Characterisation of the intracellular protozoan MPX in Scottish mussels, Mytilus edulis Linnaeus, 1758.

Ciliates have been reported as pathogens of many species of economically important bivalves. Mussel protozoan X (MPX), is an uncharacterised intracellular ciliate of mussels and has been widely reported in Mytilus spp. around the world. In order to characterise this ciliate, Mytilus edulis samples were collected from a site on the West coast of Scotland, and four different fixatives for histological examination were tested. Fresh preparations of mussel digestive glands were also examined by laser scanning confocal microscopy. Intracellular ciliates were prepared by laser capture microdissection and partial sequences of small subunit ribosomal RNA gene and of large subunit ribosomal RNA gene were generated, using Phyllopharyngea primers. Methacarn solution proved to be the best fixative for both histological and molecular characterisation. The morphological and molecular investigations confirmed that this ciliate belongs to the class Phyllopharyngea, order Rhynchodida. However, this organism does not belong to any known family, genus or species, therefore, a new description is necessary, following further morphological analyses. Most mussel samples containing MPX displayed mild to moderate infections, with no signs of necrosis or haemocytic response, although a single sample displayed a severe infection (∼103 ciliates per section). The localisation of this ciliate in tissues other than the digestive gland, the presence of necrosis in infected tissue of the most severely infected mussel and the binary fission of this ciliate have been observed here for the first time. We also report the first observation of the live ciliate isolated from tissue. Although MPX remains of unknown significance to the mussel industry, tools and protocols described here will be useful in further characterising these and other ciliates (subclass Rhynchodia) known as pathogens for bivalves.

A novel use of social media to evaluate the occurrence of skin lesions affecting wild dusky grouper, Epinephelus marginatus (Lowe, 1834), in Libyan coastal waters.

The social media network Facebook™ was used to gather information on the occurrence and geographical distribution of dusky grouper dermatitis, a skin lesion affecting the dusky grouper, Epinephelus marginatus. Dusky grouper are common targets for spear fishermen in the Mediterranean and by monitoring spearfishing activity in Libyan waters, it was possible to document skin lesions from their entries on Facebook. Thirty-two Facebook accounts and 8 Facebook groups posting from 23 Libyan coastal cities provided a retrospective observational data set comprising a total of 382 images of dusky grouper caught by spearfishing between December 2011 and December 2015. Skin lesions were observable on 57/362 fish, for which images were of sufficient quality for analysis, giving a minimal prevalence for lesions of 15.75%. Only dusky grouper exceeding an estimated 40 cm total length exhibited lesions. The ability to collect useful data about the occurrence and geographical distribution of pathological conditions affecting wild fish using social media networks demonstrates their potential utility as a tool to support epidemiological studies and monitor the health of populations of aquatic animals. To our knowledge, this represents the first time that such an approach has been applied for assessing health in a wild population of fish.

Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology.

Infectious salmon anaemia virus (ISAV) is an orthomyxovirus that has had a significant economic impact on Atlantic salmon farming in Europe, North America and Chile. Monoclonal antibodies (mAbs) were developed against Segment 3 (encoding the viral nucleoprotein, NP) of the virus. Six of the mAbs were shown to be specific to ISAV and recognised all isolates from Scotland, Norway and Canada. They reacted with ISAV in enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody technique (IFAT) and western blotting. They were also used to develop a novel detection method based on Luminex (Bio-Plex) bead-based flow cytometric technology for the detection of ISAV in the plasma of Atlantic salmon (Salmo salar L.) smolts experimentally infected with ISAV. Fish were challenged by intraperitoneal (i.p.) injection of virus at 50% Tissue Culture Infective Dose (TCID50) = 2.8 x106 per animal. Virus present in plasma of infected fish, collected at 0, 4, 8, 12, 16, 21 and 28 days post infection using a non-lethal sampling method (n = 12 at each time point), was quantified using the optimised Bio-Plex assay. The results obtained with this assay were compared with absolute quantification of the virus by RT-qPCR using SYBR Green I and TaqMan chemistries. The Bio-Plex assay developed using the NP mAbs appears to be a rapid, sensitive method for detecting and quantifying ISAV in small volumes of fish plasma and has the potential to be multiplexed for the detection of other fish pathogens (e.g. during co-infections). To our knowledge this is the first report of the use of Luminex (Bio-Plex) technology for the detection of a fish pathogen.

Ulcerative dermatitis in wild dusky grouper Epinephelus marginatus (Lowe) from Libyan waters.

