F. Real

Characterization of the probiotic strain Vagococcus fluvialis in the protection of European sea bass (Dicentrarchus labrax) against vibriosis by Vibrio anguillarum.

Aquaculture is one of the main sources of income in many countries worldwide. Intensive farms are often affected by different infectious diseases that can decrease their final production. To control this situation, several antibiotics are frequently used with known environmental consequences. The aim of this study was to analyze different bacterial strains isolated from of gilthead sea bream, sea bass, sole and meagre guts, for use as probiotics in aquaculture. The strains were evaluated in vitro through various mechanisms of selection, such as the production of antagonistic effects against pathogens, production of antibacterial substance, adhesion to the intestinal mucus, competition for nutrients or binding site, and growth in intestinal mucus. A total of 50 bacterial strains were analyzed and only one showed excellent in vitro results for consideration as a candidate to be analyzed in vivo. The strain, identified as Vagococcus fluvialis, showed good protection against Vibrio anguillarum 975-1 in vivo in the experimental challenge, showing a relative percent survival of 42.3% higher than positive control group. Therefore, in conclusion we consider this strain to be a good candidate for use as a future probiotic in aquaculture.

The in vitro effect of probiotic Vagococcus fluvialis on the innate immune parameters of Sparus aurata and Dicentrarchus labrax.

In this study we evaluated the effect of the probiotic Vagococcus fluvialis on the cellular immune unspecific system of two different fish species of great interest in aquaculture such as gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax). Leucocytes from head kidney of the two fish species were extracted and concentration adjusted to 10(7) cells ml(-1). Phagocytic and respiratory burst activity and the peroxidase content of leucocytes were observed 30 min after incubation with the probiotic Vagococcus fluvialis alive or inactivated with heat shock or UV-light at different concentrations of 10(7), 10(8), 10(9) cfu ml(-1) (final concentration 10(6), 10(7), 10(8) cfu ml(-1)). V. fluvialis produced dose-dependent increments in respiratory burst in sea bream leucocytes. The respiratory burst activity of sea bream head kidney leucocytes incubated with 10(6) cfu ml(-1) of live and inactivated bacteria was not stimulated. The highest values of peroxidase content were observed in sea bass cells with stimulation indexes higher than 1 in HK leucocytes incubated with 10(8) cfu ml(-1) of live and inactivated bacteria. Statistical analysis revealed that differences being only significant in sea bass leucocytes where 10(8) cfu ml(-1) bacteria denote statistically significant differences (P < 0.05) respect to other concentrations. Highest values of phagocytic activity were obtained in sea bass macrophages incubated with UV-light inactivated bacteria (27.33% ± 1.45), where significantly differences with sea bass HK leucocytes were detected. Our results suggest that the in vitro assays are a useful tool to optimize the effective dose of probiotic bacteria. Although in vivo studies are necessary to confirm the immunomodulatory effect of this strain.

Invasion and survival of Photobacterium damselae subsp. piscicida in non-phagocytic cells of gilthead sea bream, Sparus aurata L.

Fluorescence microscopy and gentamicin protection assays were used to investigate the ability of four Photobacterium damselae subsp. pisicida (Phdp) strains to adhere to and to invade the fish epithelial cell line, SAF-1, derived from Sparus aurata. All strains tested were detected intracellularly using both techniques, although internalization levels varied among strains. Treatment with cytochalasin D and experiments carried out at 4 degrees C demonstrated that a functional host cell cytoskeleton and active cell metabolism are necessary for bacterial internalization. Intracellular bacteria were detected for up to 7 days with a round morphology and were stained with DAPI, indicating that some bacterial cells may remain viable inside SAF-1 cells. Our in vitro findings indicate that Phdp are capable of adhering, entering and surviving within the non-phagocytic epithelial cell line SAF-1, which may be important for persistence and establishment of a carrier state in S. aurata.

Invasion and intracellular survival of Hafnia alvei strains in human epithelial cells.

Aims:  The aim of this study was to investigate the invasion and intracellular survival of different Hafnia alvei strains in HeLa cells.

Mx expression in gilthead sea bream (Sparus aurata L.) in response to poly I:C, bacterial LPS and chromosomal DNA: preliminary study.

Cells infected with a virus are stimulated to produce and secrete IFNs, which in turn induce a complex pattern of physiological changes. The IFNs provide vertebrates with a first line of defence against viral infection. Fish are known to produce molecules with IFN activity as measured by a cell protection test [1]. The induction of IFN-I includes dsRNA virus infection, LPS and some bacterial components [2]. Mx proteins interfere with virus replication by inhibiting viral polymerases in the nucleus and binding viral components in the cytoplasm [3]. Mx genes have been cloned and characterised from several other mammals, birds and fish species [4], such as rainbow trout, Atlantic salmon, Atlantic halibut, Japanese flounder, pufferfish, sea bream [5] and Senegalese sole [6]. Production of IFN-like activity and Mx gene/protein expression in fish has been demonstrated in cells, organs and serum from several fish species [7]. Viral dsRNA and synthetic dsRNA polyinosinic: polycytidylic acid (poly I:C) are very potent inducers of IFN. Most viruses produce dsRNA at some time in their replication, and it seems that animals have evolved the ability to recognise and respond to these molecules by this innate mechanism [7]. IFN-like activity can also be stimulated by other factors such as bacterial lipopolysaccharide (LPS), bacterial DNA and inactivated vaccines against bacteria [8].

