Antonio Figueras

Isolation of Vibrio alginolyticus and Vibrio splendidus from Aquacultured Carpet Shell Clam (Ruditapes decussatus) Larvae Associated with Mass Mortalities

ABSTRACT Two episodes of mortality of cultured carpet shell clams (Ruditapes decussatus) associated with bacterial infections were recorded during 2001 and 2002 in a commercial hatchery located in Spain. Vibrio alginolyticus was isolated as the primary organism from moribund clam larvae that were obtained during the two separate events. Vibrio splendidus biovar II, in addition to V. alginolyticus, was isolated as a result of a mixed Vibrio infection from moribund clam larvae obtained from the second mortality event. The larval mortality rates for these events were 62 and 73%, respectively. Mortality was also detected in spat. To our knowledge, this is the fist time that these bacterial species have been associated with larval and juvenile carpet shell clam mortality. The bacterial strains were identified by morphological and biochemical techniques and also by PCR and sequencing of a conserved region of the 16S rRNA gene. In both cases bacteria isolated in pure culture were inoculated into spat of carpet shell clams by intravalvar injection and by immersion. The mortality was attributed to the inoculated strains, since the bacteria were obtained in pure culture from the soft tissues of experimentally infected clams. V. alginolyticus TA15 and V. splendidus biovar II strain TA2 caused similar histological lesions that affected mainly the mantle, the velum, and the connective tissue of infected organisms. The general enzymatic activity of both live cells and extracellular products (ECPs), as evaluated by the API ZYM system, revealed that whole bacterial cells showed greater enzymatic activity than ECPs and that the activity of most enzymes ceased after heat treatment (100°C for 10 min). Both strain TA15 and strain TA2 produced hydroxamate siderophores, although the activity was greater in strain TA15. ECPs from both bacterial species at high concentrations, as well as viable bacteria, caused significant reductions in hemocyte survival after 4 h of incubation, whereas no significant differences in viability were observed during incubation with heat-killed bacteria.

Insights into the innate immunity of the Mediterranean mussel Mytilus galloprovincialis

BackgroundSessile bivalves of the genus Mytilus are suspension feeders relatively tolerant to a wide range of environmental changes, used as sentinels in ecotoxicological investigations and marketed worldwide as seafood. Mortality events caused by infective agents and parasites apparently occur less in mussels than in other bivalves but the molecular basis of such evidence is unknown. The arrangement of Mytibase, interactive catalogue of 7,112 transcripts of M. galloprovincialis, offered us the opportunity to look for gene sequences relevant to the host defences, in particular the innate immunity related genes.ResultsWe have explored and described the Mytibase sequence clusters and singletons having a putative role in recognition, intracellular signalling, and neutralization of potential pathogens in M. galloprovincialis. Automatically assisted searches of protein signatures and manually cured sequence analysis confirmed the molecular diversity of recognition/effector molecules such as the antimicrobial peptides and many carbohydrate binding proteins. Molecular motifs identifying complement C1q, C-type lectins and fibrinogen-like transcripts emerged as the most abundant in the Mytibase collection whereas, conversely, sequence motifs denoting the regulatory cytokine MIF and cytokine-related transcripts represent singular and unexpected findings. Using a cross-search strategy, 1,820 putatively immune-related sequences were selected to design oligonucleotide probes and define a species-specific Immunochip (DNA microarray). The Immunochip performance was tested with hemolymph RNAs from mussels injected with Vibrio splendidus at 3 and 48 hours post-treatment. A total of 143 and 262 differentially expressed genes exemplify the early and late hemocyte response of the Vibrio-challenged mussels, respectively, with AMP trends confirmed by qPCR and clear modulation of interrelated signalling pathways.ConclusionsThe Mytibase collection is rich in gene transcripts modulated in response to antigenic stimuli and represents an interesting window for looking at the mussel immunome (transcriptomes mediating the mussel response to non-self or abnormal antigens). On this basis, we have defined a new microarray platform, a mussel Immunochip, as a flexible tool for the experimental validation of immune-candidate sequences, and tested its performance on Vibrio-activated mussel hemocytes. The microarray platform and related expression data can be regarded as a step forward in the study of the adaptive response of the Mytilus species to an evolving microbial world.

Pasteurellosis in cultured gilthead seabream (Sparus aurata): first report in Spain

Abstract The first documented epizootic of pasteurellosis in juvenile gilthead seabream ( Sparus aurata ) cultured in Spain is described. The affected fish showed no apparent surface lesions and only some of them displayed slight haemorrhagic areas in the head and gills. However, fish often exhibited enlarged spleen with typical whitish tubercles. Losses of approximately 26 000 fingerlings (40% of the stock) occurred over a 4-week period (August–September 1990). Microbiological analysis of moribund fish revealed the presence in pure culture in all the organs examined of a bacterium which was characterized biochemically and serologically as Pasteurella piscicida . The isolate was sensitive to most antimicrobial agents tested, and the epizootic was effectively controlled by oral administration of chloramphenicol and oxytetracycline. The virulence assays revealed that the P. piscicida isolate was pathogenic for gilthead seabream with a LD 50 ranging from 1.6 × 10 5 to 9.5 × 10 5 (depending on the fish size), and also for turbot ( Scophthalmus maximus ) with LD 50 ≤ 1.6 × 10 4 . The failure to recover the inoculated strain from the survivor gilthead seabream indicates that the carrier state of P. piscicida in the infected population cannot be demonstrated. The histopathological changes observed in the internal organs of diseased fish are typical of a bacterial septicaemia showing extensive, acute multifocal necrosis in spleen and kidney with large masses of bacterial cells.