In the period 2013-2015, wild dusky grouper, Epinephelus marginatus (Lowe), caught in Libyan coastal waters and ranging in size from 42 to 92 cm in total length, were observed to have distinctive skin lesions of unknown aetiology. Histopathologically, the lesions comprised a multifocal, unilateral or bilateral dermatitis, involving the epidermis, superficial dermis and scale pockets, and sometimes, in severe cases, the hypodermis. Severe lesions had marked epidermal spongiosis progressing to ulceration. Healing was observed in some fish. Bacteria and fungi could be isolated from severe lesions, although they were not seen histopathologically in early-stage lesions. By contrast, metazoan parasite eggs were observed in the dermis and epidermis of some fish with mild and moderate dermatitis. Unidentified gravid digenean trematode parasites carrying similar eggs were also seen within the blood vessels of the deep and superficial dermis. The cause of this distinctive condition, termed dusky grouper dermatitis (DGD), and its potential impact upon already threatened Mediterranean wild dusky grouper populations and upon cultured grouper more widely have yet to be established.

Economic costs of protistan and metazoan parasites to global mariculture.

Parasites have a major impact on global finfish and shellfish aquaculture, having significant effects on farm production, sustainability and economic viability. Parasite infections and impacts can, according to pathogen and context, be considered to be either unpredictable/sporadic or predictable/regular. Although both types of infection may result in the loss of stock and incur costs associated with the control and management of infection, predictable infections can also lead to costs associated with prophylaxis and related activities. The estimation of the economic cost of a parasite event is frequently complicated by the complex interplay of numerous factors associated with a specific incident, which may range from direct production losses to downstream socio-economic impacts on livelihoods and satellite industries associated with the primary producer. In this study, we examine the world's major marine and brackish water aquaculture production industries and provide estimates of the potential economic costs attributable to a range of key parasite pathogens using 498 specific events for the purposes of illustration and estimation of costs. This study provides a baseline resource for risk assessment and the development of more robust biosecurity practices, which can in turn help mitigate against and/or minimise the potential impacts of parasite-mediated disease in aquaculture.

The antidepressant drug carbamazepine induces differential transcriptome expression in the brain of Atlantic salmon, Salmo salar.

Concerns are being expressed recently over possible environmental effects of human pharmaceuticals. Although the likelihood of acute toxicity is low, the continuous discharge of pharmaceuticals into the aquatic environment means that sublethal effects on non-target organisms need to be seriously considered. One-year-old Atlantic salmon parr were exposed to 7.85±0.13µgL(-1) of the antidepressant drug Carbamazepine (CBZ) for five days to investigate changes of mRNA expression in the brain by means of a custom 17k Atlantic salmon cDNA microarray. The selected concentration is similar to upper levels that can be found in hospital and sewage treatment plant effluents. After treatment, 373 features were differently expressed with 26 showing up- or down-regulation of ≥2-fold (p≤0.05). Among the mRNAs showing the highest change were the pituitary hormones encoding features somatolactin, prolactin and somatotropin, or growth hormone. Functional enrichment and network analyses of up- and down-regulated genes showed that CBZ induced a highly different gene expression profile in comparison to untreated organisms. CBZ induced expression of essential genes of the focal adhesion and extracellular matrix - receptor interaction pathways most likely through integrin alpha-6 (itga6) activation. Negative regulation of apoptotic process, extracellular matrix organization and heme biosynthesis were the most enriched biological process related GO-terms, with the simultaneous enrichment of collagen and extracellular region related cellular component GO-terms, and extracellular matrix structural constituent, hormone activity and chromatin binding molecular function related GO-terms. These results show that relatively low doses of CBZ may affect brain physiology in exposed salmon parr, targeting similar processes as in human, indicating a high degree of conservation of targets of CBZ action. However, and since the mRNAs showing most changes in expression are critical for adaptation to different stressors and life history transitions in Atlantic salmon, more research should be undertaken to assess CBZ effects to avoid impairment of normal development and maintenance of natural populations.

Role of kairomones in host location of the pennellid copepod parasite, Lernaeocera branchialis (L. 1767).

