J.O., 1996. Serum haemolytic and agglutination activity as indicators of fish immunocompetence: their suitability in stress and dietary studies. Aquacult. Int. 4, 31 – 41. Tort, L., Rotllant, J., Rovira, L., 1998a. Immunological suppression in gilthead sea bream Sparus aurata of the North-West Mediterranean at low temperatures. Comp. Biochem. Physiol. 120A, 175 – 179. Tort, L., Padro´s, F., Rotllant, J., Crespo, S., 1998b. Winter syndrome in the gilthead sea bream Sparus aurata. Immunological and histopathological features. Fish Shellfish Inmunol. 8, 37 – 47. Zapata, A.G., Varas, A., Torraba, M., 1992. Seasonal variation in the immune system of lower vertebrates. Immunol. Today 13, 142 – 147.
Functional alterations associated with ‘‘winter syndrome’’ in gilthead sea bream (Sparus aurata) ´ ngeles Gallardo a,b,*, Mo´nica Sala-Rabanal a,b, M. A Antoni Ibarz a,b, Francesc Padro´s b,c, Josefina Blasco a,b, Jaume Ferna´ndez-Borra`s a,b, Josep Sa´nchez a,b a
Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Avinguda Diagonal, 645, Barcelona E-08028, Spain b Centre de Refere`ncia i Desenvolupament en Aqu¨icultura de la Generalitat de Catalunya, Spain c Servei de Diagno`stic Patolo`gic en Peixos, Facultat de Veterina`ria, Universitat Auto`noma de Barcelona, Bellaterra 08193, Spain Received 22 April 2002; received in revised form 10 February 2003; accepted 10 February 2003
Abstract Gilthead sea bream obtained from a fish farm in the Delta de l’Ebre (Tarragona, Spain) were used to study various functional alterations associated with ‘‘winter syndrome.’’ The fish groups considered were: control fish routinely sampled 3 weeks before an outbreak (which took place in March 1998); winter syndrome symptomatic fish (assessed by eye and corroborated through histopathological analysis); and asymptomatic animals sampled from the same tank where winter syndrome fish were found. Winter syndrome animals weighed less than control and asymptomatic fish. The symptomatic animals suffered a drop in the haematocrit and number of erythrocytes, although red cell volume did not change. Furthermore, the concentration of total plasma proteins in winter syndrome fish was higher than in control animals, due to a rise in h2- and g-globulins; the a2-globulin concentration was lower and albumin and a1- and h1-globulins remained unchanged. A significant increase in the concentration of plasma amino acids, both essential and nonessential, was found in symptomatic fish. This group of animals showed a lower plasma glucose concentration. There were no differences in either the plasma sodium or chloride concentrations between control and symptomatic fish. Instead, the plasma potassium concentration in winter syndrome animals doubled that of control animals. The plasma concentrations of calcium and magnesium were lower in symptomatic animals than in control fish.
* Corresponding author. Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Avinguda Diagonal, 645, Barcelona E-08028, Spain. Tel.: +34-934021557; fax: +34-934110358. E-mail address: [email protected] (M.A. Gallardo). 0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0044-8486(03)00164-9
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Symptomatic fish showed high lipid concentration in liver and elevated plasmatic GOT activity, indicating hepatic damage. No other significant differences in the composition of liver and white muscle were found between control and symptomatic animals. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Winter syndrome; Winter disease; Sparus aurata; Haematology; Plasma determinations; Liver and muscle composition
1. Introduction The gilthead sea bream (Sparus aurata) is a fish species predominantly located in the Mediterranean Sea, but it can also be found around coasts as far north as the UK and as far south as Senegal. In the last 10 –15 years, the commercial farming of gilthead sea bream has become a common practice along the Mediterranean coastline. As with other fish species, one of the main problems associated with sea bream culture is stress due both to the fish being confined in cages, which prevents them from migrating, and handling (Flos et al., 1990). Several studies have shown immunosuppression to be a direct result of stress in this species (Page´s et al., 1995; Tort et al., 1996, 1998a), increasing their susceptibility to opportunistic pathogens and decreasing their ability to fight infections, as clearly demonstrated in other fish species (Pickering and Pottinger, 1985). The effect of temperature on the fish immune system has also been discussed (Bly and Clem, 1992; Zapata et al., 1992). Thus, after exposure to low temperatures, there is a suppression of T-cell mitogenic or antibody responses (Clem et al., 1984; Miller and Clem, 1984; Bly et al., 1986). Wild gilthead sea bream normally live in an environment whose temperature ranges from 