Immune response of zebrafish (Danio rerio) against a newly isolated bacterial pathogen Aeromonas hydrophila

A strain of Aeromonas hydrophila associated with unusual mortalities in zebrafish (Danio rerio) culture facilities was isolated, identified and characterized. In challenge experiments, adult zebrafish were susceptible to infection by intraperitoneal (i.p.) injection with viable bacteria and its extracellular products (ECPs) reaching very high mortalities in a few hours. The infection, by the viable bacteria or the ECPs, caused cell death in kidney, due to the cytotoxic and haemolytic activities of the bacterial ECPs. Moreover, the infection affected the release of oxygen (ROS) and nitrogen (NO) reactive free radicals. To determine if this A. hydrophila infection induces an inflammatory response, mRNA expression levels of tumour necrosis factor-alpha (TNFalpha), interleukin-1beta (IL-1beta), interferon-gamma (IFNgamma) and inducible nitric oxide (iNOS) were assessed by real time PCR. The expression levels of TNFalpha, IL-1beta and IFNgamma were upregulated in the kidneys of infected zebrafish with viable bacteria, heat-killed bacteria and ECPs. Expression levels of iNOS were upregulated by ECPs. Mortality rate (LD(50)) and histopathology were also determined.

Bacterial communities from shoreline environments (Costa da Morte, Northwestern Spain) affected by the Prestige oil spill

ABSTRACT The bacterial communities in two different shoreline matrices, rocks and sand, from the Costa da Morte, northwestern Spain, were investigated 12 months after being affected by the Prestige oil spill. Culture-based and culture-independent approaches were used to compare the bacterial diversity present in these environments with that at a nonoiled site. A long-term effect of fuel on the microbial communities in the oiled sand and rock was suggested by the higher proportion of alkane and polyaromatic hydrocarbon (PAH) degraders and the differences in denaturing gradient gel electrophoresis patterns compared with those of the reference site. Members of the classes Alphaproteobacteria and Actinobacteria were the prevailing groups of bacteria detected in both matrices, although the sand bacterial community exhibited higher species richness than the rock bacterial community did. Culture-dependent and -independent approaches suggested that the genus Rhodococcus could play a key role in the in situ degradation of the alkane fraction of the Prestige fuel together with other members of the suborder Corynebacterineae. Moreover, other members of this suborder, such as Mycobacterium spp., together with Sphingomonadaceae bacteria (mainly Lutibacterium anuloederans), were related as well to the degradation of the aromatic fraction of the Prestige fuel. The multiapproach methodology applied in the present study allowed us to assess the complexity of autochthonous microbial communities related to the degradation of heavy fuel from the Prestige and to isolate some of their components for a further physiological study. Since several Corynebacterineae members related to the degradation of alkanes and PAHs were frequently detected in this and other supralittoral environments affected by the Prestige oil spill along the northwestern Spanish coast, the addition of mycolic acids to bioremediation amendments is proposed to favor the presence of these degraders in long-term fuel pollution-affected areas with similar characteristics.

MytiBase: a knowledgebase of mussel (M. galloprovincialis) transcribed sequences

BackgroundAlthough Bivalves are among the most studied marine organisms due to their ecological role, economic importance and use in pollution biomonitoring, very little information is available on the genome sequences of mussels. This study reports the functional analysis of a large-scale Expressed Sequence Tag (EST) sequencing from different tissues of Mytilus galloprovincialis (the Mediterranean mussel) challenged with toxic pollutants, temperature and potentially pathogenic bacteria.ResultsWe have constructed and sequenced seventeen cDNA libraries from different Mediterranean mussel tissues: gills, digestive gland, foot, anterior and posterior adductor muscle, mantle and haemocytes. A total of 24,939 clones were sequenced from these libraries generating 18,788 high-quality ESTs which were assembled into 2,446 overlapping clusters and 4,666 singletons resulting in a total of 7,112 non-redundant sequences. In particular, a high-quality normalized cDNA library (Nor01) was constructed as determined by the high rate of gene discovery (65.6%). Bioinformatic screening of the non-redundant M. galloprovincialis sequences identified 159 microsatellite-containing ESTs. Clusters, consensuses, related similarities and gene ontology searches have been organized in a dedicated, searchable database http://mussel.cribi.unipd.it.ConclusionWe defined the first species-specific catalogue of M. galloprovincialis ESTs including 7,112 unique transcribed sequences. Putative microsatellite markers were identified. This annotated catalogue represents a valuable platform for expression studies, marker validation and genetic linkage analysis for investigations in the biology of Mediterranean mussels.