The in vitro immunomodulatory effect of extracellular products (ECPs) of Vagococcus fluvialis L21 on European sea bass (Dicentrarchus labrax) leucocytes.

The immune associated genes, interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor-α (TNF-α), ciclo-oxigenase-2 (COX-2), and Mx gene were studied by real-time PCR in head-kidney leucocytes of sea bass after incubation with the extracellular products (ECPs) of the probiotic strain Vagococcus fluvialis L21 and polyinosinic:polycytidylic acid (POLY I:C), at different times (T1.5, T6, T12, T24, T48 and T72). In general, we can observe how pro-inflammatory cytokines IL-1β, TNF-α, IL-6 and COX-2 studied displayed a strong peak after stimulation with 1.5 h of ECPs of V. fluvialis L21, significant differences (P < 0.05) exist with other periods and with the POLY I: C at the same time. Similarly to the case of IL-10 also produced a statistically significant (P < 0.05) peak of expression on leukocytes that were stimulated with the ECPs of V. fluvialis L21. In the case of Mx gene expression, we note that in almost all sampling times there is an up-regulation of the Mx gene in leucocytes incubated with ECPs and POLY I:C compared to the control and Mx expression was higher in leucocytes that were stimulated with the ECPs of V. fluvialis for all times, except in T24. With these results we can consider that the ECPs of V. fluvialis L21 have a great power of stimulating the in vitro expression of immune-related genes and may even be useful as adjuvants for vaccine in aquaculture.

Temperature influences the expression of fimbriae and flagella in Hafnia alvei strains: an immunofluorescence study

Hafnia alvei, a Gram negative bacillus related to the Enterobacteriaceae family, is considered an opportunistic pathogen of several animal species and humans. In this communication, we describe fimbrial-like structures from different strains of H. alvei that cannot be easily ascribed to any of the previously reported fimbrial types in this species (type I or type III). Polymerase chain reaction (PCR) and immunofluorescence assays were carried out to study fimbriae and flagella in H. alvei strains isolated from different sources. No correlation between the results obtained by PCR and those obtained by phenotypic methods were found, and the antibodies used gave cross or different recognition patterns of the surface structures present in these strains. We report as well that strain and growth temperature influence fimbriation and expression of flagella in human and animal isolates of H. alvei. This study also indicates that the absence of fimbriae have a significant positive influence on the initial adhesion of H. alvei to human epithelial cells.

Differential innate immune response of European seabass (Dicentrarchus labrax) against Streptococcus iniae.

Streptococcus iniae is a Gram-positive bacteria that causes invasive infections with severe septicemia and meningitis, producing high economic losses in marine and continental aquaculture. Head kidney leukocytes of European sea bass (Dicentrarchus labrax) were used to measure the differential innate immune response upon infection with S. iniae. The complete inhibition in the production of intracellular superoxide radicals and total peroxidase content was observed in infected cells. This study also elucidates changes in the relative expression of some immune-related genes. Interleukin 1β, tumor necrosis factor-α and interleukin-6 reached a peak of expression at 4-8 h post-infection, subsequently decreasing significantly up to 48 h post-infection. However, interleukin-10 and Mx protein increased over time, reaching the pick of expression at 48 h post-infection, whereas caspase-3 showed down regulation until 48 h post-infection. The in vivo study of immune related genes show the same kinetics of mRNAs expression as in vitro experience. The proinflammatory cytokines mRNA transcription levels peaked at an earlier time in vivo than in vitro system. Our findings indicate that there is a direct relationship between the dissemination of bacteria and the resulting infection-associated inhibition of respiratory burst, apoptosis, and the pro- and anti-inflammatory gene expression profiles.

Effect of lipopolysaccharides from Vibrio alginolyticus on the Mx gene expression and virus recovery from gilthead sea bream (Sparus aurata L.) experimentally infected with Nodavirus

Infections with nodavirus affect a wild and farmed fish species throughout the world, mostly from the marine environment. The aim of this work was to determine the immune status of gilthead sea bream that comes as a result of a Nodavirus infection, induced by activation of the interferon response pathway by lipopolysaccharides from Vibrio alginolyticus and the expression of interferoninduced Mx protein in liver samples. The enhancement of Mx protein gene expression was detected in liver samples of experimentally nodavirus infected fish and, furthermore, the immunostimulant LPS of V. alginolyticus decreased almost three times the virus titration with respect to no-immunized or infected with nodavirus group of fish.

Streptococcus iniae in Gilthead Seabream (Sparus aurata, L.) and Red Porgy (Pagrus pagrus, L.) Ultrastructural Analysis

Streptococcosis caused by Streptococcus iniae has become one of the most serious marine and freshwater aquatic diseases in the past decade, causing large losses in farmed and wild fish worldwide. In this study, we performed an ultrastructural study of major lesions in gilthead seabream Sparus aurata and red porgy Pagrus pagrus experimentally infected with the S. iniae IUSA-1 strain, isolated in a natural outbreak in Spain in the mentioned species. The transmission electron micrographs revealed the resistance of this pathogen inside the phagosome, indicating that the macrophage may provide a significant bacterial reservoir for continuing infection, disease dissemination, and tissue injury by crossing the blood-brain barrier.