Developmental and Comparative Immunology 36 (2012) 174–182

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Developmental and Comparative Immunology

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Infiltration and activation of acidophilic granulocytes in skin lesions of giltheadseabream, Sparus aurata, naturally infected with lymphocystis disease virus

B.S. Dezfuli a,⇑, A. Lui a, L. Giari a, G. Castaldelli a, V. Mulero b, E.J. Noga c

a Department of Biology and Evolution, University of Ferrara, St. Borsari 46, 44123 Ferrara, Italyb Department of Cell Biology and Histology, Faculty of Biology, University of Murcia, 30100 Murcia, Spainc Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, 4700 St. Hillsborough, Raleigh, NC 27606, USA

a r t i c l e i n f o

Article history:Received 31 May 2011Revised 29 June 2011Accepted 29 June 2011Available online 5 July 2011

Keywords:FishSkin diseaseIridovirusInnate immunityAcidophilic granulocytes

0145-305X/$ - see front matter � 2011 Elsevier Ltd.doi:10.1016/j.dci.2011.06.017

Abbreviations: LCDV, lymphocystis disease viruantimicrobial peptides; AGs, acidophilic granulocytesmicroscopy; EGCs, eosinophilic granule cells; IL-1b, in⇑ Corresponding author. Tel.: +39 0532 455701; fax

E-mail address: dzb@unife.it (B.S. Dezfuli).

a b s t r a c t

Light, ultrastructural and immunocytochemical investigations were carried out on the skin of giltheadseabream, Sparus aurata L., naturally infected with lymphocystis iridovirus, to assess pathology and hostcellular responses. Of 220,000 young seabream examined, 32,400 (14.7%) had clinical signs of lymphocys-tis and within 6 months of disease appearance, 45% of clinically affected fish had died. A subsample of 20S. aurata (80.0 ± 12.5 mm total length, mean ± S.D.), including 10 with lymphocystis on the skin and 10clinically normal, were examined via immunohistochemistry. Affected skin displayed macroscopic,wart-like clusters of hypertrophic fibroblasts which arose from the dermis and were covered by the epi-thelium. Clusters were encountered on the head, trunk and fins, but there was no evidence of viscerallymphocystis. The lymphocysts were surrounded by numerous granular cells that were positive for theantimicrobial peptide (AMP) piscidin 3 and underwent intense degranulation. To identify the type ofgranular cells involved in this viral disease, a double immunohistochemical staining with the monoclonalantibody G7 (mAb G7), which is specific for seabream acidophilic granulocytes (AGs), and with anti-histamine (as a marker for mast cells, MCs) was applied to the skin sections of the 10 clinically normalfish and 10 fish with lymphocystis. In infected skin, the number of G7-positive cells (i.e., AGs)(18.5 ± 10.5, mean number of cells per 20,000 lm2 ± S.D.) was significantly higher compared to their den-sity in uninfected skin (1.4 ± 2.2) (t test, p < 0.01). Notably, the AGs that infiltrated the skin lesions ofinfected animals were found to be degranulated and to produce the pro-inflammatory cytokine interleu-kin-1b. No histamine-positive granular cells (i.e., MCs) were encountered in the lymphocystis lesions. Thepresent study shows the response of skin to lymphocystis disease virus (LCDV) and provides evidencethat AGs, but not MCs, are recruited and activated in response to this skin infection.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Lymphocystis disease virus (LCDV) is a member of Iridoviridaefamily and infects at least 125 different fish species (Anders,1989; Cano et al., 2009a). Lymphocystis disease primarily affectsfibroblasts in the dermal connective tissue, causing papilloma-likehypertrophy or nodular masses (Samalecos, 1986). Nodules may besolitary or clustered, either on the skin or very rarely in the internalorgans. Each nodule is an individual, infected host cell called a lym-phocystis cell or lymphocyst (González de Canales et al., 1996;Paperna et al., 1982). Lymphocystis is usually self-limiting, with

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s; MCs, mast cells; AMPs,; TEM, transmission electronterleukin-1b.: +39 0532 455715.

the lesions resolving spontaneously and the host usually recover-ing within a few weeks (Paperna et al., 1982; Roberts, 1976). How-ever, infections can be fatal if complicated by secondary bacterialor water mold infections. For example, 90% mortality was recentlyreported in olive flounder Paralichthys olivaceus with lymphocystis(Harikrishnan et al., 2010). While the specific cause of the mortal-ities was not determined, it was most likely due to secondarymicrobial infections since uncomplicated lymphocystis lesionsare usually self-limiting.

