Transcriptome analysis reveals a complex response to the RGNNV/SJNNV reassortant Nervous Necrosis Virus strain in sea bream larvae.

The gilthead sea bream (Sparus aurata) is a marine fish of great importance for Mediterranean aquaculture. This species has long been considered resistant to Nervous Necrosis Virus (NNV), an RNA virus that causes massive mortalities in several farmed fish animals. However, the recent appearance of RGNNV/SJNNV reassortant strains started to pose a serious threat to sea bream hatcheries, as it is able to infect larvae and juveniles of this species. While host response to NNV has been extensively studied in adult fish, little attention has been devoted to early life history stages, which are generally the most sensitive ones. Here we report for the first time a time-course RNA-seq analysis on 21-day old fish gilthead sea bream larvae experimentally infected with a RGNNV/SJNNV strain. NNV-infected and mock-infected samples were collected at four time points (6 h, 12 h, 24 h, and 48 h post infection). Four biological replicates, each consisting of five pooled larvae, were analysed for each time point and group. A large set of genes were found to be significantly regulated, especially at early time points (6 h and 12 h), with several heat shock protein encoding transcripts being up-regulated (e.g. hspa5, dnaj4, hspa9, hsc70), while many immune genes were down-regulated (e.g. myd88 and irf5 at T06, pik3r1, stat3, jak1, il12b and il6st at T12). A gene set enrichment analysis (GSEA) identified several altered pathways/processes. For instance, the formation of peroxisomes, which are important anti-viral components as well as essential for nervous system homeostasis, and the autophagy pathway were down-regulated at 6 h and 24 h post infection (hpi). Finally, two custom "reactomes" (i.e. significant gene sets observed in other studies) were defined and used. The first reactome integrated the transcriptomic response to NNV in different fish species, while the second one included all genes found to be stimulated either by interferon (IFN) or by IFN and Chikungunya virus in zebrafish. Genes in both reactomes showed predominant up-regulation at 6hpi and 12hpi and a general down-regulation at 24hpi. Such evidence suggest a certain degree of similarity between the response of sea bream and that of other fish species to NNV, while the observed down-regulation of IFN- and viral-stimulated pathways argues for a possible interference of NNV against the host response.

Development of a real-time PCR assay for rapid detection and quantification of Photobacterium damselae subsp. piscicida in fish tissues.

The availability of a rapid and accurate method for the diagnosis of Photobacterium damselae subsp. piscicida (Phdp), able to discriminate its strictly correlated subsp. damselae (Phdd), formally known as Vibrio damsela, is essential for managing fish pasteurellosis outbreaks in farmed fish. A single-step, high-sensitivity real-time PCR assay for simultaneous detection and quantification of P. damselae was designed targeting partial of the sequence of the bamB gene and tested for specificity and sensitivity on laboratory-generated samples as well as on experimentally infected seabream tissue samples. With a limit of detection (LOD) of one copy in pure bacterial DNA, the sensitivity was higher than all methods previously reported. Validation in target and non-target bacterial species proved the assay was able to discriminate Phdd-Phdp subspecies from diverse hosts/geographical origins and between non-target species. In addition, two SNPs in the target amplicon region determine two distinctive qPCR dissociation curves distinguishing between Phdp-Phdd. This is the first time that a molecular method for P. damselae diagnosis combines detection, quantification and subspecies identification in one step. The assay holds the potential to improve the knowledge of infection dynamics and the development of better strategies to control an important fish disease.

Efficacy of domestic cooking inactivation of human hepatitis A virus in experimentally infected manila clams (Ruditapes philippinarum).

AIM: The aim of this work was to evaluate the efficacy of domestic cooking in inactivating Manila clams experimentally infected with human hepatitis A virus (HAV). METHODS AND RESULTS: Electronic temperature probes were positioned to measure the internal temperature of Manila clams during domestic cooking. Two batches were infected with 10(7) and 10(5) TCID50  ml(-1) of HAV. The infected whole-in-shell clams were divided into three replicates and cooked on a conventional stove both singularly and in group and removed from the pan at fixed intervals. Pools of three digestive glands were examined by virus isolation for three blind passages and cell culture supernatant tested with real-time PCR. CONCLUSION: Results showed that 2-min cooking by a traditional domestic method at a temperature close to 100°C, after the opening up of the valves of all the clams, can completely devitalize the HAV in high viral load-infected clams. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study on inactivation of HAV in experimentally infected Manila clams subjected to domestic cooking. At present, labelling all lagoon products as 'requiring cooking before consumption' is highly recommended, but no specifications are given on how long and at what temperature they should be cooked. Considering the high commercial value of Manila clams, our results can provide both the producers and the consumer with useful indications on how to cook clams to prevent the risk of HAV foodborne illness.

