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Acute Aeromonas salmonicida infection in turbot(Scophthalmus maximus L.). Histopathological andimmunohistochemical studies
Germán Alberto Coscelli a,b, Roberto Bermúdez c, Ana Paula Losada a, Luis Daniel Faílde a,Ysabel Santos d, María Isabel Quiroga a,⁎a Departamento de Ciencias Clínicas Veterinarias, Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus Universitario s/n, 27002, Lugo, Spainb Cátedra de Patología General, Anatomía y Fisiología Patológicas, Facultad de Ciencias Clínicas Veterinarias, Universidad Nacional de Rosario, Bv. Ovidio Lagos y Ruta 33, 2170, Casilda, Argentinac Departmento de Anatomía and Production Animal, Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus Universitario s/n, 27002, Lugo, Spaind Departmento de Microbiología y Parasitología, Centro de Investigaciones Biológicas (CIBUS), Facultad de Biología, Universidad de Santiago de Compostela, Campus Sur, 15782,Santiago de Compostela, Spain
Abbreviations: A. salmonicida, Aeromonas salmonicida smunohistochemistry; hpi, h post-inoculation; PBS, phosphloendothelial system; TSA-1, tryptic soy agar with 1% NaC⁎ Corresponding author. Tel.: +34 982822304; fax: +3
Aeromonas salmonicida subsp. salmonicida (A. salmonicida) is a highly pathogenic bacterium for turbot culture,which represents a great threat in fish farming. In this study, gross and histological lesions in turbot experimen-tally inoculatedwith A. salmonicidawere analysed. In addition, the distribution of the bacterial antigen in tissueswas investigated by using an immunohistochemical method specifically developed for this purpose.A. salmonicida caused septicaemia in challenged fish, characterized by vascular and necrotic changes, and thepresence of bacterial colonies in several organs. Immunoreactivity against the bacterial antigen was evidencedin the coelomic cavity, blood vessels, and associated to the necrotic foci. These findings indicated the rapidonset of bacteraemia and of the tissue colonization by A. salmonicida and a direct relation between the presenceof the bacteria and the development of lesions. Immunoreactivity against bacterial antigenwas also located in thecytoplasm in both circulating and tissue monocyte/macrophages, suggesting the phagocytosis of the bacteriumduring the initial phase of the infection. To our knowledge, this is the first description of the morphohistologicalchanges induced by acute A. salmonicida infection in turbot. The immunohistochemicalmethod represents a newtool for the diagnosis of the disease and to gain insight in some aspects of its pathogenesis.
Aeromonas salmonicida subsp. salmonicida (A. salmonicida), theetiologic agent of classical furunculosis, is one of the most importantfish pathogen in aquaculture worldwide (Austin and Austin, 2007;Toranzo et al., 2005). A. salmonicida affects mainly both farmed andwild salmonid species, among which Atlantic salmon (Salmo salar) isconsidered particularly susceptible (Diamanka et al., 2013; Pedersenet al., 2008; Saleh et al., 2011). A. salmonicida is able to enter into thesusceptible hosts via the injured skin and gills as well as through themucosa of gastrointestinal tract (Effendi and Austin, 1995; Fergusonet al., 1998; Ringo et al., 2004; Svendsen et al., 1999). In salmonids,
different clinical presentations of the disease, varying from acute tochronic as well as subclinical form, have been described (Austin andAustin, 2007; Bernoth, 1997; Hiney et al., 1994). The acute form is themost common presentation of the disease in both juvenile and adultfish, resulting in a hemorrhagic septicaemia with high mortality rates(Fernández et al., 1995; Ogut and Reno, 2005; Orozova et al., 2009).Pathological findings include exophthalmia, skin haemorrhages andulcers, haemorrhages and necrosis in the muscle and different internalorgans, mainly in spleen and kidney (Burr et al., 2005; Diamankaet al., 2013).
A. salmonicida has also been isolated from awide variety of commer-cially valuable non-salmonids species, including turbot (Scophthalmusmaximus), halibut (Hippoglosus hippoglosus), cod (Gadus morhua) andSenegalese sole (Solea senegalensis) (Bricknell et al., 1999; Lago et al.,2012; Lillehaug et al., 2003; Magariños et al., 2011).
