Moving rom Histopathology to Molecular Tools in the Diagnosis of Molluscs Diseases of Concern under EU Legislation

10 páginas, 2 tablas.-- This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these termsOne of the main factors limiting molluscs production is the presence of pathogens and diseases. Disease agent transfer via transfers of live molluscs has been a major cause of disease outbreaks and epizootics. Because of that, the European Union has adopted several decisions and directives, the last in 2006 (2006/88/EC) to control movements of marine organisms over the European countries. Once the disease is established in a determined area its eradication is a complicated task because life cycle of pathogens are not completely known and only a good and early diagnosis of the disease could be the most appropriate way to deal with it. Besides, molluscs do not have an adaptive immune response and vaccination strategies are not possible. Molluscs listed diseases under EU legislation are mainly protozoan parasites, that's why histological techniques are recognized for their diagnosis. However, molecular techniques are being increasingly used primarily as confirmatory techniques of the presence of the pathogens but also in disease monitoring programs. Research perspectives are mainly focussed in the optimization, of the already described techniques to gain in sensitivity and sensibility and in the development of new molecular biology techniques (quantitative real time PCRs), that are faster and easier to apply and that allow a positive diagnosis even in early stages of infection. However, molecular tools detect DNA sequences of the pathogen which does not imply that pathogen is viable in the cell host and the infection is established. Consequently, it needs to be validated against other techniques, such as histology or in situ hybridization, so that its reliability can be determinedAuthors want to thanks the Ministerio de Agricultura, Alimentacion y Medio Ambiente, to CSIC (project 201640E024) and to Ministerio de Economía y competitividad (project AGL2015-65705-R) for their financial supportPeer reviewe

In vitro culture of Perkinsus atlanticus, a parasite of the carpet shell clam Ruditapes decussatus

8 pages, 8 figures.The ability to mass-culture parasites in vitro facilitates biological, metabolic and morphological research that would otherwise be difficult to accomplish. In vitro continuous culture of the protistan Perkinsus atlanticus from carpet-shell clam was established and variables including culture medium, inoculum size, temperature and salinity, which affect parasite proliferation, were studied. This parasite can adapt to very different culture media and salinity and temperature conditions, and the inoculum density does not affect the final cellular concentration attained in vitro. The morphology of cultured P. atlanticus is described and its optimum in vitro growth conditions were compared with those for P. marinus, a parasite of the eastern oyster Crassostrea virginica. Cryopreservation of cultured P. atlanticus was developed to assure the long-term storage of this parasite.The work was partially funded by CICYT (Spain, Acción especial MAR 95-1862-E). M.C.O. thanks Xunta de Galicia for a research fellowship.Peer reviewe

Cell mediated immune response of the Mediterranean sea urchin Paracentrotus lividus after PAMPs stimulation

9 páginas, 5 figuras, 1 tablaThe Mediterranean sea urchin (Paracentrotus lividus) is of great ecological and economic importance for the European aquaculture. Yet, most of the studies regarding echinoderm's immunological defense mechanisms reported so far have used the sea urchin Strongylocentrotus purpuratus as a model, and information on the immunological defense mechanisms of Paracentrotus lividus and other sea urchins, is scarce. To remedy this gap in information, in this study, flow cytometry was used to evaluate several cellular immune mechanisms, such as phagocytosis, cell cooperation, and ROS production in P. lividus coelomocytes after PAMP stimulation. Two cell populations were described. Of the two, the amoeboid-phagocytes were responsible for the phagocytosis and ROS production. Cooperation between amoeboid-phagocytes and non-adherent cells resulted in an increased phagocytic response. Stimulation with several PAMPs modified the phagocytic activity and the production of ROS. The premise that the coelomocytes were activated by the bacterial components was confirmed by the expression levels of two cell mediated immune genes: LPS-Induced TNF-alpha Factor (LITAF) and macrophage migration inhibitory factor (MIF). These results have helped us understand the cellular immune mechanisms in P. lividus and their modulation after PAMP stimulationThis work has been funded by the National Project A/026000/09AECID, 2010–2012 “Respuesta inmune de invertebrados marinos”Peer reviewe

Evaluation of immunomodulatory effects of lactic acid bacteria in turbot (Scophthalmus maximus)