Betanodavirus ability to infect juvenile European sea bass, Dicentrarchus labrax, at different water salinity.

Viral encephalopathy and retinopathy (VER) is one of the most devastating and economically relevant diseases for marine aquaculture. The presence of betanodavirus in freshwater fish is recorded, but very little is known about VER outbreaks in marine species reared in freshwater. Our study investigated the ability of betanodavirus to cause disease in European sea bass, Dicentrarchus labrax, reared at different salinity levels. Fish were challenged with RGNNV or mock infected by bath at different salinity levels (freshwater, 25‰ and 33‰). Fish were checked twice a day and the dead ones were examined by standard virological techniques, by rRT-PCR and by histochemical and immunohistochemical analyses. All the infected groups showed a significant higher mortality rate than the one of the mock-infected group. VERv presence was confirmed by rRT-PCR. Histochemical and immunohistochemical analyses highlighted the typical lesions associated with VER. Our results highlight that salinity does not affect the ability of betanodavirus to induce clinical signs and mortality in European sea bass infected under experimental conditions. These results underline the great adaptation potential of VERv, which in combination with its already known high environmental resistance and broad host range, may explain the diffusion of this disease and the threat posed to aquaculture worldwide.

Quantitative immunoenzymatic detection of viral encephalopathy and retinopathy virus (betanodavirus) in sea bass Dicentrarchus labrax.

Viral encephalopathy and retinopathy disease caused by betanodavirus, genus of the family Nodaviridae, affects marine, wild and farmed species including sea bass, one of the most important farmed species in Europe. This work describes a reliable and sensitive indirect ELISA assay to detect betanodavirus in biological samples using a polyclonal antiserum (pAb 283) against the 283/I09 virus strain, the most common red-spotted grouper nervous necrosis virus (RGNNV) genotype in the Mediterranean area, and a capture-based ELISA using a monoclonal antibody (mAb 4C3) specific to a common epitope present on the capsid protein. Using adsorbed, purified VERv preparation, the detection limit of indirect ELISA was 2 µg mL(-1) (3 × 10(5) TCID50 per mL), whereas for capture-based ELISA, the sensitivity for the antigen in solution was 17 µg mL(-1) (35 × 10(5) TCID50 per mL). The capture-based ELISA was employed to detect VERv in brain homogenates of in vivo infected sea bass and resulted positive in 22 of 32 samples, some of these with a high viral load estimates (about 1.1 × 10(8)  TCID50 per mL). The ELISA system we propose may be helpful in investigations where coupling of viral content in fish tissues with the presence of circulating VERv-specific IgM is required, or for use in samples where PCR is difficult to perform.

The effectiveness of domestic cook on inactivation of murine norovirus in experimentally infected Manila clams (Ruditapes philippinarum).

AIM: The aim of this work was to evaluate the efficacy of domestic cooking in inactivating Manila clams experimentally infected with murine norovirus (MNV). METHODS AND RESULTS: A cooking pan was modified to enable electronic temperature probes to be positioned to record both flesh and environment temperature. Manila clams were infected with 10(4) TCID 50% ml(-1) of MNV. The infected whole-in-shell clams, divided into three replicates, were cooked on an electric stove, and groups of nine clams were removed from the pan at fixed intervals. Pools of three digestive glands were examined by virus isolation to ascertain residual viral load. CONCLUSION: Results showed that 10 min of cooking by a traditional domestic method at a temperature close to 100°C, for at least 2 min, can completely devitalize the MNV in infected clams. This is generally the time needed for the majority of valves to open up. SIGNIFICANCE AND IMPACT OF THE STUDY: At present, it is highly recommended to label all lagoon products as 'requiring cooking before consumption', but no specifications are given on how long and at what temperature they should be cooked. Our results can provide the consumer with useful indications on how to cook clams to prevent any risk of foodborne illness.

Expression of 8-OHdG in Zosterisessor ophiocephalus from the Venetian lagoon, Italy.