Turbot aquaculture is an important economical activity in severalcountries of Europe, Asia and South America, and epizootic outbreaksof acute furunculosis in turbot farms have been reported in Spain,Portugal, Denmark, France and Norway (Lago et al., 2012; Lillehaug
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et al., 2003; Nougayrede et al., 1990; Pedersen et al., 1994; Toranzo andBarja, 1992). While A. salmonicida infection in turbot generally resultsin an acute fatal disease (Farto et al., 2011; Perez et al., 1996), chronical-ly infected specimens show multifocal granulomatous dermatitis(Coscelli et al., 2014). Nevertheless, up to date, there is a paucity ofdata about the pathogenesis and pathology of the A. salmonicida infec-tion in turbot.
The aim of this work was to experimentally reproduce the acuteform of A. salmonicida infection in turbot, in order to obtain a completedescription of the disease from a morphopathological point of view.Moreover, an immunohistochemical (IHC) technique was specificallydeveloped to detect A. salmonicida antigen in host tissues, in order tostudy the pathogenesis of the disease as well as provide a useful toolfor diagnosis of the acute infection.
2. Material and methods
2.1. Bacterial strain and antibody production.
A. salmonicida strain TO96 7.1, isolated in 1996 from diseased turbotcultured in Portugal and recently passaged through turbot, was used forthe production of antiserum and the pathogenicity tests. The identity ofthe isolatewas verified by phenotypic and serological tests as previouslydescribed by Santos et al. (2005). Stock cultures of the strains werestored at −70 °C in MicrobankTM tubes (Prolab Diagnostics) untilpassage on Tryptic Soy agar with 1% NaCl (TSA-1) added, immediatelybefore use. Bacterial cultureswere incubated at 18 ºC for 48 h. Polyclonalantiserum was produced in rabbit against formol-fixed A. salmonicidastrain TO96 7.1 (adjusted to 1 × 109 cells mL−1, McFarland standardno. 3) as previously described by Santos et al. (1995). Pre-immuneserum was previously collected from the rabbit. Anaesthetized rab-bits were bled by ear venepuncture, and serum recovered wasaliquoted, undiluted and frozen at −20 °C until use. The immuno-globulin G fraction (IgG) from antiserum was purified through aprepacked protein A Sepharose affinity chromatography column(HiTrapTM Protein A HP, GE Healthcare) according to themanufacturer's protocol. The reactivity of affinity-purified TO96 7.1IgG with whole-cell antigens was verified using slide agglutinationand Dot Blot assays as previously described by Santos et al. (1995).Purified antibodies were stored at −20 °C.
2.2. Fish and challenge procedure
Turbot were obtained from a farm located in Northwestern Spain.Before their use in challenge experiments, fishwere subjected tomicro-biological analysis in order to verify their health status. For challenge,bacterial suspension of virulent TO96 7.1 strain was prepared in sterilesaline solution and adjusted to contain 1 × 109 cells mL−1 (McFarlandstandard No. 3). Cell viability of bacterial suspension was verified bythe plate dilution method using TSA-1 plates and counting the bacterialcolonies produced. A total of 140 clinically healthy juvenile turbot witha mean weight of 68 g (±10.97 g) and a mean total length of 15 cm(±0.62 cm) were divided into two equal experimental groups. Bothgroups were intracoelomic injected. Challenged fish received 0.1 mL ofbacterial suspension [1 × 108 colony forming units (CFU) per fish].Fish from control groupwere injected with 0.1 mL of sterile saline solu-tion. Turbot were kept in plastic tanks (length 115 × width 96 × depth65 cm) under controlled conditions of water quality and ventilation andfed ad libitumwith a commercial diet until the end of the assays. Duringthe course of the experiments water temperature was maintained at18 °C ± 1.
2.3. Sampling
Challenged and control fish were euthanized by overexposure totricaine methane sulfonate (MS-222, Sigma) and necropsy was
performed. Samples from head kidney and/or spleen obtained from ex-perimentally infected and control fish were streaked on the surface ofTSA-1 plates and incubated at 18 °C for up to 7 days. Pure cultures re-covered on TSA-1 were identified using phenotypic and serologicalmethods as above described. Tissue samples were also analysed forthe presence of A. salmonicida by using Dot Blot assay following the pro-cedure described by Gonzalez et al. (2004).