7 pages, 4 figures.In the present work, the effects of several lactic acid bacteria on the immune response of turbot (Scophthalmus maximus) macrophages have been studied both in vitro and in vivo. Out of six lactic acid bacterial strains tested, only heat-killed Lactococcus lactis significantly increased the turbot head kidney macrophage chemiluminescent (CL) response after 24 h of incubation. Nitric oxide (NO) was also significantly enhanced by this bacterium after 72 h of incubation with either viable (103 and 106 cells/ml) or heat-killed (106 cells/ml) bacteria. Viable Leuconostoc mesenteroides (106 cells/ml) was also capable of significantly increasing NO production. Since L. lactis proved to be the strain with more effects on the host immune function, further in vivo and in vitro experiments were conducted with this bacterium. The in vitro capacity of L. lactis to adhere to turbot intestinal mucus was positively confirmed. When orally administered, L. lactis significantly increased the macrophage CL response and the serum NO concentration after 7 days of daily administration. The antibacterial effect of the extracellular products from the six LAB strains against the fish-pathogenic bacterium Vibrio anguillarum was also demonstrated in vitro.This work was partially supported by the project 1FD97-0044-C03-03 from FEDER funds and a grant from Caixa Galicia (Spain). L. Villamil acknowledges the University of Vigo for a research fellowship. C. Tafalla acknowledges the Consejo Superior de Investigaciones Científicas (CSIC) for a research fellowship.Peer reviewe

Extending Immunological Profiling in the Gilthead Sea Bream, Sparus aurata, by Enriched cDNA Library Analysis, Microarray Design and Initial Studies upon the Inflammatory Response to PAMPs

21 páginas, 7 figuras, 3 tablas.-- Sebastian Boltaña ... et al.-- This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) licenseThis study describes the development and validation of an enriched oligonucleotide-microarray platform for Sparus aurata (SAQ) to provide a platform for transcriptomic studies in this species. A transcriptome database was constructed by assembly of gilthead sea bream sequences derived from public repositories of mRNA together with reads from a large collection of expressed sequence tags (EST) from two extensive targeted cDNA libraries characterizing mRNA transcripts regulated by both bacterial and viral challenge. The developed microarray was further validated by analysing monocyte/macrophage activation profiles after challenge with two Gram-negative bacterial pathogen-associated molecular patterns (PAMPs; lipopolysaccharide (LPS) and peptidoglycan (PGN)). Of the approximately 10,000 EST sequenced, we obtained a total of 6837 EST longer than 100 nt, with 3778 and 3059 EST obtained from the bacterial-primed and from the viral-primed cDNA libraries, respectively. Functional classification of contigs from the bacterial- and viral-primed cDNA libraries by Gene Ontology (GO) showed that the top five represented categories were equally represented in the two libraries: metabolism (approximately 24% of the total number of contigs), carrier proteins/membrane transport (approximately 15%), effectors/modulators and cell communication (approximately 11%), nucleoside, nucleotide and nucleic acid metabolism (approximately 7.5%) and intracellular transducers/signal transduction (approximately 5%). Transcriptome analyses using this enriched oligonucleotide platform identified differential shifts in the response to PGN and LPS in macrophage-like cells, highlighting responsive gene-cassettes tightly related to PAMP host recognition. As observed in other fish species, PGN is a powerful activator of the inflammatory response in S. aurata macrophage-like cells. We have developed and validated an oligonucleotide microarray (SAQ) that provides a platform enriched for the study of gene expression in S. aurata with an emphasis upon immunity and the immune responseThis study was supported by the Consolider-Ingenio Programme 2010, project CSD2007-0002 funded by the Spanish Ministry of Science and Education, Spain to SM, and FONDAP (15110027) and FONDECYT 1150585 awarded by CONICYT-Chile to Sebastian BoltañaWe acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).This study was supported by the Consolider-Ingenio Programme 2010, project CSD2007-0002 funded by the Spanish Ministry of Science and Education, Spain to SM, and FONDAP (15110027) and FONDECYT 1150585 awarded by CONICYT-Chile to Sebastian Boltaña.Peer reviewe

Characterisation, expression and ontogeny of interleukin-6 and its receptors in zebrafish (Danio rerio)