In gilthead seabream, lymphocystis was first reported in RedSea fish from Israel (Paperna et al., 1982); it was later observedin this species in France (Le Deuff and Renault, 1993) and Spain(Borrego et al., 2001; Cano et al., 2006, 2009a,b; González deCanales et al., 1996; Sarasquete et al., 1998). While Sparus auratais the most important cultured marine fish in Italy, with 6800 ton-nes being produced in 2009 (see MIPAF, 2010), there is only oneprevious report of lymphocystis in this species in Italy (Masoeroet al., 1986).

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The initial response of a host in defending against a pathogen isvia innate immunity (Beutler, 2004). Mast cells (MCs) occur in skin,gills, and digestive tract of several species of fish, and changes intheir number have been observed in response to pathogen (espe-cially parasite) invasion (Alston and Lewis, 1994; Andrews et al.,2010; Dezfuli et al., 2003, 2008). Mast cells have been recognizedas important components of non-specific immune defence in manyvertebrates (Colorni et al., 2008; Jia et al., 2000; Silphaduang andNoga, 2001).

Mast cells and acidophilic granulocytes (AGs) of fish oftenexpress antimicrobial peptides (AMPs) (Mulero et al., 2008a). Anti-microbial peptides are host defense effector molecules that occurin virtually all life forms and are a key component of innate defense(Zasloff, 2002). The piscidin family of AMPs have potent, broad-spectrum antibacterial, antifungal, antioomycete and antiparasiticactivity (Andrews et al., 2010; Colorni et al., 2008; Dezfuli et al.,2010; Mulero et al., 2008a; Salger et al., 2011; Zahran and Noga,2010). In addition, they are active against certain viral infections(Chinchar et al., 2004). Piscidins are present in MCs of wide rangeof teleost taxa, including the families Moronidae, Sciaenidae,Siganidae, Belontidae, Cichlidae, Percichthyidae and Latridae(Andrews et al., 2010; Corrales et al., 2010; Dezfuli et al., 2010;Mulero et al., 2008a; Salger et al., 2011; Silphaduang and Noga,2001; Silphaduang et al., 2006).

In the gilthead seabream, acidophilic granulocytes are function-ally equivalent to the neutrophils of higher vertebrates. The acido-philic granulocytes (AGs) of the teleost gilthead seabream, despitetheir staining pattern, display similar functions to human neutro-phils. For example, they are the most abundant circulating granu-locytes and are recruited rapidly from the head-kidney to theinfection site (Chaves-Pozo et al., 2005; López-Ruiz et al., 1992;Meseguer et al., 1993, 1994). In addition, their main role is thephagocytosis of bacteria, which is accomplished by coordinatingtheir attachment and internalization of bacteria to their releaseof reactive oxygen intermediates into the phagocytic vacuole(Sepulcre et al., 2002). Notably, AGs seem to be involved in the reg-ulation of the inflammatory response, since they are able to pro-duce interleukin-1b upon in vitro activation (Sepulcre et al.,2007) as well as in vivo following bacterial infection (Muleroet al., 2008a). In addition, AGs of gills, intestine and head kidneyfrom both clinically normal gilthead seabream and individualsexperimentally infected with Vibrio anguillarum contain piscidins,which are released into the bacteria-containing phagosome uponphagocytosis (Mulero et al., 2008a). It is now well-known that inthe order Perciformes, histamine is stored in MCs, is biologicallyactive, and can regulate inflammation by acting on AGs (Muleroet al., 2007). As both AGs and MCs of gilthead seabream have eosin-ophilic granules, G7 and anti-histamine antibodies have been usedto unequivocally distinguish AGs (G7+/histamine�) from MCs (G7�/histamine+) (Mulero et al., 2007).

The main purpose of our study was to examine the dermal re-sponse of S. aurata to lymphocystis. Evidence is presented for therole of piscidin 3-expressing AGs in response to the infection, aswell as evidence of degranulation of AGs in the lesions. We also re-late observations made on histopathology and ultrastructure of theimmune cells in infected skin.

