Imanol Ruiz-Zarzuela

Changes in intestinal microbiota and humoral immune response following probiotic administration in brown trout ( Salmo trutta )

We studied the effect of several lactic acid bacteria (LAB) on the humoral response of brown trout (Salmo trutta). LAB groups (Lactococcus (Lc.) lactis ssp. lactis, Lactobacillus (Lb.) sakei and Leuconostoc (Leu.) mesenteroides) were administered orally at 10(6) colony-forming units/g feed to brown trout for 2 weeks, after which fish were switched to an unsupplemented feed. Blood and intestinal samples were taken from the onset of feeding supplemented diets at 1, 2, 3 and 4 weeks. During the LAB-feeding period, Lc. lactis ssp. lactis, Lb. sakei and Leu. mesenteroides persisted in the fish intestines, but the number of LAB slowly decreased in the intestines after changing to the unsupplemented diet. Only Lb. lactis ssp. lactis and Leu. mesenteroides were detected at levels above 1 x 10(2) colony-forming units/g at the end of the fourth week. In comparison to untreated control fish, the alternative complement activity in the serum was found to be significantly greater in all LAB groups at the end of the second week. Groups supplemented with Lc. lactis ssp. lactis and Leu. mesenteroides exhibited an elevated level of lysozyme activity at the end of the third week, but the group supplemented with Lb. sakei did not exhibit any significant change in lysozyme activity. Serum immunoglobulin levels were higher compared with the control group, but there was no significant difference between the LAB and control groups.

Host-microbiota interactions within the fish intestinal ecosystem.

Teleost fish are in direct contact with the aquatic environment, and are therefore in continual contact with a complex and dynamic microbiota, some of which may have implications for health. Mucosal surfaces represent the main sites in which environmental antigens and intestinal microbiota interact with the host. Thus, the gut-associated lymphoid tissues (GALT) must develop mechanisms to discriminate between pathogenic and commensal microorganisms. Colonization of intestinal mucosal surfaces with a normal microbiota has a positive effect on immune regulatory functions of the gut, and disturbance in these immune regulatory functions by an imbalanced microbiota may contribute to the development of diseases. Significant attention has therefore been recently focused on the role of probiotics in the induction or restoration of a disturbed microbiota to its normal beneficial composition. Given this, this article explores the fascinating relationship between the fish immune system and the bacteria that are present in its intestinal microbiota, focusing on the bacterial effect on the development of certain immune responses.

Expression of immune-related genes in rainbow trout (Oncorhynchus mykiss) induced by probiotic bacteria during Lactococcus garvieae infection

The aim of the present study was to investigate the effect of lactic acid bacteria (LAB) on the control of lactococcosis as well as to assess the impact of probiotics on the expression of immune-related genes in the head kidney and intestine of rainbow trout (Oncorhynchus mykiss). Lactobacillus plantarum, Lactococcus lactis and Leuconostoc mesenteroides, were administered orally at 10⁶ CFU g⁻¹ feed to fish for 36 days. Twenty-one days after the start of the feeding period, fish were challenged with Lactococcus garvieae. Only the fish fed the diet containing Lb. plantarum showed significantly (P < 0.05) improved protection against L. garvieae compared to the control. Subsequently, real-time PCR was employed to determine the mRNA levels of IL-1β, IL-8, IL-10 and TNF-α in the head kidney, and IL-8, Tlr5 and IgT in the intestine of the control and Lb. plantarum groups. IL-1β, IL-10 and TNF-α gene expression were significantly up-regulated by Lb. plantarum. Moreover, the mRNA levels of IL-10, IL-8 and IgT were significantly higher in the Lb. plantarum group after L. garvieae infection, suggesting that Lb. plantarum can stimulate the immune response of rainbow trout. PCR-DGGE revealed no detectable levels of the probiotics or the pathogen present on the distal intestinal mucosa. These findings demonstrate that direct probiotic-host interactions with the intestine are not always necessary to induce host stimulatory responses which ultimately enhance disease resistance. Furthermore, as L. garvieae did not colonise the intestinal tract, and therefore likely did not infect via this route, the antagonistic properties of the probiotic candidate towards L. garvieae were likely of little influence in mediating the improved disease resistance which could be attributed to the elevated immunological response.

Lactococcus lactis subsp. tructae subsp. nov. isolated from the intestinal mucus of brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss)

The species Lactococcus lactis currently includes three subspecies; L. lactis subsp. lactis and L. lactis subsp. cremoris, isolated from milk sources, and L. lactis subsp. hordniae, isolated from the leafhopper Hordnia circellata. In this study, three strains, designated L105(T), I3 and L101, were isolated from the intestinal mucus of brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss). These strains were closely related to members of the species Lactococcus lactis. Strain L105(T) showed 99.4 % 16S rRNA gene sequence similarity to that of the type strains L. lactis subsp. lactis NCDO 604(T) and L. lactis subsp. hordniae NCDO 2181(T) and showed 99.9 % similarity to the type strain Lactococcus lactis subsp. cremoris NCDO 607(T). Analysis of two housekeeping genes, rpoB and recA, confirmed the close relationship between the novel strains and L. lactis subsp. cremoris with similarities of 99.3 and 99.7 %, respectively. The three strains could, however, be differentiated from their closest relatives on the basis of several phenotypic characteristics, as was the case for L. lactis subsp. lactis and L. lactis subsp. hordniae, which were also closely related on the basis of 16S rRNA, rpoB and recA gene sequence similarities. The strains isolated in this study represent a new subspecies, for which the name Lactococcus lactis subsp. tructae subsp. nov. is proposed. The type strain is L105(T) ( = LMG 24662(T)  = DSM 21502(T)).