The life cycle of the parasitic copepod Lernaeocera branchialis involves 2 hosts, typically a pleuronectiform host upon which development of larvae and mating of adults occurs and a subsequent gadoid host, upon which the adult female feeds and reproduces. Both the copepodid and adult female stages must therefore locate and identify a suitable host to continue the life cycle. Several mechanisms are potentially involved in locating a host and ensuring its suitability for infection. These may include mechano-reception to detect host movement and chemo-reception to recognize host-associated chemical cues, or kairomones. The aim of this study was to identify the role of kairomones in host location by adult L. branchialis, by analysing their behaviour in response to fish-derived chemicals. Experiments demonstrated that water conditioned by immersion of whiting, Merlangius merlangus, elicited host-seeking behaviour in L. branchialis, whereas cod- (Gadus morhua) conditioned water did not. Lernaeocera branchialis are considered a genetically homogeneous population infecting a range of gadoids. However, their differential response to whiting- and cod-derived chemicals in this study suggests that either there are genetically determined subspecies of L. branchialis or there is some form of environmental pre-conditioning that allows the parasite to preferentially recognize the host species from which it originated.

Structural differentiation of apical openings in active mitochondria-rich cells during early life stages of Nile tilapia (Oreochromis niloticus L.) as a response to osmotic challenge.

This study examines the structural differentiation of the apical crypts of mitochondria-rich cells (MRCs) in Nile tilapia as a response to osmotic challenge. Larvae were transferred from freshwater at 3 days post-hatch to 12.5 and 20 ppt and were sampled at 24- and 48-h post-transfer. Scanning electron microscopy allowed quantification of MRCs, based on apical crypt appearance and surface area, resulting in a morphological classification of 'sub-types', that is, Type I or absorptive (surface area range 5.2-19.6 µm(2)), Type II or active absorptive form (surface area range 1.1-15.7 µm(2)), Type III or weakly functioning form (surface area range 0.08-4.6 µm(2)) and Type IV or active secreting form (surface area range 4.1-11.7 µm(2)). Mucus cell crypts were discriminated from those of MRCs based on the presence of globular extensions and quantified. Density and frequency of MRCs and mucus cells varied significantly according to the experimental salinity and time post-transfer; in freshwater-adapted larvae, all types were present except Type IV but, following transfer to elevated salinities, Type I and Type II disappeared and appeared to be replaced by Type IV crypts. Type III crypt density remained constant following transfer. Transmission electron microscopy with immunogold labelling, using a novel pre-fixation technique with anti-Na(+)/K(+)-ATPase, allowed complementary ultrastructural visualisation of specific localisation of the antibodies on active MRCs, permitting a review of MRC apical morphology and related Na(+)/K(+)-ATPase binding sites.

Stocking methods and parasite-induced reductions in capture: modelling Argulus foliaceus in trout fisheries.

Argulus foliaceus is a macroparasite which can have a significant impact on yield in recreational trout fisheries, partly by increasing fish mortalities but also by reducing the appetite of infected fish, making them less likely to respond to bait. The aim of this paper is to determine the impact of four commonly used fish stocking methods both on the parasite dynamics, and on fisheries' yields. The wider consequences of the resultant reduction in host feeding are also of interest. To this end four different stocking methods were incorporated into Anderson and May's macroparasite model, which comprises three differential equations representing the host, attached parasite and free-living parasite populations. To each of these a reduction in the fish capture rate, inversely linked to the mean parasite burden, is added and the effects interpreted. Results show that (1) the common practise of increasing the stocking rate as catches drop may be counterproductive; (2) in the absence of any wild population of reservoir hosts, the parasite will be unable to survive if the stocking rate does not exceed the rate of capture; (3) compensatory stocking to account for fish mortalities can have disastrous consequences on yield; and (4) the parasite can, under certain circumstances, maintain the host population by preventing their capture.

Development of a light microscopy stain for the sclerites of Gyrodactylus von Nordmann, 1832 (Monogenea) and related genera.

Recent developments in semi-automated identification techniques and the increasing ability to rapidly access digital images and taxonomic descriptions offer to increase the range of individuals capable of performing taxonomic identifications. The present study details methodological approaches undertaken in developing a dedicated stain for the visualisation of monogenean haptoral skeletal elements and reproductive sclerites. The histochemical protocols centre around the use of fluorescent dyes and standard light and laser scanning confocal microscopy to support studies of the functional morphology of these hard structures in small, relatively uncompressed specimens, making these structures more amenable to semi-automated analysis and identification techniques. Staining of the sclerites was achieved using a tissue digestion step to remove the tegument and tissues enclosing the sclerites and then staining them in situ with 40 mM chromothrope 2R (C2R) containing 3 mM phosphotungstic acid (PTA) and 0.5% acetic acid (AA) at room temperature for up to 2 days. Visualisation of the armature of the male copulatory organ of warm water Gyrodactylus species was achieved using 40 mM C2R containing 3 mM PTA for 3 days, whilst cold water species were best stained in 6.4 mM C2R for 1 day without an NaOH pre-treatment. The developed techniques allow for good visualisation of the skeletal elements in a number of monogenean groups and promise to assist the preparation and identification/description of specimens. The 2D/3D digital images of specimens prepared in this manner should provide a useful resource for taxonomists and others needing material to assist specimen identification.