2. Material and methods

2.1. Animals

Gilthead sea bream were obtained from a local semi-intensivefish farm, ‘‘Valle Ca’ Zuliani’’ (Pila di Porto Tolle, Rovigo, Italy) inOctober 2008. On this site, sea bream are artificially spawned inthe hatchery and the fry are grown in indoor concrete tanks. A sub-

sample of 20 fish (80.0 ± 12.5 mm total length, mean ± S.D.;7.4 ± 1.7 g, mean ± S.D.), including 10 with lymphocystis on theskin and 10 clinically normal, were transported live to the labora-tory where they were anesthetized using MS222 (Sandoz, Basel,Switzerland) and observed macroscopically with the aid of stere-omiscroscope. Skin, gills, and internal organs (spleen, liver, kidney,gonads) were examined in search of lymphocystis. Tissues of theabove 20 fish were used for histopathological, immunohistochem-ical and ultrastructural examination.

2.2. Histology and electron microscopy

The spinal cord was severed and skin samples were fixed in cold(4 �C) Bouin’s fluid for 8 h. The samples were then paraffin-embed-ded following standard procedures and 5 lm sections were stainedwith either haematoxylin-eosin, Alcian Blue-PAS, Giemsa, or Gramfollowing Giunti (Gram Kit for identification of bacteria in histolog-ical sections, Code 010221, Diapath, Martinengo, Italy). For lightand electron microscopy, pieces of skin less than 7 mm in diameterwere fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 3 hat 4 �C before post-fixing them in 1% osmium tetroxide in the samebuffer for 3 h. The specimens were dehydrated through a gradedacetone series before being embedded in Epoxy resin (DurcupanACM, Fluka). Semi-thin sections (1.5 lm) were cut on a ReichertOm U 2 ultramicrotome using glass knives and then stained withtoluidine blue. Ultra-thin sections (90 nm) were stained with a4% uranyl acetate solution in 50% ethanol and Reynold’s lead cit-rate and examined using a Hitachi H-800 electron microscope.

Light photomicrographs were taken using a Nikon Eclipse 80imicroscope (Nikon, Tokyo, Japan). Images of skin sections showingthe distribution of lymphocystis lesions, their dimensions, and thethickness of their capsules (40 lymphocystis cells total) were ob-tained using computerized image analysis software (Nis ElementsAR 3.0, Nikon, Tokyo, Japan).

2.3. Immunohistochemistry (IHC)

Anti-piscidin 3 (anti-HAGR) antibody was produced by a com-mercial laboratory (Bethyl Laboratories, Montgomery, TX) usingthe company’s standard procedures (detailed in Dezfuli et al.,2010). Briefly, a synthetic peptide constituting the C-terminus ofpiscidin 3 (HAGRSIGRFLTG) was conjugated to keyhole limpethemocyanin (KLH) and then injected into rabbits. The antiserumwas then affinity purified by running over a column having the12-mer piscidin fragment conjugated to cyanogen bromide-activated agarose as an immunosorbent. The resulting titer of theaffinity-purified antibody was 1:13,000 via ELISA. The peptide-specific antibody had less than 1% cross-reactivity by ELISA, where1% cross-reactivity is 100 times more antibody than is required toproduce the same optical density with either free KLH, conjugatedKLH, or free peptide that shares less than three amino acids in thesequence. Piscidin 3-positive cells were detected in skin histologi-cal sections using an indirect immunohistochemical method.Briefly, sections (5 lm) were deparaffinised in xylene, rehydrated,then non-specific staining was blocked in goat normal serum(1:20) (30 min). After incubation with the primary antibody(anti-HAGR diluted 1:400) for 3 h at room temperature, sectionswere incubated for 1 h at room temperature with an anti-rabbitIgG (whole molecule)-alkaline phosphatase antibody produced ingoat (Sigma) diluted 1:50. The alkaline phosphatase activity wasrevealed by incubation for 5 min at room temperature with FastRed TR/Naphthol AS-MX (Sigma). The sections were mounted inglycerine and immediately examined and photographed. Non-immune rabbit serum and diluent-only sections were used as neg-ative controls. Positive control tissue was striped bass (Moronesaxatilis) intestine.