11 jC in winter to 23 jC in summer, without any apparent problems related to the temperature changes. There is also evidence that sea bream migrate to greater depths (warmer waters) when surface temperatures start to decline (Davis, 1988). However, the problem of low temperature may be critical in intensive cultured gilthead sea bream because the fish are unable to move to warmer waters. In fact, when the temperature drops to 8– 10 jC, the plasma cortisol of exposed sea bream rises as a stress response (Rotllant et al., 2000) and, below 12 jC, the species stops feeding (Padro´s et al., 1999). ‘‘Winter syndrome’’ or ‘‘winter disease’’ is a pathology exclusively affecting cultured gilthead sea bream in the Mediterranean sea, being more severe in the northern areas. Other fish species, such as sea bass, that are frequently reared in the same facilities seem not to be affected. Winter syndrome causes chronic mortality during the coldest months and acute mortality episodes when the temperature rises (Tort et al., 1998a; Sarusic, 1999). Mortality rates are usually around 7– 10% of the fish stock, although in some very acute cases, they may be as high as 80% (Padro´s et al., 1998). The causes of winter syndrome are not understood and cannot be explained as the result of only one factor (Tort et al., 1998b); although deaths are associated with reduced temperatures, this may not be the only factor affecting mortality as the majority of fish survive through cold periods. Winter syndrome increases plasma cortisol, decreases the complement and lysozyme activities, and reduces circulating lymphocytes (Tort et al., 1998a), therefore reducing the
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ability of fish to resist attacks by opportunistic parasites. Different bacterial and viral agents (Bovo et al., 1995; Doimi, 1996), particularly Pseudomonas anguilliseptica (Berther et al., 1995; Domenech et al., 1997, 1999), have often been isolated from different outbreaks of winter syndrome, although these isolations were not consistent from year to year and no specific pathogen was found (Padro´s et al., 1998). In contrast, infections in other fish species exposed to low temperatures are normally due to specific pathogens (Evensen et al., 1991; Holt et al., 1993; Roberts, 2001). The visible signs of winter syndrome-affected fish are lethargy, sideways or even backwards swimming, and minimal reaction to external stimuli. A variety of significant tissue lesions have been described in symptomatic animals, such as granular degeneration and necrosis in white muscle fibres, atrophy of the exocrine pancreas, pale and friable liver, and a fatty degeneration in hepatocytes (Galeotti et al., 1998; Padro´s et al., 1998; Tort et al., 1998b; Contessi et al., 2000). The digestive tract appears distended and filled with a clear liquid, indicating a potentially reduced nutrient absorption, and many symptomatic fish also have white fibromucous materials in the intestine. Moreover, intestinal mucosa displays a significant hyperplasia of mucous cells and, frequently, the lamina propia shows oedema (Bovo et al., 1995; Padro´s et al., 1998; Tort et al., 1998b; Contessi et al., 2000). Less consistent lesions have also been observed in the brain and kidney (Padro´s et al., 1998). In some cases, blood sampled from symptomatic animals has a clearer appearance due to low red blood cell counts (Padro´s et al., 1999). Most studies related to winter disease are devoted to descriptions of histopathological lesions and immunological variations, but few have shown changes in other aspects of fish physiology. The aim of the present study was to investigate changes in certain physiological parameters related, on one hand, to the homeostasis of animals (proteins, amino acids, glucose, and ions in plasma) and, on the other hand, to haematology and tissue composition. Plasmatic GOT was also analysed as an indicator of hepatic injury. The study of these parameters could help to reveal the aetiology of winter syndrome.
2. Materials and methods 2.1. Animals Gilthead sea bream (S. aurata) were obtained from fish farms located in the Delta de l’Ebre (Tarragona, Spain), where they were maintained in shallow pools with brackish water. Twenty control fish (C) were sampled in February 1998 from a routine seasonal sampling. Twenty-four days later, winter syndrome appeared in the pond and 20 visually symptomatic (WS) fish, along with 10 apparently asymptomatic (A) fish, were sampled. 2.2. Blood collection, morphometric analysis, and tissue sampling Blood was collected by caudal puncture. Lithium EDTA, at a final concentration of 5 mgml 1 blood, was used as an anticoagulant. Fish were killed by severing their spinal cord and were subsequently weighed and measured. Samples of liver and white muscles were taken and immediately frozen in liquid nitrogen for further analysis.