An assessment of the use of drug and non-drug interventions in the treatment of Ichthyophthirius multifiliis Fouquet, 1876, a protozoan parasite of freshwater fish.

Infection by the ciliate protozoan Ichthyophthirius multifiliis Fouquet, 1876 causes significant economic losses in freshwater aquaculture worldwide. Following the ban on the use of malachite green for treating food fish, there has been extensive research aimed at identifying suitable replacements. In this paper we critically assess drug and non-drug interventions, which have been tested for use or have been employed against this parasite and evaluate possibilities for their application in farm systems. Current treatments include the administration of formaldehyde, sodium chloride (salt), copper sulphate and potassium permanganate. However, purportedly more environmentally friendly drugs such as humic acid, potassium ferrate (VI), bronopol and the peracetic acid-based products have recently been tested and represent promising alternatives. Further investigation, is required to optimize the treatments and to establish precise protocols in order to minimize the quantity of drug employed whilst ensuring the most efficacious performance. At the same time, there needs to be a greater emphasis placed on the non-drug aspects of management strategies, including the use of non-chemical interventions focusing on the removal of free-swimming stages and tomocysts of I. multifiliis from farm culture systems. Use of such strategies provides the hope of more environmentally friendly alternatives for the control of I. multifiliis infections.

Ontogenetic changes in location and morphology of chloride cells during early life stages of the Nile tilapia Oreochromis niloticus adapted to fresh and brackish water.

Ontogenetic changes in the location, size, density and morphology of chloride cells in the Nile tilapia Oreochromis niloticus adapted to fresh and brackish water are described using Na(+) /K(+) -ATPase immunohistochemistry, light microscopy (LM), scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). The pattern of chloride cell distribution changed during development under both treatments, with chloride cell density decreasing significantly from hatch to 7 days post-hatch, but appearing on the inner opercular area at 3 days post-hatch and increasing significantly thereafter (P < 0·05). Chloride cells were always denser in fresh- than in brackish-water larvae. In both treatments, chloride cells located on the outer operculum and tail showed a marked increase in size with age, but cells located on the abdominal epithelium of the yolk sac and the inner operculum showed a significant decrease in size (P < 0·05). Chloride cells from brackish-water adapted larvae from 1 day post-hatch onwards were always significantly larger (P < 0·05) than those from freshwater-adapted larvae. SEM revealed structural differences in chloride cell apical morphology according to environmental conditions. There appears to be clearly defined temporal staging of the appearance of adaptive mechanisms that confer an ability to cope with varying environmental conditions during early development.

Determining the age of individual Lepeophtheirus salmonis (Krøyer, 1837) copepodids by measuring stored lipid volume; proof of principle.

Confocal microscopy has facilitated measurement of stained lipid volume in Lepeophtheirus salmonis copepodid larvae. Quantity of lipid, location and morphology of vesicles may allow an estimate of age and viability.

Multi-centre testing and validation of current protocols for the identification of Gyrodactylus salaris (Monogenea).