The aim of the present work was to evaluate the expression of 8-OHdG (8-hydroxydeoxyguanosine) in the benthic fish Zosterisessor ophiocephalus collected in two differently polluted sites of the Venetian lagoon (Porto Marghera and Caroman). We compared our data on 8-OHdG with those of CYP1A (Cytochrome P450, family 1, subfamily A, polypeptide 1), which is a well known biomarker for detoxification of contaminants. Immunohistochemistry with an antibody to 8-OHdG showed immunopositivity in nuclei of hepatocytes as well as in melanomacrophage centres of spleen and kidney, whereas an anti-CYP1A antibody exhibited positive immunostaining in the liver, kidney and ovary. The liver of males showed higher expression of both proteins than females. In animals from Porto Marghera site, the enzymatic assay for 8-OHdG exhibited higher levels in liver of males than in females. Western Blot analysis using the antibody anti-CYP1A recognized the presence of a band of about 60 kDa in the liver of males and females. Males exhibited a strong band, whereas in females the band showed a lower intensity. By using Real-Time PCR, the mRNA expression of CYP1A did not show any differences between males and females from each site, but it was at borderline significance level. Comparing the two sites, mRNA expression of CYP1A was significantly higher in the liver of both males and females from Porto Marghera than that of Caroman. The present data suggest that pollutants are bio-available as demonstrated by our biomarker analyses and may have a harmful effect on aquatic organisms such as Z. ophiocephalus. We report that the highest levels of hepatic 8-OHdG and CYP1A expression were detected in males, showing clear gender specificity.

Expression of heat shock protein 70 in the liver of extensively and intensively kept heavy pigs.

The objective of this work was to investigate the expression of heat shock protein 70 (HSP70) by Western blot (WB) in swine liver. Subsequently, the study aimed to apply this method to two experimental groups of heavy pigs raised in different confinement systems: intensive/indoor (Group A) and extensive/outdoor (Group B). Thirty-six crossbred commercial heavy pigs were divided as follows: Group A (eight castrated males and eight females) was equally distributed into two single-sex indoor pens (1.02 m²/pig); Group B (11 castrated males and nine females) was kept in one single (partially grassy and partially wooded) open area of about 6000 m². Group A was slaughtered at 41 weeks of age (170 ± 9 kg) and Group B at 48 weeks of age (172 ± 13 kg). At the abattoir the livers of all the animals were collected and analyzed by WB assay in order to quantify the levels of HSP70. Moreover, a further liver sample was taken from the same animals in order to investigate the cellular localization of HSP70 by immunohistochemistry (IHC). The interaction between sex and group resulted statistically significant (P = 0.001). When stratified by sex, Group A showed significantly higher HSP70 values compared with Group B for both male and female subjects (P < 0.001). Stratifying by group, males showed significantly higher HSP70 values than females in Group A (P < 0.001), whereas no statistical differences were observed between sexes for Group B (P = 0.653). The IHC results evidenced cytoplasmic immunoreactivity in a granular pattern in both groups. The different expression pattern observed by WB could prove to be a useful tool in the assessment of pig health and welfare.

Seasonal effects on hematological and innate immune parameters in sea bass Dicentrarchus labrax.

The temperate aquatic environment is affected by two primary components of season, temperature and photoperiod, during the annual cycle. Many organisms respond to seasonal change physiologically, behaviorally or both. The aim of this study was to investigate the effect of seasonality on cortisol, hematological and innate immune parameters in European sea bass reared under traditional semi-intensive aquaculture. Sea bass (Dicentrarchus labrax) were reared in an outdoor pond and serum cortisol, hematocrit, leucocrit, serum lysozyme activity and total glutathione were bimonthly monitored over a 14-months period. The effect of seasonality was observed for all parameters carried out, with generally higher values in summer and lower in winter. These results could improve the understanding of the influence of seasonal cues on the immune system and hematological parameters in fish in order to optimize the husbandry practices.

Immunohistochemical localization of IGF-I, IGF-II and MSTN proteins during development of triploid sea bass (Dicentrarchus labrax).

The cellular localization of IGF-I, IGF-II and MSTN proteins was investigated during ontogenesis of triploid sea bass (Dicentrarchus labrax) by an immunohistochemical approach. The results were compared with those observed in diploids. IGF-I immunostaining was mainly observed in skin, skeletal muscle, intestine and gills of both diploids and triploids. From day 30 of larval life, IGF-I immunoreactivity observed in skeletal muscle, intestine, gills and kidney was stronger in triploids than in diploids. At day 30, triploids exhibited a standard length significantly higher than the one of diploids. Although IGF-II and MSTN immunoreactivity was detectable in different tissues and organs, no differences between diploids and triploids were observed. The spatial localization of IGF-I, IGF-II and MSTN proteins detected in this study is in agreement with previous findings on the distribution of these proteins in diploid larvae and fry. The highest IGF-I immunoreactivity observed in triploids suggests a possible involvement of ploidy in their growth performance.