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2.3. Histology Liver, muscle, kidney, digestive tract, and gill samples were fixed in neutral 10% phosphate-buffered formalin for histological studies. Samples were processed in paraffin and stained with hematoxylin – eosin. 2.4. Haematology Haematocrit was measured by using a HaemofugeR microcentrifuge (Heraeus-Christ, Osterode, Germany). Total red blood cell counts and volumes were determined by means of the Coulter principle (Melamed et al., 1990; Shapiro, 1995), using a Multisizer particle counter (Coulter, Hialeah, FL, USA) equipped with a 100-AM orifice. 2.5. Plasmatic determinations The remaining blood was rapidly centrifuged and the obtained plasma was kept at 80 jC until use for analysis. Total protein concentration was determined by the Lowry technique using bovine serum albumin as standard. Protein fractions were obtained by electrophoresis on cellulose acetate bands (CellogelR; Biosystems, Barcelona, Spain), using a Pre´fe´renceR photodensitometer (Sebia, Paris, France) to read the transparent bands. The amino acid content of deproteinized plasma samples was determined by ionic exchange chromatography, following Canals et al. (1992), using an Alpha Plus II autoanalyzer (Pharmacia LKB Biotechnology, Uppsala, Sweden). Plasmatic glucose was determined by means of the glucose oxidase reaction, using a GlucofixR colorimetric kit (Menarini, Florence, Italy). Plasmatic potassium was analysed by flame atomic absorption spectroscopy, using a PU 9200XR spectrometer (Phillips, Eindhoven, the Netherlands), whereas sodium, calcium, and magnesium were determined by inductively coupled plasma spectrometry using a Polyscan 61ER spectrometer (Thermo Jarrell Ash, Franklin, MA, USA). Chloride was quantified by the ClorofixR colorimetric kit (Menarini), based on the mercury thiocyanate reaction. Plasma GOT concentration was determined by a modified version of Beuther (1984) method. In this assay, GOT activity was coupled, through the action of malic dehydrogenase (MDH), to the oxidation of NADH, which was measured spectrophotometrically at 340 nm. 2.6. Liver and white muscle composition The samples of liver and muscle were powdered in liquid nitrogen and separated into three fractions. One fraction was used to determine gravimetrically the tissue water content, drying the sample at 105 jC for 24 h. The protein percentage was determined from another fraction, measuring the elemental nitrogen by burning the sample in a chromatograph (EA 1108 CHNS-O; Carlo Erba Instruments, Milan, Italy). To convert the elemental nitrogen into a protein percentage, the typical correction factor (6.25) was used. From the last fraction, the lipid content was measured after two extractions with chloro-
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Fig. 1. Haematocrit (bars) and number of erythrocytes (dot) in the three experimental groups: (C) control, (A) asymptomatic, and (WS) winter syndrome symptomatic fish. Values are shown as mean F S.E.M. Group differences ( p < 0.05) for each parameter are marked with different letters.
form/methanol (2:1, vol/vol), according to the procedure of Folch et al. (1957), using butylated hydroxy-toluene (BHT; 0.01%) as an antioxidant. The final washed lipid extract was dried on a Buchi rotavap (Bu¨chi Rotavapor R-114, Postfach, Switzerland) and lipid mass was determined gravimetrically. 2.7. Data and statistical analysis Results are given as mean F S.E.M. Differences among groups were tested by one-way ANOVA and the Tukey test using a computerized package (SigmaStat 2.01).
3. Results The diagnosis of fish visually classified as symptomatic for winter syndrome was corroborated though histological analysis, according to the signs described by Padro´s et al. Table 1 Concentrations (gl
Total plasma protein Albumin a1-Globulins a2-Globulins h1-Globulins h2-Globulins Fibrinogen g-Globulins
1
) of total plasma proteins and their fractions in the three experimental groups Control fish
Asymptomatic fish
Symptomatic fish
42.1 F 1.0a 2.4 F 0.7 2.1 F 0.3 10.6 F 0.5a 7.1 F 0.4a 10.0 F 0.4a 1.8 F 0.2 8.1 F 0.5a
48.4 F 0.9b 2.8 F 1.5 1.9 F 0.3 8.2 F 0.7a,b 9.7 F 0.5b 11.9 F 0.7b 2.5 F 0.3 11.4 F 0.6b
46.1 F 1.0b 2.9 F 0.8 2.2 F 0.3 6.7 F 0.3b 8.1 F 0.8a,b 11.9 F 0.5b 1.6 F 0.3 12.7 F 0.9b
Values are shown as mean F S.E.M. Group differences ( p < 0.05) for each parameter are marked with different letters.