For histological and IHC studies, samples of gastrointestinal tract,liver, spleen, pancreas, gonads, heart, thymus, gills, brain, skin and skel-etal muscle were taken and examined. Tissue samples were fixed inBouin's liquid for 12 h. Challenged fish were sampled at 3, 6, 24, 48and 96 h post-inoculation (hpi), and control fish were sampled at 24,48 and 96 hpi. Ten fish from each group were randomly taken atevery sampling point.
2.4. Histology
Bouin's fixed tissues were processed by routine methods and paraf-fin embedded. Tissue sections were cut at 2–3 μm in thickness andstained with haematoxylin–eosin (H–E) and Gram stain.
2.5. Immunohistochemistry
Tissue sections from both control and challenged fish were air driedovernight at room temperature (RT) and then were de-waxed andrehydrated. All incubation steps were performed at RT in a humidchamber and slides were washed with 10 mM phosphate-buffered sa-line (PBS) 0.5% Tween 20, pH 7.4, in three successive immersionsof 5 min between every step. Endogenous peroxidase was quenchedby Peroxidase Blocking Solution (Dako, Denmark) for 1 h. Tissue sec-tions were incubated for 2 h with the affinity-purified rabbit anti-A. salmonicidaTO96.1 (anti-A. salmonicida, diluted 1:30,000), washedand then incubated for 30 min with an anti-rabbit EnVision + SystemLabelled Polymer-HRP (Dako). Sections were developed employing3,3 diaminobenzidine tetrahydrocloride as chromogen (DAB, Dako). Fi-nally the immunolabelled tissue sections were counterstained withhaematoxylin. For negative controls, the primary antibodywas replacedby PBS or an irrelevant polyclonal antibody. In addition, in order to ruleout cross-reactivity with other common pathogenic bacteria affectingturbot, the antibody anti-A. salmonicida was tested in tissue samplesobtained from turbot experimentally injected with Tenacibaculummaritimum and Vibrio anguillarum.
3. Results
3.1. Gross and microscopic findings
Significant gross lesions were not observed in either challenged orcontrol fish. At light microscopy, the most significant lesions showedby challenged fish occurred in the coelomic cavity. At 3 hpi, a proteina-ceous, clear eosinophilic material with a scarce number of extravasatederythrocytes, compatible with serous exudates, was observed into thecoelomic cavity.
At 6 hpi, turbot developed a mild to moderate coelomitis character-ized by the presence of reactivemesothelial cells (Fig. 1a) and diffuse in-filtrates of inflammatory cells (mainly composed by monocyte/macrophages) on the connective tissue of coelomic cavity as well ason the serosal surface of organs. Moreover, from 6 hpi onwards, chal-lenged fish showed generalized vascular congestion.
From 24 hpi, turbot exhibited lesions in kidney. These changesconsisted in perivascular oedema together with mild to extensiveperitubular haemorrhages, as well as, multifocal areas of necrosis ofthe lympho-haematopoietic parenchyma (Fig. 1b and c).
From 48 hpi, random foci of necrosis jointly with mild haemorrhagein the parenchyma of the liver and spleen were seen. A high number ofcirculating leukocytes as well as leukocytes adhered to the vascular
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endothelium were observed in small vessels from internal organs(Fig. 1d). Fibrin deposits were present in small vessels (hyaline throm-bi) and in the ventricular cavity of the spongy layer of the myocardium(mural thrombi) (Fig. 1d and e).
At 96 hpi, numerous Gram negative bacterial clusters were dissemi-nated on the serosal surface of coleomic organs and in the parenchymaand blood vessels of all sampled tissues (except the brain and gastroin-testinal tract) (Fig. 1f, g and h). The tissue surrounding bacterial coloniesshowed necrotic changes, particularly evident in kidney, liver, spleenand pancreas, while mild necrosis was detected in gills, thymus andgonads (Fig. 1 g and h). Strikingly, no evident leukocyte infiltrates asso-ciated with the microcolonies were detected.
Neither bacteria nor histological changes were detected in tissuesfrom control fish.
3.2. A. salmonicida isolation/identification
Themicrobiological analysis and Dot Blot assay in kidney and spleentissues allowed the detection of A. salmonicida from the challenged fishfrom 3 hpi to 96 hpi.