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Other sections were subjected to a double IHC method using theG7 mouse monoclonal antibody, which is specific to sea breamAGs, together with a commercial rabbit polyclonal antibodyagainst histamine (Sigma). After deparaffinisation and rehydrata-tion of sections, the endogenous peroxidase activity and non-spe-cific staining were blocked, respectively in 3% H2O2 (10 min) andin goat normal serum (1:20) (30 min). The slides were incubatedwith the G7 antibody (diluted 1:100) for 2 h at room temperatureand then for 1 h with an anti-mouse IgG (whole molecule)-alkalinephosphatase antibody produced in goat (Sigma) diluted 1:50. Thealkaline phosphatase activity was revealed by incubation for5 min at room temperature with Fast Red TR/Naphthol AS-MX (Sig-ma). The same sections were then incubated with the anti-hista-mine primary antibody (diluted 1:100) for 1 h at roomtemperature. After washing with PBS, slides were incubated for30 min with biotinylated goat anti-rabbit serum (Vector, Burlin-game, USA) followed by avidin-conjugated horseradish peroxidase(Vector, Burlingame, USA). The enzyme activity was detected usingDAB (3,30-diaminobenzidine). The slides were mounted in glycer-ine and immediately examined and photographed. Positive controltissues were rat stomach (as indicated by the data sheet of theantibody) and gills of S. aurata on which this antibody was success-fully tested by Mulero et al. (2007).

Another double IHC was performed using the G7 mouse mono-clonal antibody and a monospecific rabbit polyclonal antibodyraised against the C-terminal of gilthead seabream interleukin-1b(Mulero et al., 2008a,b). Sections were exposed to the G7 mAb asdescribed above. After identification of the G7 positive cells inslides mounted in glycerine (immediately examined and photo-graphed), a second immunostaining reaction was performed onthe same sections, which were then incubated with a 1:100 dilu-tion of anti-ILl-1b antibody for 1 h at room temperature. After that,the sections were exposed for 30 min to biotinylated goat anti-rab-bit serum (Vector, Burlingame, USA) followed by avidin-conjugatedhorseradish peroxidase (Vector, Burlingame, USA). The peroxidaseactivity was revealed by incubation for 5 min at room temperaturewith DAB or with the cromogen VIP (Code SK-4600 Vector, Burlin-

Fig. 1. Skin of Sparus aurata. (a) Fish with lymphocystis nodules on the caudal fin (thick athrough uninfected skin; note the integrity of the dermis (thick arrow) and epithelium (E)enlarged, virus-infected, dermal fibroblasts (asterisks), with an undisplaced nucleus (cepithelium (E); Haematoxylin and Eosin, bar = 50 lm. (d) Intense host cellular reaction ngranular cells stained eosinophilic (thin arrows), Haematoxylin and Eosin, bar = 10 lm.

game, USA). Finally, the sections were dehydrated (the red stainingfrom the alkaline phosphatase activity is lost after dehydration),mounted in Canada balsam, re-examined and photographed.

2.4. Statistical analysis

The number of cells positive for G7 (all of these cells were alsopositive for piscidin 3) was counted in the skin via light micros-copy, using computerised image analyser software (Nis ElementsAR 3.0). For comparative purposes, the cells were screened at400x magnification in one randomly selected area (20.000 lm2)for each fish of the two groups: clinically normal fish (n = 10)and fish with lymphocystis (n = 10). A two-tailed t-test was per-formed to determine if there was a significant difference in thenumber of G7-positive cells between the clinically normal versuslymphocystis-affected fish. A p < 0.01 level of significance wasselected.

3. Results

Investigation of a semi-intensive fish farm during September2008 showed that 32,400 of 220,000 (14.7%) gilthead seabream heldin concrete tanks had lymphocystis infection on the body and fins.According to farm veterinarian report, six months after the initialexamination, 14,580 of the 32,400 affected fish (45%) had died. Nonecropsies were performed on these fish, so the specific cause ofdeath was unknown; however, in most survivors, the skin lesionswere not fully healed and scar tissue remained. In 50 fish withlymphocystis on the skin, there were no visible lesions on eitherthe gills or internal organs (liver, kidney, spleen, intestine).