10 páginas, 8 figuras, 2 tablasInterleukin-6 (IL-6) is one of the most pleiotropic cytokines due to its importance in both innate and adaptive immune responses and other physiological processes. In this study, we identified the zebrafish (Danio rerio) IL-6 homologue by investigating the synteny between the human (Homo sapiens), the fugu (Takifugu rubripes) and the zebrafish genome. Although zebrafish IL-6 showed a low sequence homology with other IL-6 sequences in other species, it presented a high structural similarity to human IL-6. We also analysed IL-6 expression in several different tissues, along with analysis of the expression of the genes that form the IL-6 receptor complex, IL-6R and gp130. After treatment with bacterial or viral stimuli, zebrafish IL-6 expression was modulated in a manner similar to that of other proinflammatory molecules, such as IL-1β and TNF-α. The expression of IL-6, IL-6R and gp130 was also studied during the ontogeny of zebrafish larvae using quantitative PCR and in situ hybridisation. Our results indicated that the transcripts were detected very early, increased during the first week of life and were predominantly expressed in the head, epidermis and neuromasts of the anterior and posterior lateral line system, suggesting their involvement in the normal development of these tissues.We want to thank the funding from the project CSD2007-00002 “Aquagenomics” of the program Consolider-Ingenio 2010 from the Spanish Ministerio de Ciencia e Innovación. M. Varela gratefully acknowledges the JAE Program, co financed by CSIC and European Social Funds, for a predoctoral grant.Peer reviewe

Zebra Fish Lacking Adaptive Immunity Acquire an Antiviral Alert State Characterized by Upregulated Gene Expression of Apoptosis, Multigene Families, and Interferon-Related Genes

16 páginas, 5 figuras, 4 tablas.-- Pablo García-Valtanen et al.--This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY)To investigate fish innate immunity, we have conducted organ and cell immune-related transcriptomic as well as immunohistologic analysis in mutant zebra fish (Danio rerio) lacking adaptive immunity (rag1−/−) at different developmental stages (egg, larvae, and adult), before and after infection with spring viremia carp virus (SVCV). The results revealed that, compared to immunocompetent zebra fish (rag1+/+), rag1−/− acquired increased resistance to SVCV with age, correlating with elevated transcript levels of immune genes in skin/fins and lymphoid organs (head kidney and spleen). Gene sets corresponding to apoptotic functions, immune-related multigene families, and interferon-related genes were constitutively upregulated in uninfected adult rag1−/− zebra fish. Overexpression of activated CASPASE-3 in different tissues before and after infection with SVCV further confirmed increased apoptotic function in rag1−/− zebra fish. Concurrently, staining of different tissue samples with a pan-leukocyte antibody marker showed abundant leukocyte infiltrations in SVCV-infected rag1−/− fish, coinciding with increased transcript expression of genes related to NK-cells and macrophages, suggesting that these genes played a key role in the enhanced immune response of rag1−/− zebra fish to SVCV lethal infection. Overall, we present evidence that indicates that rag1−/− zebra fish acquire an antiviral alert state while they reach adulthood in the absence of adaptive immunity. This antiviral state was characterized by (i) a more rapid response to viral infection, which resulted in increased survival, (ii) the involvement of NK-cell- and macrophage-mediated transcript responses rather than B- and/or T-cell dependent cells, and (iii) enhanced apoptosis, described here for the first time, as well as the similar modulation of multigene family/ interferon-related genes previously associated to fish that survived lethal viral infections. From this and other studies, it might be concluded that some of the characteristics of mammalian trained immunity are present in lower vertebratesThis work was supported by INIA project RTA2013-00008-00-00, CICYT project AGL2014-51773-C3, AGL2014-53190 REDC, BIO2011- 23400, and BIO2014-52655-R of the Ministerio de Economía y Competitividad of Spain.Peer reviewe

A Status Assessment of Perkinsiosis, Bonamiosis, and Mateiliosis in Commercial Marine Bivalves from Southern Brazil