Effect of Lactococcus lactis CLFP 100 and Leuconostoc mesenteroides CLFP 196 on Aeromonas salmonicida Infection in Brown Trout (Salmo trutta)

Aeromonas salmonicida is the etiological agent of furunculosis in salmonid fish. This pathogen is important from an epizootic perspective because fish surviving an outbreak can remain lifelong asymptomatic carriers, serving as reservoirs of infection. As a result, the early detection and the control of infection are essential to prevent the spread of new furunculosis outbreaks. We have thus analyzed the effect of probiotic administration on the incidence of A. salmonicida in brown trout (Salmo trutta), that were subjected to temperature stress. Treatment with probiotic strains (Lactococcus lactis CLFP 100 and Leuconostoc mesenteroides CLFP 196) resulted in a higher survival rate after challenge, activation of phagocytic cells in the head kidney, and a lower rate of pathogen proliferation in the intestine as determined by real-time PCR.

Detection and Identification of tdh- and trh-Positive Vibrio parahaemolyticus Strains from Four Species of Cultured Bivalve Molluscs on the Spanish Mediterranean Coast

ABSTRACT Presented here is the first report describing the detection of potentially diarrheal Vibrio parahaemolyticus strains isolated from cultured bivalves on the Mediterranean coast, providing data on the presence of both tdh- and trh-positive isolates. Potentially diarrheal V. parahaemolyticus strains were isolated from four species of bivalves collected from both bays of the Ebro delta, Spain.

Experimental uptake and retention of pathogenic and nonpathogenic Vibrio parahaemolyticus in two species of clams: Ruditapes decussatus and Ruditapes philippinarum

Aims:  To describe uptake and retention of pathogenic and nonpathogenic Vibrio parahaemolyticus in Ruditapes decussatus and Ruditapes philippinarum.

Effect of fish farming on the water quality of rivers in northeast Spain.

The effects of fish farming activities on the aquatic environment were evaluated by studying the water quality of twelve rivers located in northeast Spain. Two sampling sites were used for each river: the first sampling point was located just upstream from the fish farming facilities and the second one was downstream from fish farm effluent discharge point. In order to avoid any misinterpretation due to watershed location and seasonality, a stratified statistical analysis was performed. The results show significant decreases in pH and dissolved oxygen, in contrast to chemical oxygen demand, ammonia, phosphates and microbiological parameters, which significantly increased downstream from the fish farm discharges. Other significant variations were also found for conductivity and temperature. According to the European and local regulations concerning to support fish populations, our results fell within the allowable limits for salmonid waters. Nevertheless, we suggest that further investigations should be carried out to study the ecological interactions between farmed and wild fish populations.

Persistence of sucrose fermenting and nonfermenting vibrios in tissues of Manila clam species, Ruditapes philippinarum, depurated in seawater at two different temperatures.

Effective post-harvest treatment to eliminate toxigenic vibrios is an important measure to reduce risk associated with seafood consumption. In the present study, we examined whether natural populations of sucrose nonfermenting (SNFV) and sucrose fermenting vibrios (SFV) persisted in Ruditapes philippinarum, after depuration at two different temperatures. Two experiments (one in Spring and one in Summer) were performed. Clams were depurated in duplicate tanks in an open-circuit seawater system using filtration and ultraviolet treatment. Clams were sampled daily (9 days), individually processed and inoculated on thiosulphate-citrate-bile salts-sucrose (TCBS) agar plates to estimate the density of SNFV and SFV. Results showed that depuration was less effective in Summer than in Spring, for SNFV and SFV. Despite an initial lower presence in the Summer experiment, clearance of SNFV was less efficient than in Spring, with 22.2% of clams presenting SNFV on day 8 and without significant changes on day 9. When compared, daily proportion of presence of SNFV and SFV in Spring, it was observed that SFV were more frequent than SNFV on days 8 and 9. In Summer these differences were significant on days 7-9. Present study demonstrated that SNFV (involved in food poisoning) are purged, at any temperature, faster than SFV.

Quantitative analysis of bacterial adhesion to fish tissue.

Adhesion to host tissue represents a first crucial step in most bacterial infections. Both specific adhesion-ligand as well as hydrophobic interactions may be involved. The adhesion of Aeromonas salmonicida subsp. salmonicida, Lactococcus garvieae, and Yersinia ruckeri strains to fish tissue cells was assessed. To determine whether the observed bacterial adhesion to fish tissue cells was caused by non-specific interactions, adhesion to bovine serum albumin (BSA) and polystyrene was also tested. Our results demonstrated that non-specific adhesion such as hydrophobic interactions are only partially involved in the binding process since adhesion to BSA was low, and there was no correlation between adhesion to polystyrene and adhesion to fish tissue cells.