Lymphocystis presented as extensive nodules on the pectoral,dorsal and caudal fins, as well as on the operculum, around theeyes and on the trunk (Fig. 1a). The nodules typically coalesced intolarge masses of hypertrophied cells and had a granular appearance(Figs. 1a and 2a,c). In contrast with clinically normal skin (Fig. 1b),lymphocystis-affected dermis had hypertrophic (lymphocystis)cells which occupied a prominent portion of the dermis (Fig. 1c)

rrow) and lateral side of the body (thin arrows), bar = 6 mm. (b) Histological section; Haematoxylin and Eosin, bar = 50 lm. (c) Lymphocystis lesions showing massivelyurved arrow) with nucleolus, irregular inclusions (arrowheads), and capsule (C);ear a lymphocysts’s hyaline capsule (thick arrows); note the presence of numerous

Fig. 2. Lymphocystis in the skin of Sparus aurata. (a) Histological section through four nodules (arrows), each composed of multiple lymphocysts; Haematoxylin and Eosin,bar = 200 lm. (b) High magnification of a nodule with several lymphocysts (asterisks), Haematoxylin and Eosin, bar = 50 lm. (c) AGs (arrows) near a dermal capillary, withone cell in the lumen (arrowhead); asterisk shows a lymphocystis cell. Haematoxylin and Eosin, bar = 10 lm. (d) Intact AGs (arrows) near a lymphocyst (asterisk) and intensedegranulation of other AGs (curved arrows), Haematoxylin and Eosin, bar = 10 lm.

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and altered its integrity, especially in severe infections (Fig. 2a andb). Lymphocystis cells had an intact capsule with a large, roundnucleolus, intracytoplasmic inclusions, and ‘‘ground-glass’’ cyto-plasm. The major axis of the lymphocystis cells ranged from112.9 to 311.6 lm (229.1 ± 45.1 lm, mean ± S.D.); the minor axisranged from 75.5 to 237.9 lm (154.2 ± 42.2 lm, mean ± S.D.) (Figs.1c and 2b). Sub-peripheral cytoplasm contained globular inclu-sions and was filled with granules or aggregates of different sizes.The cytoplasm outside the inclusion was densely packed with virusparticles; indeed, the cytoplasm had a uniformly granular texture(Figs. 1c and 2b). Lymphocystis cells were surrounded by a hyalinecapsule, and the thickness of the capsule ranged from 1.3 to 9.6 lm(4.5 ± 2.3, mean ± S.D.) (Fig. 1c and d).

In the sections of skin with lymphocystis stained with the Grammethod, Gram-positive bacteria (bacilli and cocci) were present inthe mucus and in the epidermal surface, whilst near the viral le-sions no bacterial proliferation was noticed (data not shown).

Hypertrophic cells were surrounded by dermal connective tis-sue; accordingly, a large number of irregularly-shaped granularcells stained eosinophilic (Figs. 1c,d and 2c,d). Moreover, thesecells were close to and inside the capillary (Fig. 2c) or within theaffected dermis (Fig. 2d). Lymphocystis lesions treated with anti-piscidin 3 antibody showed immunopositive cells close to the lym-phocystis cells (Fig. 3a and b). Piscidin-positive cells were alsoadjacent to capillaries and in some instances, within them(Fig. 3c and d). Serial sections treated with double IHC (G7/hista-mine) revealed that the piscidin 3-positive granular cells aroundthe lymphocystis cells were G7-positive (which is specific for sea-bream AGs) and negative for histamine (which is typically ex-pressed by MCs of perciform fish) (Mulero et al., 2007) (data notshown). In affected skin, the number of AGs (G7-positive cells)(18.5 ± 10.5, mean ± S.D., N = 10) was significantly higher in com-parison to clinically normal skin (1.4 ± 2.2, mean ± S.D., N = 10)(t-test, p < 0.01). AGs were observed within the thickness of thedermis very close to the lymphocystis cells (Fig. 4a–d). In some in-stances, AGs appeared to encircle the lymphocystis cells (Fig. 4b),or were on one side of the infected cell (Fig. 4c). Using a double

IHC method with the G7 antibody together with anti-IL-1b, itwas observed that a few acidophilic granulocytes (G7-positivecells) around the lymphocysts were immunostained with theanti-IL-1b (Supplementary Fig. S1).