Control of Vibrio alginolyticus in Artemia culture by treatment with bacterial probiotics

Abstract In order to determine the influence of six lactic acid bacterial (LAB) strains, lactic acid and major extracellular products (ECPs) of LAB on the bacterial load of Artemia culture, two experiments were carried out. In both experiments, the dominant bacterial species were identified as Vibrio alginolyticus. The treatment with Lactobacillus brevis (1×108 bacteria/ml) and lactic acid (0.1 g/l) reduced the load of this bacterium in the Artemia culture water. Biochemical and morphological characteristics of the V. alginolyticus strains isolated from Artemia culture were determined. For comparative purposes, another V. alginolyticus strain isolated from diseased turbot larvae was also included. The in vitro antibacterial activity of extracellular products (ECPs) from LAB, as well as lactic acid (0.1 and 0.05 g/l), was assessed against both V. alginolyticus strains. ECPs from LAB were able to inhibit the growth of V. alginolyticus, especially those from L. brevis. In the case of lactic acid treatments, no significant inhibition of V. alginolyticus growth was observed.

Zebrafish fin immune responses during high mortality infections with viral haemorrhagic septicemia rhabdovirus. A proteomic and transcriptomic approach

BackgroundDespite rhabdoviral infections being one of the best known fish diseases, the gene expression changes induced at the surface tissues after the natural route of infection (infection-by-immersion) have not been described yet. This work describes the differential infected versus non-infected expression of proteins and immune-related transcripts in fins and organs of zebrafish Danio rerio shortly after infection-by-immersion with viral haemorrhagic septicemia virus (VHSV).ResultsTwo-dimensional differential gel electrophoresis detected variations on the protein levels of the enzymes of the glycolytic pathway and cytoskeleton components but it detected very few immune-related proteins. Differential expression of immune-related gene transcripts estimated by quantitative polymerase chain reaction arrays and hybridization to oligo microarrays showed that while more transcripts increased in fins than in organs (spleen, head kidney and liver), more transcripts decreased in organs than in fins. Increased differential transcript levels in fins detected by both arrays corresponded to previously described infection-related genes such as complement components (c3b, c8 and c9) or class I histocompatibility antigens (mhc1) and to newly described genes such as secreted immunoglobulin domain (sid4), macrophage stimulating factor (mst1) and a cluster differentiation antigen (cd36).ConclusionsThe genes described would contribute to the knowledge of the earliest molecular events occurring in the fish surfaces at the beginning of natural rhabdoviral infections and/or might be new candidates to be tested as adjuvants for fish vaccines.

The C1q domain containing proteins of the Mediterranean mussel Mytilus galloprovincialis: A widespread and diverse family of immune-related molecules

The key component of the classical complement pathway C1q is regarded as a major connecting link between innate and acquired immunity due to the highly adaptive binding properties of its trimeric globular domain gC1q. The gC1q domain also characterizes many non-complement proteins involved in a broad range of biological processes including apoptosis, inflammation, cell adhesion and cell differentiation. In molluscs and many other invertebrates lacking of adaptive immunity, C1q domain containing (C1qDC) proteins are abundant, they most probably emerged as lectins and subsequently evolved in a specialized class of pattern recognition molecules through the expanding interaction properties of gC1q. Here we report the identification of 168 C1qDC transcript sequences of Mytilus galloprovincialis. The remarkable abundance of C1qDC transcripts in the Mediterranean mussel suggests an evolutionary strategy of gene duplication, functional diversification and selection of many specific C1qDC variants. A comprehensive transcript sequence survey in Protostomia also revealed that the C1qDC family expansion observed in mussel could have occurred in some specific taxa independently from the events leading to the establishment of a large complement of C1qDC genes in the Chordates lineage.

β-Glucan administration enhances disease resistance and some innate immune responses in zebrafish (Danio rerio)

The present study was conducted to investigate the effect of beta-glucan (derived from Saccharomyces cerevisiae) on the immune response and its protection against an infection of the bacterial pathogen Aeromonas hydrophila in zebrafish (Danio rerio). Zebrafish received beta-glucan by intraperitoneal injection at three different concentrations (5, 2 and 0.5 mgml(-1)) at 6, 4 and 2 days prior the challenge. On challenge day the control and beta-glucan pretreated zebrafish were intraperitoneally injected with A. hydrophila and mortality was recorded for 4 days. Intraperitoneal injection of 5 mgml(-1) of beta-glucan significantly reduced the mortality. A single injection of 5 mgml(-1) of beta-glucan 6 days before challenge also enhanced significantly the survival against the infection. The treatment with beta-glucan increased the myelomonocytic cell population in the kidney at 6h postchallenge with A. hydrophila. Moreover it enhanced the ability of kidney cells to kill A. hydrophila. beta-glucan did not affect the expression of TNFalpha or IL-1 beta but seemed to modulate IFNgamma and chemokine expression in kidney.