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Fig. 2. Changes in the a-amino acid concentration in the three experimental groups. Total a-amino acids (n), essential amino acids ( ), nonessential amino acids ( ), and the essential/nonessential amino acid ratio (.). Values are shown as mean F S.E.M. Group differences ( p < 0.05) for each parameter are marked with different letters.
(1998) and Tort et al. (1998b). All symptomatic fish showed granular degeneration and necrosis in white muscle fibres, different degrees of atrophy (not necrosis) in exocrine pancreas, pale and friable liver, and fatty degeneration in hepatocytes. They also had a distended and liquid-filled digestive tract, hyperplasia of the mucous cells, and oedema in the submucosa. Moreover, white fibrous materials were often found in the intestines. Control and asymptomatic fish did not show any of these signs. Body weights of both control (151 F 2 g) and asymptomatic (153 F 6 g) animals were significantly higher ( p V 0.05) than those of symptomatic fish (125 F 1 g). In contrast, no significant differences were found between the length of asymptomatic and symptomatic fish (192.0 F 5.8 and 188.6 F 2.8 cm, respectively). Some haematological and plasmatic parameters were studied. Fig. 1 shows the haematocrit and the number of erythrocytes (RBC) for the three groups of fish. A significant fall in the haematocrit of symptomatic fish (WS) compared to control (C)
Fig. 3. Concentration of L-alanine (.) and other nonessential amino acids (o) in the three experimental groups. Values are shown as mean F S.E.M. Group differences ( p < 0.05) for each parameter are marked with different letters.
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Table 2 Nonessential a-amino acid and taurine levels (AM) in the plasma of the three experimental groups Control fish Alanine Asparagine Aspartic acid Glycine Glutamic acid Proline Ornitine Serine Tyroxine Taurine
a
222 F 40 46 F 5a 25 F 3 390 F 26 32 F 4a 49 F 12 56 F 7 94 F 7 131 F 14 449 F 37a
Asymptomatic fish a,b
409 F 31 27 F 4a,b 32.0 F 6 392 F 58 53 F 12a,b 35 F 7 25 F 2 98 F 11 164 F 5 777 F 36a,b
Symptomatic fish 803 F 130b 20 F 3b 33 F 3 498 F 77 55 F 7b 91 F 20 54 F 14 116 F 12 182 F 22 818 F 89b
Values are shown as mean F S.E.M. Group differences ( p < 0.05) for each parameter are marked with different letters.
and asymptomatic (A) animals was observed; this seems to be related to a significant drop in RBC number because no changes in RBC volume among C, A, and WS fish were found (around 85 Am3). The concentration of total plasma proteins and their fractions were also measured (Table 1). There was a significant increase in the total protein concentration of winter symptomatic (WS) and asymptomatic (A) fish compared to controls (C), the globulin fraction being responsible for most of the change, whereas the albumin fraction remained constant. The different globulin fractions did not change uniformly. h2- and g-globulins increased significantly in WS and A fish, while the concentration of a2-globulins was lower in WS animals than in both controls. Additionally, the levels of h1-globulins in A animals were higher than in C and WS fish. Finally, the levels of a1-globulins and fibrinogen did not change. A significant increase in plasma amino acid concentration was also observed in the WS group (Fig. 2). Although this rise affects both essential and nonessential amino acids and the essential/nonessential ratio did not differ significantly between the C and WS groups (Fig. 2), the nonessential amino acids increased more than essential amino acids (82.5% Table 3 Essential amino acid levels (AM) in the plasma of the three experimental groups
Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine
Control fish
Asymptomatic fish
Symptomatic fish
58 F 10 61 F 5a 83 F 6a 189 F 10 148 F 12 24 F 3 71 F 9 75 F 5 209 F 14
85 F 20 106 F 25a,b 95 F 11a,b 174 F 11 170 F 23 23 F 12 82 F 11 81 F 18 218 F 21
152 F 61 117 F 8b 154 F 17b 244 F 27 202 F 26 41 F 10 72 F 8 96 F 15 285 F 34
Values are shown as mean F S.E.M. Group differences ( p < 0.05) for each parameter are marked with different letters.