3.3. Distribution of A. salmonicida antigens
A. salmonicida antigenwas identified by IHC in different tissues fromchallenged fish at all sampling times. From 3 to 96 hpi, the bacterial an-tigen was present on the serosal surface of coelomic organs, not only inan extracellular position, but alsowithin themacrophages of the inflam-matory infiltrates (Fig. 2a). Inside macrophages, the immunoreactivityagainst A. salmonicida varied from small positive granules to diffuse im-munostaining (Fig. 2b and c).
From 6 hpi onwards, the A. salmonicida antigen was seen in thelumen of small blood vessels and capillaries, as well as associatedto the endothelium and occasionally in the subendothelial tissue(Fig. 2d). In the heart, strong positive reaction was observed in mono-cyte/macrophages in the lumen of cavities and related to the endocardi-al surface (Fig. 2e and f). In spleen, the bacterial antigen was identifiedinside the ellipsoidal sheath cells (Fig. 2 g). Positivity associated to ne-crotic foci was detected in kidney and liver from 24 and 48 hpi, respec-tively (Fig. 2 h).
At 96 hpi, the bacterial clusters were strongly positive forA. salmonicida (Fig. 2i). Immunoreactivity was also detected within thecytoplasm of large, mononuclear, macrophage-like cells, as well as ne-crotic/apoptotic cells, surrounding the bacterium colonies in kidney(Fig. 2j and k). In addition, the A. salmonicida antigen was observed inthe lumen of the blood vessels and perivascular tissue of the myoseptaof skeletal muscle.
In tissues from control fish no bacterial antigen was detected. In ad-dition, tissues from fish infected with T. maritimum and V. anguillarumdid not show immunoreactivity with anti-A. salmonicida serum.
4. Discussion
The present study describes the morphopathologic profile of theacute infection by A. salmonicida in turbot. In addition, the distributionand tissue localization of the bacterium were assessed by use of IHC.
The first microscopic changes corresponded to the proteinaceousmaterial seen in the coelomic cavity, which probably represented boththe injected suspension and the acute intracoelomic serous exudates.The development of coelomitis as well as the IHC detection ofA. salmonicida in the coelomic cavity was due to the route of injectionof the bacterium (Chaves-Pozo et al., 2005; Do Vale et al., 2002;Mutoloki et al., 2006; Weeks-Perkins and Ellis, 1995).
The main histological lesions consisted on generalized vascularchanges (intense vascular congestion, oedema,microvascular thrombo-sis, widespread haemorrhages and endothelial activation) as well asmultifocal coagulative necrosis of tissues affecting several organs. The
vascular disturbances, jointly with intravascular mobilization of leuko-cytes, and the spreading andmultiplication of the pathogen in target tis-sues are compatible with a septicaemia and systemic inflammatoryresponse (Do Vale et al., 2007; Peters et al., 2003; Remick, 2007;Slauson and Cooper, 2002). These findings were similar to thoseobserved in salmonid species infected with A. salmonicida and othersepticaemic diseases caused by Aeromonas hydrophila, atypicalA. salmonicida or V. anguillarum (Björnsdóttir et al., 2005; Burr et al.,2005; Ferguson, 2006; Noga, 2010; Rey et al., 2009; Roberts, 2012;Treasurer et al., 2007).
The early immunostaining of the bacteria in the lumen of blood ves-sels suggested that A. salmonicida, after body penetration, rapidlyreaches the bloodstream and from there, it spreads systemically as bac-terial emboli to the target organs. These results agree with studieswhere A. salmonicida was detected in several internal organs from 2 to12 h after exposition by bath challenge in Atlantic salmon and turbot(Farto et al., 2011; Svendsen et al., 1999).
The visualization of A. salmonicida antigen associated with bloodvessels and necrotic areas supports a strong relationship between le-sions and the presence of bacteria. Tissue necrosis probably resultedfrom cellular hypoxia due to vascular damage, as well as direct damagecaused by the action of toxins and enzymes secreted by A. salmonicida(Braun et al., 2002; Lago et al., 2012; Magnadóttir et al., 2002; Orozovaet al., 2009; Peters et al., 2003; Reith et al., 2008; Swain et al., 2008;Vanden Bergh et al., 2013a). The critical involvement of extracellularproducts in the development of lesions and clinical signs related toacute furunculosis has been reported in rainbow trout and Atlanticsalmon (Gunnlaugsdottir and Gudmundsdóttir, 1997; Lee and Ellis,1991; Orozova et al., 2009).