Via transmission electron microscopy (TEM), the cytoplasm oflymphocysts was filled with viral particles (Fig. 5a), which hadan elongated, hexagonal profile (Fig. 5b). The capsid was very thinand had two adherent osmophilic layers separated by a very nar-row, electron-lucent zone (not shown). The cytoplasm was in anadvanced stage of degeneration, reduced to an electron-lucent ma-trix with membranous and other less definable residues (Fig. 5aand c). Mitochondria, rough endoplasmic reticulum, ribosomes,and vacuoles were present mainly in the periphery of the cell(Fig. 5a and c). The cytoplasmic border was deeply indented(Fig. 5d).

The cellular response to the infection mainly involved AGs,which via TEM appeared as large cells having an irregular outlineand a cytoplasm filled with many electron-dense granules(Fig. 6a), major axis (6.84 ± 1.02 lm, mean ± S.D, n = 30), minoraxis (5.03 ± 0.79 lm, mean ± S.D, n = 30). The AGs were often adja-cent to infected cells and/or capillaries (Fig. 6b). The electron-dense granules were round to oval, and filled with a round, elec-tron-dense core (Fig. 6c). The AG cytoplasm had numerous freeribosomes and well-developed rough endoplasmic reticulum; thelatter was often in close contact with numerous electron-densegranules (Fig. 6d).

Presumptive degranulation of AGs was visible by light micros-copy (Fig. 2d) and was confirmed by TEM (Fig. 6b); AGs exhibitedhigher rates of degranulation when adjacent to or within capillar-ies near lymphocystis cells (Fig. 2d).

4. Discussion

Understanding the gilthead seabream’s immune response tolymphocystis could allow a better assessment of not only hostdamage but also provide a better understanding of how this fish

Fig. 3. Lymphocystis virus-infected S. aurata skin treated with anti-piscidin 3 antibody. (a) Localization of piscidin 3 in infected skin. Positive AGs (arrows) are visible nearlymphocysts (asterisks); epithelium (E), bar = 20 lm. (b) High magnification of Fig. 2a showing immunopositive AGs (arrows) near lymphocysts (asterisk), which appear asbig size cells; epithelium (E), bar = 10 lm. (c). Immunopositive AGs (arrows) within dermal connective tissue near a capillary (asterisks); epithelium (E), bar = 10 lm. (d)Positive AGs (thick arrows) are inside a capillary (asterisk) and others (arrows) are nearby; epithelium (E), bar = 10 lm.

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defends itself against this pathogen, so that potential mitigationstrategies may eventually be developed. The proliferation of mac-rophages and epithelioid cells around lymphocystis cells has beenpreviously described as an immune response to LCDV in plaice,Pleuronectes platessa (Roberts, 1976), in Sciaenops ocellatus (Colorniand Diamant, 1995) and in the sea bass, Sebastes schlegeli (Shenget al., 2007). The present investigation was focused on the skin ofS. aurata naturally infected with lymphocystis iridovirus. Althoughit was not possible to determine the stage of the infection of eachspecimen, the data reported here are novel and provide new in-sights into the response of innate immune cells to lymphocystis.

Lymphocystis is usually a self-limiting disease; the lesions gen-erally disappear and fish recover after several weeks (González deCanales et al., 1996; Paperna et al., 1982; Roberts, 1976). But, veryrecently, 90% mortality was observed in olive flounder with lym-phocystis. While fish treated with Jenoclean had much lower mor-tality (Harikrishnan et al., 2010), this was probably due to thecontrol of secondary microbial infections. The integumental tissuesare important as a primary site of attachment and replication ofviruses in susceptible fish; at the same time, the integument acts

as a barrier to infection in resistant fish (Dorson and Torchy,1993). Under normal (unstressed) conditions, the fish maintainsa healthy state by defending itself against potential invaders by acomplex system of innate defence mechanisms (Ellis, 2001).

Innate immunity involves various cell types, and one of themost common is the mast cell (Murray et al., 2003). A detailed re-view of piscine mast cell morphology, staining properties, func-tional responses and relationship with eosinophilic granule cells(EGCs) was provided in Reite and Evensen (2006). The designationeosinophilic granule cell was introduced by Roberts et al. (1971)but there has been a tendency in recent years to use the term mastcell because these cells have functional and morphological similar-ities to mammalian MC. In fact, the EGCs described in tissues ofmany species of teleostean fish (e.g., cyprinids and salmonids)are actually MCs and can be placed in MC categories based on samecriteria accepted for mammalian MCs (Reite, 1998).

