An Enriched European Eel Transcriptome Sheds Light upon Host-Pathogen Interactions with Vibrio vulnificus

Infectious diseases are one of the principal bottlenecks for the European eel recovery. The aim of this study was to develop a new molecular tool to be used in host-pathogen interaction experiments in the eel. To this end, we first stimulated adult eels with different pathogen-associated molecular patterns (PAMPs), extracted RNA from the immune-related tissues and sequenced the transcriptome. We obtained more than 2 x 10(6) reads that were assembled and annotated into 45,067 new descriptions with a notable representation of novel transcripts related with pathogen recognition, signal transduction and the immune response. Then, we designed a DNA-microarray that was used to analyze the early immune response against Vibrio vulnificus, a septicemic pathogen that uses the gills as the portal of entry into the blood, as well as the role of the main toxin of this species (RtxA13) on this early interaction. The gill transcriptomic profiles obtained after bath infecting eels with the wild type strain or with a mutant deficient in rtxA13 were analyzed and compared. Results demonstrate that eels react rapidly and locally against the pathogen and that this immune-response is rtxA13-dependent as transcripts related with cell destruction were highly up-regulated only in the gills from eels infected with the wild-type strain. Furthermore, significant differences in the immune response against the wild type and the mutant strain also suggest that host survival after V. vulnificus infection could depend on an efficient local phagocytic activity. Finally, we also found evidence of the presence of an interbranchial lymphoid tissue in European eel gills although further experiments will be necessary to identify such tissue

Nitrosylation of Myoglobin and Nitrosation of Cysteine by Nitrite in a Model System Simulating Meat Curing

Demand is growing for meat products cured without the addition of sodium nitrite. Instead of the direct addition of nitrite to meat in formulation, nitrite is supplied by bacterial reduction of natural nitrate often added as vegetable juice/powder. However, the rate of nitrite formation in this process is relatively slow, and the total ingoing nitrite is typically less than in conventional curing processes. The objective of this study was to determine the impact of the rate of addition of nitrite and the amount of nitrite added on nitrosylation/nitrosation reactions in a model meat curing system. Myoglobin was preferentially nitrosylated as no decrease in sulfhydryl groups was found until maximum nitrosylmyoglobin color was achieved. The cysteine–myoglobin model retained more sulfhydryl groups than the cysteine-only model (p \u3c 0.05). The rate of nitrite addition did not alter nitrosylation/nitrosation reactions (p \u3e 0.05). These data suggest that the amount of nitrite but not the rate of addition impacts the nitrosylation/nitrosation reactions this syste

Characterisation and expression analysis of the Atlantic halibut (Hippoglossus hippoglossus L.) cytokines: IL-1β, IL-6, IL-11, IL-12β and IFNγ

Genes encoding the five Atlantic halibut (Hippoglossus hippoglossus L.) cytokines; interleukin (IL)-1β, IL-6, IL-11b, IL-12βc, and interferon (IFN) γ, were cloned and characterised at a molecular level. The genomic organisation of the halibut cytokine genes was similar to that seen in mammals and/or other fish species. Several mRNA instability motifs were found within the 3′-untranslated region (UTR) of all cytokine cDNA sequences. The putative cytokine protein sequences showed a low sequence identity with the corresponding homologues in mammals, avian and other fish species. Nevertheless, important structural features were presumably conserved such as the presence, or absence in the case of IL-1β, of a signal peptide, secondary structure and family signature motifs. The relative expression pattern of the cytokine genes was analyzed in several halibut organs, revealing a constitutive expression in both lymphoid and non-lymphoid organs. Interestingly, the gills showed a relatively high expression of IL-1β, IL-12βc and IFNγ. The real time RT-PCR data also showed that the mRNA level of IL-1β, IL-6, IL-12βc and IFNγ was high in the thymus, while IL-11b was relatively highly expressed in the posterior kidney and posterior gut. Moreover, the halibut brain showed a relatively high level of IL-6 transcripts. Anterior kidney leucocytes in vitro stimulated with imiquimod showed a significant increase in mRNA level of the five halibut cytokine genes. The sequence and characterisation data presented here will be useful for further investigation of both innate and adaptive immune responses in halibut, and be helpful in the design of vaccines for the control of various infectious diseases

Melanogenesis in Visceral Tissues of Salmo salar. A link between Immunity and Pigment production?

The Atlantic salmon (Salmo salar L.) is an ichthyic breeding species of considerable economic importance. The accumulation of pigments in salmon visceral organs and musculature (1) adversely affects the quality of fish products and is a problem for the aquaculture industry. Moreover, a close relationship between nutrition products and human health has been widely demonstrated. Liver, spleen, kidney and other organs of lower vertebrates harbour a visceral pigment cell system with an embryonic origin that differs from that of melanocytes (2). In teleosts, melanin-containing cells, named melano-macrophages, occur in the reticulo-endothelial system and are mainly located in kidney and spleen. In these organs, the pigment content may increase in different physiological conditions and as a response to pathological and inflammatory states (2). With the aim to reveal novel functions and behaviour of the poorly understood teleost extracutaneous pigment system, we undertook the investigation of aspects of the melanogenic systems of Atlantic salmon tissues as well as of leukocyte salmon head-kidney cells (SHK-1 cells). SHK-1 cells is a long-term cell line derived from macrophages of the Atlantic salmon head-kidney (3). Using a radiometric method we demonstrated that a tyrosinase activity is present in SHK-1 cells and salmon tissues. Western blotting showed the presence of a tyrosinase protein. Moreover differing melanin-producing activities in salmon tissues were demonstrated suggesting that pigmented cells in the organs undergo a tissue-specific differentiation. These results were confirmed by a tyrosinase protein expression in lymphoid organs, also highlighted by immunohistochemical analysis. As teleosts lack lymph nodes and Peyer’s patches, the head-kidney and spleen are regarded as the most important secondary lymphoid organs (4). The detection of tyrosinase activity in lymphoid organs indicates that a link may exist between the extracutaneous pigmentary system and the immune system in salmon. The extracutaneous melanogenetic system could be used as a biomarker for the health status of farmed fishes. References: 1. Koppang, E.O., Haugarvoll, E., Hordvik, I., Aune, L., Poppe, T. (2005) Journal of Fish Diseases 28: 13-22. 2. Agius, C., Roberts, R.J. (2003) Journal of Fish Diseases 26: 499-509. 3. Dannevig, B., Falk, K., Narmork, E. (1995) Journal of General Virology 76: 1353-1359. 4. Zapata, A., Amemiya, CT. (2000) Curr Top Microbiol Immunol 248: 67-107