The observation of A. salmonicida inside the blood vessels of themyosepta at 96 hpi suggested the potential capacity of the bacteriumto invade the skeletal muscle, which could finally result in the charac-teristic focal necrotic–hemorrhagic lesions affecting the musculaturereported in salmonids (Bruno and Poppe, 1996; Diamanka et al.,2013; Young et al., 2009). Similarly, Farto et al. (2011) found thatA. salmonicida colonized the musculature of turbot at 7 days post-exposition to the pathogen (later stages of infection) by bathchallenge.
According to reports of acute furunculosis in salmonids, chal-lenged turbot also did not show inflammatory cell infiltrates aroundmicrocolonies (Bernoth, 1997). In this work, the apparent lack of in-flammatory cells surrounding bacterial colonies was likely a result ofthe rapid development of the septicaemic condition. On the otherhand, A. salmonicida possesses diverse virulence factors with immuno-suppressive properties, which may contribute to the relative absenceof inflammatory exudates since the septicaemia has been established(Dautremepuits et al., 2006; Ellis, 1991; Fast et al., 2009; Merino et al.,1994; Vanden Bergh et al., 2013b). However, the inflammatory infil-trates cannot be completely excluded in tissueswith abundant amountsof leukocytes such as the splenic or renal parenchyma.
The localization of the bacterial antigen in the cytoplasm of mono-cytes/macrophages indicates that these cells were involved in therapid uptake of the live bacteria. Taking into account that these phago-cytic cells are a component of the reticuloendothelial system (RES) inteleosts, thisfinding suggests their participation in the early immune re-sponse against A. salmonicida infection (Dalmoet al., 1996, 1997; EkmanandNorrgren, 2003; Zapata et al., 1996). These results are in accordancewith other studies that showed A. salmonicida components trapped inellipsoids of turbot and kidney macrophages in Atlantic salmon afterintracoelomic injection (Dalmo et al., 1998; Stensvåg et al., 1999).
In line with the removal function of pathogens, the diffuse intracel-lular immunostaining in the turbot phagocytes might be compatiblewith degraded bacterial components, while positive small granulesfound inside macrophages would represent clusters of recently phago-cytosed bacteria. This findingmay also indicate intracellular persistenceof the bacteria for at least 96 hpi. The microbiological analysis and Dot
Fig. 1. Histological changes in turbot challenged with Aeromonas salmonicida subspecies salmonicida, H–E staining. a, Spleen, reactive mesothelium surrounding the spleen parenchyma(S),withmarked hyperplastic proliferation and hypertrophy of individualmesothelial cells (arrowhead), 6 hpi. Bar=20 μm. b, Kidney, generalized peritubular haemorrhages (*) extend-ing into lympho-haematopoietic tissue, 24 hpi. Bar=100 μm. c, Kidney, coagulative necrosis of the haematopoietic tissue (arrow) accompanied by haemorrhages and oedema (*), 24 hpi.Bar = 100 μm. d, Liver, margination of leukocytes (arrows) and deposition of eosinophilic fibrinous-like material (arrowhead) within the lumen of the hepatic sinusoids, 48 hpi. Bar =25 μm. e, Heart, the ventricular cavity showed a fibrin thrombi adhered to the endocardium, containing enmeshed erythrocytes and leukocytes, 48 hpi. Bar = 25 μm. f, Heart, bacterialemboli in the lumen of the ventricular cavity (arrowhead). The bacterial colonies also colonized the myocardium (arrow), 96 hpi. Bar = 100 μm. g, Spleen, bacterial colonies (arrows)in the lumen of ellipsoids and necrosis of surrounding tissue (arrowheads), 96 hpi. Bar = 100 μm. h, Kidney, large bacterial colonies (arrows) associated with necrosis of thehaematopoietic tissue (arrowheads), 96 hpi. Bar = 100 μm. Inset: necrosis of tubular cells with pyknotic nuclei (arrow) adjacent to bacterial colony (arrowhead). Bar = 50 μm.
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Blot assay, which detected the bacterium after only 3 h of the infectionand up to 96 h post infection, supported this hypothesis. The capacity ofA. salmonicida strains for internalization and potential survival in fishmacrophages and other cells based on in vitro and in vivo studies hasbeen demonstrated (Daly et al., 1996; Fast et al., 2009; Garduño and
Kay, 1995; Garduño et al., 1992, 2000) and it was suggested thatA. salmonicida may use macrophages as a mechanism of evasion of theimmune response.