In fish, different type of cells are involved in phagocytic defence,but, the specific lineage/identity of these cells is not clearly estab-lished because first, there is lack of specific cell markers, and sec-ond, there is considerable morphological heterogeneity among

Fig. 4. Lymphocystis virus-infected S. aurata skin treated with monoclonal antibody G7 (mAb G7). (a) Immunopositive AGs (arrows) near lymphocysts (asterisks); epithelium(E), bar = 50 lm. (b) Positive AGs (arrows) near lymphocysts (asterisk), bar = 20 lm. (c) High magnification of a lymphocyst (asterisk) with several adjacent immunopositiveAGs (arrows); bar = 20 lm. (d) Positive AGs (arrows) within the dermis of infected skin; epithelium (E), bar = 50 lm.

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leucocytes, especially granulocytes (Mulero et al., 2008a; Sepulcreet al., 2002). The AGs of gilthead seabream are abundant in im-mune tissues and can phagocytize the fish-pathogenic bacteriumV. anguillarum (Sepulcre et al., 2002). AGs may be functionallyequivalent to the neutrophils of higher vertebrates (Chaves-Pozoet al., 2005; López-Ruiz et al., 1992; Meseguer et al., 1993, 1994;Sepulcre et al., 2002). In our study, the granular cells surroundingthe lymphocysts were positive for G7 and negative for histamine,which defines them as AGs (Mulero et al., 2007), and some of theseAGs appeared to contain IL-1b. The low number of IL-1b-positiveAGs in infected sea breams could be explain by the fact that thisprotein is rapidly release upon production (Olavarría et al., 2010).In fact, a similar low number of AGs were immunostained withthe anti-IL-1b antibody in V. anguillarum-infected sea bream (Mule-ro et al., 2008b). It was suggested that AGs are involved in the reg-ulation of the inflammation through the production of IL-1b uponactivation following infection (Sepulcre et al., 2007; Mulero et al.,2008a). In addition, we observed AGs in perivascular sites andwithin capillaries. The close relationship between AGs and the cir-culatory system of gilthead seabream also suggests that theirmigration through blood vessels might function in regulating the

inflammatory response of skin immune cells against LCDVinfection.

Many AMPs have been isolated from fish (Bao et al., 2006;Chang et al., 2005; Corrales et al., 2010; Smith and Fernandes,2008) and they appear to have a significant role in protecting fishfrom many infectious diseases (Corrales et al., 2010; Silphaduangand Noga, 2001). AMPs are increasingly recognized as a critical firstline defence against pathogens (Fernandes et al., 2010; Smith et al.,2008). A major category of AMPs in fish are piscidins (Silphaduangand Noga, 2001; Colorni et al., 2008; Corrales et al., 2010; Salgeret al., 2011) which have potent, broad-spectrum antiviral (Chin-char et al., 2004), antibacterial (Mulero et al., 2008a), antifungal(Sung et al., 2008), antioomycete (Zahran and Noga, 2010) andantiparasitic (Andrews et al., 2010; Colorni et al., 2008; Dezfuliet al., 2010) activity. Interestingly, piscidins have been identifiedin MCs of several fish species (Corrales et al., 2010; Dezfuli et al.,2010; Lauth et al., 2002; Patrzykat et al., 2003; Silphaduang andNoga, 2001; Silphaduang et al., 2006; Sun et al., 2007) and inAGs of gilthead seabream (Mulero et al., 2008a). Piscidin 3 is deliv-ered to the bacteria-containing phagosome of AGs upon phagocy-tosis; this finding suggests a dual role for these AMPs (i.e., in the

Fig. 5. Transmission electron micrographs of lymphocystis in the skin of S. aurata. (a) TEM of virus particles in the cytoplasm of a lymphocystis virus-infected skin cell; roughendoplasmic reticulum (arrow) and membranous residues (arrowheads); bar = 0.6 lm. (b) High magnification of the elongated, hexagonal profile of virus particles (arrows);note the capsid around each particle; bar = 0.1 lm. (c) Cytoplasm of an infected cell, with mitochondria (curved arrows), rough endoplasmic reticulum (arrows) andmembranous residues (arrowheads); bar = 0.5 lm. (d) Periferal region of a lymphocyst, with indented cytoplasm (thick arrows), hyaline cell wall (asterisk) and skin tissue(arrow); bar = 2.8 lm.