Despite routine screening requirements for the notifiable fish pathogen Gyrodactylus salaris, no standard operating procedure exists for its rapid identification and discrimination from other species of Gyrodactylus. This study assessed screening and identification efficiencies under real-world conditions for the most commonly employed identification methodologies: visual, morphometric and molecular analyses. Obtained data were used to design a best-practice processing and decision-making protocol allowing rapid specimen throughput and maximal classification accuracy. True specimen identities were established using a consensus from all three identification methods, coupled with the use of host and location information. The most experienced salmonid gyrodactylid expert correctly identified 95.1% of G. salaris specimens. Statistical methods of classification identified 66.7% of the G. salaris, demonstrating the need for much wider training. Molecular techniques (internal transcribed spacer region-restriction fragment length polymorphism (ITS-RFLP)/cytochrome c oxidase I (COI) sequencing) conducted in the diagnostic laboratory most experienced in the analysis of gyrodactylid material, identified 100% of the true G. salaris specimens. Taking into account causes of potential specimen loss, the probabilities of a specimen being accurately identified were 95%, 87% and 92% for visual, morphometric and molecular techniques, respectively, and the probabilities of correctly identifying a specimen of G. salaris by each method were 81%, 58% and 92%. Inter-analyst agreement for 189 gyrodactylids assessed by all three methods using Fleiss' Kappa suggested substantial agreement in identification between the methods. During routine surveillance periods when low numbers of specimens are analysed, we recommend that specimens be analysed using the ITS-RFLP approach followed by sequencing of specimens with a "G. salaris-like" (i.e. G. salaris, Gyrodactylus thymalli) banding pattern. During periods of suspected outbreaks, where a high volume of specimens is expected, we recommended that specimens be identified using visual identification, as the fastest processing method, to select "G. salaris-like" specimens, which are subsequently identified by molecular-based techniques.

Lipid and fatty acid composition of parasitic caligid copepods belonging to the genus Lepeophtheirus.

Sea lice are copepod ectoparasites that constitute a major barrier to the sustainability and economic viability of marine finfish aquaculture operations worldwide. In particular, the salmon louse, Lepeophtheirus salmonis, poses a considerable problem for salmoniculture in the northern hemisphere. The free-swimming nauplii and infective copepodids of L. salmonis are lecithotrophic, subsisting principally on maternally-derived lipid reserves. However, the lipids and fatty acids of sea lice have been sparsely studied and therefore the present project aimed to investigate the lipid and fatty acid composition of sea lice of the genus Lepeophtheirus obtained from a variety of fish hosts. Total lipid was extracted from eggs and adult female L. salmonis obtained from both wild and farmed Atlantic salmon (Salmo salar L.) sampled at two time points, in the mid 1990s and in 2009. In addition, L. salmonis from wild sea trout (Salmo trutta L.) and L. hippoglossi from wild Atlantic halibut (Hippoglossus hippoglossus L.) were sampled and analyzed. The lipids of both females and egg strings of Lepeophtheirus were characterized by triacylglycerol (TAG) as the major neutral (storage) lipid with phosphatidylcholine and phosphatidylethanolamine as the major polar (membrane) lipids. The major fatty acids were 22:6n-3 (DHA), 18:1n-9 and 16:0, with lesser amounts of 20:5n-3, 22:5n-3 and 18:0. L. salmonis sourced from farmed salmon was characterized by higher levels of 18:2n-6 and 18:3n-3 than lice from wild salmon. Egg strings had higher levels of TAG and lower DHA compared to females, whereas L. hippoglossi had lower levels of TAG and higher DHA than L. salmonis. The results demonstrate that the fatty acid compositions of lice obtained from wild and farmed salmon differ and that changes to the lipid and fatty acid composition of feeds for farmed salmon influence the louse compositions.

The susceptibility of Atlantic salmon fry to freshwater infectious pancreatic necrosis is largely explained by a major QTL.

Infectious pancreatic necrosis (IPN) is a viral disease with a significant negative impact on the global aquaculture of Atlantic salmon. IPN outbreaks can occur during specific windows of both the freshwater and seawater stages of the salmon life cycle. Previous research has shown that a proportion of the variation seen in resistance to IPN is because of host genetics, and we have shown that major quantitative trait loci (QTL) affect IPN resistance at the seawater stage of production. In the current study, we completed a large freshwater IPN challenge experiment to allow us to undertake a thorough investigation of the genetic basis of resistance to IPN in salmon fry, with a focus on previously identified QTL regions. The heritability of freshwater IPN resistance was estimated to be 0.26 on the observed scale and 0.55 on the underlying scale. Our results suggest that a single QTL on linkage group 21 explains almost all the genetic variation in IPN mortality under our experimental conditions. A striking contrast in mortality is seen between fry classified as homozygous susceptible versus homozygous resistant, with QTL-resistant fish showing virtually complete resistance to IPN mortality. The findings highlight the importance of the major QTL in the genetic regulation of IPN resistance across distinct physiological lifecycle stages, environmental conditions and viral isolates. These results have clear scientific and practical implications for the control of IPN.

A description of the origins, design and performance of the TRAITS-SGP Atlantic salmon Salmo salar L. cDNA microarray.