On the other hand, the A. salmonicida antigen observed within de-generate cells around the bacterial colonies in the kidney suggested
Fig. 2. Photomicrographs of immunohistochemistry for Aeromonas salmonicida subspecies salmonicida in challenged turbot. a, Bacteria were phagocytized bymacrophage-like cells locat-ed on the stomach serosa (st), 6 hpi. Bar = 50 μm. Insert: macrophage with strong immunostaining in the cytoplasm. Note the extracellular localization of free bacteria in the coelomiccavity (arrows). Bar= 2.5 μm. b and c, Macrophages showing a diffuse reactivity in the cytoplasm (b), while in others, the immunohistochemistry revealed the presence of spherical-liketo oval structures (arrows) in the cytoplasm (c), 96 hpi. Bar = 5 μm. d, Gill, strong positive reaction into the lumen of the blood vessels (arrows) of the secondary lamellae, 6 hpi. Bar =25 μm. e and f, Heart, single circulating monocyte (e) andmacrophages associated to endocardial lining (f) showing cytoplasmic immunoreactivity, 6 hpi. Bar= 25 μm. g, Spleen, evidentimmunostaining in the cytoplasm of the ellipsoidal macrophages (arrows), 6 hpi. Bar= 25 μm. h, Kidney, immunopositivity displaying spherical to ovoid shapes (short rods) disposed inshort chainswithin the necrotic tissue in the lympho-haematopoietic parenchyma, 48 hpi. Bar=10 μm. i, Liver, immunoreactive bacterial colony surrounded by necrotic tissue (*), 96 hpi.Bar=50 μm. j, Kidney,macrophage-like cells around the large bacterial colonieswithin the haematopoietic tissue, showing strong positive staining in the cytoplasm, 96 hpi. Bar=25 μm.k, Kidney, positive immunostaining in apoptotic/necrotic cells around the bacterial colonies (arrowheads), 96 hpi. Bar = 25 μm.
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that A. salmonicida may also induce necrosis of infected cells (VandenBergh et al., 2013a). This assumption is consistent with in vitro studiesthat demonstrate the capacity of A. salmonicida to induce cytopathic ef-fect in macrophages and other cells after internalization in them(Garduño and Kay, 1992; Garduño et al., 2000).
In summary, in this study, pathological findings showed that intra-coelomic injection of A. salmonicida in turbot resulted in a septicaemiacompatible with the acute form of furunculosis described in salmonids.Furthermore, IHC detection of A. salmonicida antigens demonstrated therapid blood distribution of the bacteria from the coelomic cavity to tar-get organs and the development of the lesions. On the other hand, cellsof RES were able to phagocyte the live bacteria, likely as part of themechanism of host innate resistance.
To our knowledge, this is the first description of the morpho-histological changes induced by A. salmonicida infection in turbot. More-over, we have standardized an IHC method to specifically identifyA. salmonicida in paraffin embedded tissues, which represents a newtool for the diagnosis of the disease. This method was found to be usefulfor identifying the pathogen from the first stage of the disease and there-by, differentiate acute A. salmonicida infection fromothers septicemic dis-orders. In addition, histopathology and IHC were especially valuable togain insight into some aspects of the pathogenesis of the acute diseasecaused by A. salmonicida in turbot.
Contributors
MIQ, RB and YS lead and supervised the study. YS also obtained andpurified A. salmonicida antibody and carried out the infectivity assays inturbot and the microbiological and Dot Blot analysis of fish tissues. APLand LF have collaborated in sampling and processing the tissues for lightmicroscopy studies. GC has contributed substantially processing sam-ples for immunohistochemistry study, taking microscopic images anddoing the analysis and interpretation of data. The paper was initiallywritten by GC and after that revised and corrected by MIQ, RB and YS.All authors read and approved the final version of the manuscript.
Acknowledgements
The authorswould like to thankMaría del CarmenCarreira and SandraMaceiras for their technical assistance. This work was funded by the Pro-jects from Xunta de Galicia (08MMA011200PR, PGIDT07MMA012CT andINCITE 09E2R208063ES).
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