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killing of both extracellular and intracellular pathogenic bacteria)(Mulero et al., 2008a). Moreover, it is striking that histamine wasfound only in MCs of perciform fish (Mulero et al., 2007).

Several investigations have showed that piscine MCs degranu-late in response to a variety of known degranulating agents andpathogens, with release of their contents (Dezfuli and Giari,2008; Dezfuli et al., 2008; Ellis, 1985; Mulero et al., 2007; Murrayet al., 2007; Vallejo and Ellis, 1989). But, there is no previous recordof degranulation of AGs. We observed intense degranulation ofAGs; the AGs exhibited higher rates of degranulation in perivascu-lar sites and/or inside capillary vessels in proximity to lympho-cysts. This observation, together with the production of IL-1b,suggests the activation of these cells by LCDV and the possibleinvolvement of fish granulocytes in antiviral responses. This isnot surprising, since AGs are armed with different effector mole-cules, such as piscidin 3, which displays potent antiviral activity(Chinchar et al., 2004). In addition, mammalian neutrophils canalso release their granular content to the extracellular milieu (Lacyand Eitzen, 2008), where they play an essential role in the directkilling of bacteria and in the recruitment and activation of macro-phages and dendritic cells (Soehnlein et al., 2009). Whether a sim-

ilar crosstalk between AGs and macrophages exists in the giltheadseabream is unknown, but it would be worthy of study in order tounderstand the coordinated action of different innate immune celltypes to infectious pathogens in fish. Piscidins have been previ-ously shown to have potent antiviral activity over a wide temper-ature range that is physiologically relevant for a poikilothermicanimal. In this regard, they differ from mammalian AMPs such asdefensins, which only function within a much more narrow tem-perature range (Chinchar et al., 2004). As is true for most antiviralAMPs, activity is strongest against enveloped viruses. Potent anti-viral activity was demonstrated against a herpesvirus, channel cat-fish virus, while activity against a non-enveloped virus (frog virus3, an iridovirus) was less strong (Chinchar et al., 2004). Since LCDVis a non-enveloped virus, it is uncertain as to whether piscidins aredirectly toxic to this pathogen. It might be possible that an anti-lymphocyst response (toxicity by piscidins) might be modulatedvia recognition of these cells as ‘‘foreign’’ rather than directly inac-tivating the virus. In some affected skin sections, we noticed a fewcells which seemed to be necrotic, but this will require furtheranalysis. In addition, it remains to be determined whether thestrong degranulation of AGs and the release of IL-1b in

Fig. 6. Transmission electron micrographs of lymphocystis virus-infected skin cells. (a) Some AGs (arrows) in the vicinity of a lymphocyst (not shown); bar = 1.9 lm. (b) TwoAGs (thin arrows) in close proximity to a capillary (thick arrows) in the dermal interstitium; degranulation (curved arrows) is present; bar = 2.0 lm. (c) High magnification ofelectron-dense granules (arrows) of an AG; note the round, electron-dense core inside each granule; N = nucleus, bar = 0.2 lm. (d) Enlarged view of AGs showing well-developed rough endoplasmic reticulum (curved arrows) in contact with electron-dense granules (thin arrows); abundant free ribosomes (thick arrows) are visible;N = nucleus, bar = 0.2 lm.

B.S. Dezfuli et al. / Developmental and Comparative Immunology 36 (2012) 174–182 181

lymphocysts contributes to viral clearance/lesion healing or, alter-natively, if it further promotes skin lesions. Whatever the case, ourdata pave the way for future studies aimed at determining the roleplayed by fish professional phagocytic granulocytes in the antiviralresponse of this group of animals.

Acknowledgments

We are indebted to Silvia Fabbri from providing fish, AndreaMargutti and Samantha Squerzanti from the University of Ferrarafor technical assistance. This study was supported by grants fromthe Italian Ministry of the University and Scientific Research andTechnology and the Spanish Ministry of Science and Technology(Grant BIO2008-01379).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.dci.2011.06.017.

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