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Fig. 4. Changes in the plasmatic concentration of minority cations in the three experimental groups: potassium (bars), calcium (.), and magnesium (o). Values are shown as mean F S.E.M. Group differences ( p < 0.05) for each parameter are marked with different letters.
versus 46.3%). Among the nonessential amino acids, the rise in L-alanine concentration (260%) was notable (Fig. 3 and Table 2). However, there was not a single entity responsible for the change in essential amino acids (Table 3). Finally, plasma taurine rose significantly in WS fish compared with control animals (Table 2). Another change found in the plasma of symptomatic fish was a significant ( p < 0.05) fall in the glucose concentration, in winter syndrome animals (3.7 F 0.2 mM) being 21% and 33% lower with respect to control (4.7 F 0.2 mM) and asymptomatic fish (5.5 F 0.3 mM), respectively. The ionic composition of plasma was also analysed. Main ions such as sodium (172.1 F 3.0, 178.0 F 4.0, 166.8 F 2.2 mM in C, A, and WS, respectively) and chloride (116.1 F 3.1, 113.6 F 2.9, 114.9 F 2.2 mM in C, A, and WS, respectively) did not change significantly. In contrast, the potassium concentration increased by 100% in A and WS fish
Fig. 5. Lipid percentage in liver (bars) and plasmatic GOT activity (dot) in the three experimental groups. Values are shown as mean F S.E.M. Group differences ( p < 0.05) for each parameter are marked with different letters.
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Table 4 Liver and muscle composition (%) of the three experimental groups
Liver
Muscle
Water Lipid Protein Water Lipid Protein
Control fish
Asymptomatic fish
Symptomatic fish
71.0 F 0.5 8.4 F 0.6a 14.6 F 0.3 73.6 F 0.4 3.8 F 0.2 19.8 F 0.3
70.7 F 1.6 10.4 F 2.0a,b 14.9 F 0.6 76.2 F 0.5 3.3 F 0.2 19.8 F 0.6
70.5 F 0.9 12.2 F 0.7b 14.1 F 0.2 75.5 F 0.4 3.9 F 0.2 20.7 F 0.3
Values are shown as mean F S.E.M. Group differences ( p < 0.05) for each parameter are marked with different letters.
compared with C animals; the concentrations of calcium and magnesium were the same in C and A animals, but decreased by 10% and 34%, respectively, in WS animals (Fig. 4). The greatest change in the liver morphology of symptomatic fish was the increase of friability of the organ, probably due to a rise in lipid content (Fig. 5 and Table 4). As a result of apparent alterations in liver morphology, the GOT plasmatic concentration was measured as an indicator of hepatic damage. Plasmatic GOT levels in WS fish showed a dramatic increase (Fig. 5), confirming the existence of liver damage in these animals. On the other hand, no significant differences among groups were found in liver water and protein contents (Table 4). Neither were significant differences found among C, A, and WS fish with respect to water, protein, and lipid percentages in white muscle (Table 4).
4. Discussion As stated above, most studies related to winter syndrome in gilthead sea bream describe histopathological changes (Galeotti et al., 1998; Padro´s et al., 1998; Tort et al., 1998b; Contessi et al., 2000) and immunological variations (Tort et al., 1998a,b; Contessi et al., 2000), but few provide other functional information (Galeotti et al., 1999; Padro´s et al., 1999). Identifying modifications in certain physiological parameters would offer a more complete understanding of the pathogenesis of this syndrome. From an epidemiological point of view, a fall in water temperature seems to be the single common factor associated with the development of the disease, although there are obviously other factors involved as the majority of fish survive through cold periods (Tort et al., 1998b). One of most noticeable changes in affected fish is lipid accumulation in liver. It has been argued that for some fish, this change represents an adaptive mechanism in response to a drop in water temperature (Giardina et al., 1998), as it helps oxygen to diffuse into the mitochondria. However, a potential intoxication related to bacterial or other toxins should not be ruled out (Pascale et al., 1989; Sornaraj et al., 1995). In gilthead sea bream, feeding stops at low temperatures ( < 12 jC) (Padro´s et al., 1999) and, therefore, the liver lipid accumulation cannot be due to lipid absorption from the gut. Symptomatic fish may present an imbalance between the entry and exit of liver nutrients, possibly as a result of a hepatic dysfunction. However, in spite of the raised liver lipid percentage and the
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pathological levels of GOT in plasma (Galeotti et al., 1999 and present data), it is not clear if this situation induces a general failure in this organ functioning. This is because some hepatic-dependent systems remain active; for instance, symptomatic animals show an increase in h2-globulin concentration, although other plasmatic protein fractions, such as a2-globulins, are reduced in these fish. No significant differences were found in the albumin fraction concentration among the three groups studied. This is not consistent with the fall described by Galeotti et al. (1999) in symptomatic animals. Observation of a seasonal cycle showed that albumin normally reaches its minimum value in winter (data not published), suggesting that this difference