The origins, design, fabrication and performance of an Atlantic salmon microarray are described. The microarray comprises 16 950 Atlantic salmon-derived cDNA features, printed in duplicate and mostly sourced from pre-existing expressed sequence tag (EST) collections [SALGENE and salmon genome project (SGP)] but also supplemented with cDNAs from suppression subtractive hybridization libraries and candidate genes involved in immune response, protein catabolism, lipid metabolism and the parr-smolt transformation. A preliminary analysis of a dietary lipid experiment identified a number of genes known to be involved in lipid metabolism. Significant fold change differences (as low as 1.2x) were apparent from the microarray analysis and were confirmed by quantitative real-time polymerase chain reaction analysis. The study also highlighted the potential for obtaining artefactual expression patterns as a result of cross-hybridization of similar transcripts. Examination of the robustness and sensitivity of the experimental design employed demonstrated the greater importance of biological replication over technical (dye flip) replication for identification of a limited number of key genes in the studied system. The TRAITS (TRanscriptome Analysis of Important Traits of Salmon)-salmon genome project microarray has been proven, in a number of studies, to be a powerful tool for the study of key traits of Atlantic salmon biology. It is now available for use by researchers in the wider scientific community.

Microbial protein production in activated suspension tanks manipulating C:N ratio in feed and the implications for fish culture.

The present experiment investigated the possibility of microbial protein production in 250 l indoor tanks by manipulating C:N ratio in fish feed applied. Two different levels of protein feed (35% and 22% CP) resulting in C:N ratio of 8.4 and 11.6, respectively, were applied at 25 g daily in each tank. Tanks were aerated and agitated continuously using a dome diffuser. The experiment was carried out for eight weeks. The biofloc development in terms of VSS and BOD5 was better in the low protein fed tanks than in the high protein fed tanks. An estimated biofloc productivity ranged 3-5 g Cm(-3)day(-1). A 3-D image stained with DAPI indicates that the biofloc is comprised of hundreds of bacterial nuclei, size being ranged from 100 to 200 microm. Biofloc quality was independent of the quality of feed applied and contained more than 50% crude protein, 2.5% crude lipid, 4% fibre, 7% ash and 22 kJ g(-1) energy on dry matter basis. The dietary composition and size of biofloc can be considered as appropriate for all omnivorous fish species. The underlying ecological processes are explained through factor analysis. The potential of using biofloc in fish culture is also discussed.

Interferon type I and type II responses in an Atlantic salmon (Salmo salar) SHK-1 cell line by the salmon TRAITS/SGP microarray.

Interferons (IFNs) are cytokines that have proinflammatory, antiviral, and immunomodulatory effects and play a central role during a host response to pathogens. The IFN family contains both type I and type II molecules. While there are a number of type I IFNs, there is only one type II IFN. Recently both type I and type II IFN genes have been cloned in salmonid fish and recombinant proteins produced showing IFN activity. We have stimulated an Atlantic salmon cell line (SHK-1) with both type I and type II recombinant salmonid IFNs and analyzed the transcriptional response by microarray analysis. Cells were exposed to recombinant IFNs for 6 or 24 h or left unexposed as controls. RNA was hybridized to an Atlantic salmon cDNA microarray (salmon 17K feature TRAITS/SGP array) in order to assess differential gene expression in response to IFN exposure. For IFN I and II, 47 and 72 genes were stimulated, respectively; most genes were stimulated by a single IFN type, but some were affected by both IFNs, indicating coregulation of the IFN response in fish. Real-time PCR analysis was employed to confirm the microarray results for selected differentially expressed genes in both a cell line and primary leukocyte cultures.

Identification of B-cell epitopes on the betanodavirus capsid protein.

The pepscan procedure was used to identify betanodavirus B-cell epitopes recognized by neutralizing mouse monoclonal antibodies (MAbs) and serum samples obtained from sea bass, Dicentrarchus labrax, naturally infected with betanodavirus. Pepscan was performed with a panel of thirty-four 12-mer synthetic peptides that mimicked the entire betanodavirus capsid protein. Sea bass serum samples reacted strongly with three regions of the capsid protein comprising amino acid residues 1-32, 91-162 and 181-212. The latter region was also recognized by neutralizing MAbs and coincided with a region of high antigenic propensity identified by an antigen prediction algorithm. These data suggest that a region of the betanodavirus capsid protein spanning amino acid residues 181-212 may represent a neutralization domain that could potentially be used to inform the development of nodavirus vaccines and immunodiagnostic reagents.