14 páginas, 1 figura, 3 tablas.-- Simone Sühnel ... et al.The protozoans Perkinsus marinus, Perkinsus olseni, Bonamia ostreae, Bonamia exitiosa, and Marteilia refringens are responsible for some of the most detrimental diseases in the production of cultivated shellfish worldwide and are classified as notifiable diseases by the World Organization for Animal Health (OIE). This study examined the general health status of wild and cultured bivalves from southern Brazil and included diagnostic tests for the presence of Perkinsus sp., Bonamia sp., andMarteilia sp. Cultured bivalves included the mangrove oyster Crassostrea gasar (syn. Crassostrea brasiliana), the brown mussel Perna perna, the lion's paw scallop Nodipecten nodosus, andwing pearl oyster Pteria hirundo. Themangrove oyster Crassostrea rhizophorae and the carib pointed venus clam Anomalocardia brasiliana (syn. Anomalocardia flexuosa) were collected from wild populations. A variety of parasitic or commensal organisms were detected by histology including Ancistrocoma-like and Spenophrya-like organisms; Bucephalus genus, Nematopsis sp., Steinhausia sp., and Tylocephalum sp.; unidentified trematode; unknown protozoan and metazoans; and an amoeba parasite. Hemocytic infiltration was most commonly associated with parasitized animals. Histological, culture and molecular diagnostic tests did not find any evidence of the presence of OIE-listed pathogens or related species in this study. Although the current survey did not identify any pathogens or diseases of concern, it provides baseline health assessment data for these species against which any future disease developments or significant changes in population health can be compared. These data are also valuable with respect to the development and implementation of public policies related to aquatic animal healthWe would like to thank to the Ministry of Fisheries and Aquaculture, Brazil, for the research support provided to the Fundaçao de Amparo à Pesquisa do Estado de Santa Catarina (FAPESC), Brazil; the National Council of Technological and ScientificDevelopment, Brazil (CNPq-558222/2008-0), for supporting themacro- andmicroscopical analysis and for providing a grant (CNPq-301072/2007-8); the Coordenaçao de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES), Brazil, for post-doctoral scholarship support for SS; the Pacific Biologic Station and Vancouver Island University (VIU) in Canada for supporting the molecular studiesPeer reviewe

Settlement and post-larvae behaviour of Mytitus galtoprovincialis: field and laboratory experiments

11 pages, 6 figures, 3 tables.Field sampling carried out in the Rfa de Vigo (NW Spain) from 1991 to 1993 showed that Mytilus galloprovincialis settle directly from the plankton onto substrates exposed to various environmental conditions: exposed rocky shore; protected rocky shore; exposed, raft mussel-culture area; and protected, raft mussel-culture area. For direct settlement, competent larvae may use a wide variety of substrates: filamentous nylon ropes; the byssus and intricately arranged material in the bottom of mussel beds; filamentous, thallus and membranous algae; and rugosities on adult mussel shells. The peak of settlement occurred from spring to early autumn and differences in the settlement abundance among localities were influenced by currents. After this peak, the settlement of larger post-larvae continued, associated with the increase in storms during autumn that detach them from their original substrates. This dispersion phase allows for the possibility of colonising, or recruiting on other areas, even during the post-spawning season when the presence of small post-larvae is at its minimum. Laboratory experiments carried out with post-larval stages from 0.250 to 2.000 mm showed that under static water conditions they crawl and form clumps, but do not search for a specific substrate. If they are not disturbed, they may remain in their original place of settlement. Conversely, under moving water conditions they attach to a wide variety of substrates, particularly to byssal filaments and thalli of red algae Ceramium rubrum. The contact and attachment to substrates is carried out with a long mucous thread that also aids in forming clumps. The use of this mucus to settle results in a 'preference' for natural filamentous substrates but also in settlement on rugous hard surfaces. An alternative hypothesis to the primary and secondary settlement pattern previously described in the literature for Mytilus edulis is suggested.J.C.-M. was supported by a grant from the Consejo Nacional de Ciencia y Tecnologia (CONACyT). Mexico, and by the Consejo Superior de Investigacion Cientifica (CSIC), Spain. J.A.F.R. has a grant from the Xunta de Galicia.Peer reviewe

Cell mediated immune response of the Mediterranean urchin Paracentrotus lividus to PAMPs stimulation

Póster presentado en el 13th International Society of Developmental and Comparative Immunology Congress, Murcia, 28 junio - 3 julio 2015The Mediterranean sea urchin (Paracentrotus lividus) is of great ecological and economic importance for the European aquaculture. Few studies explain how this animal interacts with pathogens and which immune mechanisms are induced to overcome the diseases. The immune system involves humoral and cellular components. The immune cells are coelomocytes and move in the coelomic spaces. There is not a single standard classification of coelomocytes for all echinoderms since they are heterogeneous in morphology and size. The immune functions of each type of coelomocyte are still not totally understood, but it is postulated that amoeboid-phagocytic cells and spherule cells are the only cellular components of the immune systemPeer reviewe