Investigations on immunoglobulin from Antarctic skates

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Abstracts / Fish & Shellfish Immunology 34 (2013) 1692–1752

(MMCs), while their distribution in infected turbot was diffuse and generalized in the parenchyma. Some fish showed positive reaction in the MMCs and ellipsoids. The number of iNOS+ cells was significantly higher in kidney of infected fish compared with control group at all sampling points. In spleen, the iNOS activity was also clearly increased in infected fish respect the control group. These findings suggest that innate immunesystem cells are actively involved in the response against ASS. In agreement with other studies, the iNOS expression was increased at early stages of infection or sepsis, and remained present until the end of experiment. The IHC against iNOS demonstrated to be a useful tool to the study of the pathogenesis of furunculosis and septic shock in fish. This work was funded by the Xunta of Galicia project 08MMA011200PR. * Corresponding author. E-mail address: [email protected] (G. Coscelli)

P-415. Investigations on immunoglobulin from Antarctic skates M.R. Coscia*, E. Cocca, S. Giacomelli, F. Cuccaro, U. Oreste. Institute of Protein Biochemistry, CNR, Via P. Castellino 111, Naples, Italy Abstract At present, investigations on Immunoglobulins (Ig) in skates remain scarce. This is particularly true in the case of Antarctic species, despite the skates are dominant component of the chondrichthyan fauna in Antarctic water. We focused on IgM, the most abundant heavy chain isotype, of the Antarctic species (Order Rajiformes) Bathyraja eatonii, Bathyraja albomaculata, Bathyraja brachyurops (subfamily Arhynchobatinae), and Amblyraja georgiana (subfamily Rajinae) which live at high latitudes in the Southern Ocean, and at very low temperature. mRNA from the spleen of individuals of each species was subjected to RT-PCR experiments using forward and reverse primers designed on the alignment of various chondrichthyan IgM heavy chain sequences deposited in GenBank. The PCR products of about 1400 nt were then cloned and sequenced. The nucleotide sequence identities between constant region domains ranged from 91.1% to 99.7% within individual species, and from 88.5% to 97.5% between species. Sequences were aligned, including also sequences of a temperate skate, Raja erinacea, and a distance tree was generated. The tree showed two well-supported major branches, one containing Arhynchobatinae sequences, the other one Rajinae sequences. Four putative D gene segments were identified in the region of the VH/D/JH recombination; two different D segments were often found in the same sequence. Multiple copies of CH genes were shown by Southern blotting, indicating that the Antarctic skates investigated exhibit a cluster type genomic organization similar to that described for other cartilaginous fish. The positional diversity of B. eatonii amino acid sequences was investigated by Shannon entropy analysis, showing CH4 as the most conserved domain, and CH3 as the most variable one. We analyzed CDR3 length distribution in the Antarctic skate B. eatonii in comparison with the temperate skate Leucoraja eglanteria. Antarctic skate CDR3 were longer, the mean length (13.4 aa) being greater than that of L. eglanteria CDR3 (7.7 aa). Taken together these results may contribute to fill the knowledge gap relative to Antarctic Rajiformes in the field of fish immunology. * Corresponding author. E-mail address: [email protected] (M.R. Coscia)

P-335. Antinutrients alone or in combination modulate immune mechanisms in gilthead seabream B. Costas 1, *, A. Couto 1, 2, R. Azeredo 1, 2, Å. Krogdahl 3,4, A. Oliva-Teles 1, 2. 1

CIMAR/CIIMAR – Centro Interdisciplinar de Investigação Marinha e Ambiental, 4050-123 Porto, Portugal; 2 Departamento de Biologia, Faculdade de Ciências da Universidade do Porto, 4169-007 Porto, Portugal;

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Aquaculture Protein Centre, Centre of Excellence, Ås, Norway; Department of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science, P.O. Box 8146 Dep., N-0033 Oslo, Norway 4

Abstract Soybean meal (SBM) is among the most common ingredients used to replace fish meal. However, its use is limited as it may induce development of enteritis. The specific agents causing such condition are not yet known, but one or more of the antinutrients found in SBM, such as lectins, protease inhibitors, saponins and phytosterols, are likely involved. A comparison of immunological parameters in fish fed a regular diet and fish exposed to antinutrients represents a unique tool for the investigation of the immunological mechanisms that are involved in the protection of the gut and the organism as a whole. Therefore, the present study intends to evaluate the effects of two purified antinutrients, soy saponins and phytosterols, in an important species for Mediterranean aquaculture. For this purpose, gilthead seabream (Sparus aurata) were fed six experimental diets containing two levels of the antinutrients, alone or in combination, and a control diet, to apparent visual satiation under controlled conditions. Blood and head-kidney were collected at 7 and 14 days following first feeding in order to evaluate humoral parameters and the expression of immune-related genes. Alternative complement pathway activity increased in fish fed antinutrients in a dose dependent manner compared to fish fed the control diet at both 7 and 14 days. While plasma lysozyme activity increased in fish fed saponins regardless of dietary inclusion at 7 days, lysozyme values augmented similarly in fish fed both antinutrients and levels compared to fish fed the control diet at 14 days. Moreover, plasma peroxidase activity and total immunoglobulins were not different among treatments at both times. Results suggest that both antinutrients may act as antigens and thus appear to boost an immune response, which seems to be earlier when fish are fed saponins. This hypothesis is in agreement with other studies that recently have proposed the existence of unidentified antigens in SBM. Some authors have also identified the involvement of T lymphocytes during the development of SBM-induced enteritis (20% increase in SBM compared to a fish meal based diet), as well as an alteration of the NF-kB signaling pathway, which is considered a general cellular response to a variety of stimuli including various stressors, infection and antigens. In the present study, several immune-related genes are now being analysed and their expression profile may give further insights regarding the mechanisms that these particular antinutrients are activating. * Corresponding author. E-mail address: [email protected] (B. Costas)

P-443. Antimicrobial responses of Senegalese sole (Solea senegalensis) primary head-kidney leucocytes against Tenacibaculum maritimum B. Costas 1, *, I. Simões 1,2, M. Castro-Cunha 3, A. Afonso 1, 2. 1

CIMAR/CIIMAR – Centro Interdisciplinar de Investigação Marinha e Ambiental, 4050-123 Porto, Portugal; 2 ICBAS – Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal; 3 A. Coelho & Castro Lda., Praça Luís de Camões, Póvoa de Varzim, Portugal Abstract Tenacibaculum maritimum has been described as the etiological agent of tenacibaculosis in marine fish. This disease is nowadays among the most threatening bacterial infections limiting the culture of many species of commercial value, including the Senegalese sole (Solea senegalensis). However, little is known regarding Senegalese sole ability to cope with this pathogen, and information on the interactions between T. maritimum and host phagocytes is still scarce. Therefore, the aim of the present study was to contribute to this endeavor by investigating the response of Senegalese sole head-kidney leucocytes against T. maritimum. Primary cell cultures in 96-well plates were used to analyze the respiratory burst by quantifying extracellular superoxide anion and nitric oxide (NO) levels following incubation with UV killed T. maritimum (strains ACC6.1 and ACC13.1; 2  105