may be the reason for this discrepancy. In February – March, the analysed fish (C, A, and WS) showed a particularly low albumin concentration. The increase in the g-globulin fraction in WS and A animals suggests that fish are responding to an infectious process, although this increase was not found by Galeotti et al. (1999) in symptomatic winter syndrome animals. Additionally, a2-globulin concentration was the only plasma protein fraction that varied in symptomatic fish but not in asymptomatic animals. This latter group showed a h1-globulin concentration significantly higher that did the C and WS groups; no explanation for this change can be found, unless the h1-globulin fraction contains a protective protein. Neither the a2- or h1-globulins were homogeneous fractions and no further analysis was carried out to assess which entity(ies) was actually responsible for the observed changes. Due to the heterogeneity of plasma proteins, the total protein concentration cannot be considered as a good indicator of this disease. In spite of lipid accumulation in the liver and the elevated gluconeogenic substrate in plasma (L-alanine), the symptomatic gilthead sea bream could not maintain the glucose level; this fall in glucose concentration in affected animals is in agreement with the findings of Galeotti et al. (1999). These data confirm the idea that there is a partial liver failure in fish affected by winter syndrome. Although winter disease is associated with a granular degeneration of white muscle (Padro´s et al., 1998; Tort et al., 1998a,b), nonsignificant changes were found in tissue percentages of water, lipids, and proteins. However, muscle destruction may be reflected in the observed rise in the concentration of plasmatic amino acids. Proteolysis may be detected earlier in plasma than in muscle, due to the differences in size of the two compartments. Fasting, as a simple cause of this increase, can be ruled out because control and asymptomatic animals were subjected to the same water temperature ( < 12 jC) and did not eat either. In addition, although starvation can induce changes in muscle related to catabolic reactions, granular degeneration was not observed in C and A animals. The dramatic increase in L-alanine, the only nonessential amino acid that increased significantly, could be related to the rise in branched-chain amino acid oxidation, which would supply amino acid groups for L-alanine synthesis, as has been observed in fasting fish (Blasco et al., 1991, 1992). Apart from the observed fall in the number of erythrocytes in symptomatic fish, the drop in haematocrit could be partially due to haemodilution, but this can be ruled out because of the increase in the total concentration of plasma proteins. In some cases, sea bream affected by winter syndrome also showed a clear hypoplasia of the haematopoietic tissue (Padro´s, personal communication) and this could also be associated with the fall in erythrocytes; however, this hypoplasia was not found in the WS group.
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The rise in potassium plasma levels detected in WS fish may be related to the cellular destruction in muscles and liver, and haemolysis can be ruled out as cause because no free haemoglobin was detected in plasma. WS fish also presented an imbalance in the concentrations of magnesium and calcium. The fall in these ions could have important consequences for the metabolism of symptomatic animals because these cations are involved in metabolic processes, such as muscular activity or coenzymes. Asymptomatic fish were a heterogeneous group for some parameters. These animals also showed intermediate values, between control and winter syndrome fish, on some of the analysed parameters, such as number of red blood cells, amino acids, or a2-globulins. This could be due to the fish living under compromising conditions and some of them developing winter syndrome subsequently. Moreover, some variations between C and A animals may be due to seasonal changes, such as the variation in the concentration of h2globulins or the number of erythrocytes. The number of erythrocytes of gilthead sea bream is subject to a seasonal cycle that reaches its minimum in winter (data not published). This evolution has already been described for some species of fish, such as trout (Lane, 1979), as being due to a fall of erythropoiesis in winter (Cossins and Kilbey, 1989). However, if the described changes in h1-globulins between C and A fish are due to seasonal variation, then WS animals must be unable to maintain the levels of this protein fraction. Winter syndrome appears to affect only the weakest farmed gilthead sea bream, either the smallest fish or those most sensible to stress factors or toxins. Symptomatic animals show partial liver failure, either as cause or effect.
Acknowledgements This work has been supported by a grant from the CICYT (MAR97-0408-00-02) of the Spanish Government, and was partially funded by the Comisio´ Interdepartamental de Recerca i Innovacio´ Tecnolo`gica (CIRIT) of the Generalitat de Catalunya. M. SalaRabanal and A. Ibarz received fellowships from the Generalitat de Catalunya and the Spanish Government, respectively. We wish to thank Joana Valentı´n for technical help.
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