Daniel Vendrell

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.

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.

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.

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.

Accumulation and depletion kinetics of erythromycin in rainbow trout (Oncorhynchus mykiss)

Erythromycin is an antimicrobial agent recommended for the control and treatment of diseases caused by gram-positive bacteria. Few studies, however, have determined the metabolic and pharmacokinetic aspects of this antimicrobial agent in fish. The aim of the present study, therefore, was to determine the accumulation and depletion time of erythromycin after administration of medicated feed containing 52 mg kg(-1) body weight day(-1) for 8 days in rainbow trout (Oncorhynchus mykiss). Results were analyzed following the European Agency for Evaluation of Medicinal Products guidelines. We measured a withdrawal time of 187°C-day (°C-day=water temperature×days), lower than the value (500°C-day) recommended by Council Directive 2004/28/EC for veterinary medicinal products. Our results provide data to establish therapeutic regimens for the use of erythromycin in aquaculture.

A new real time PCR-based assay for diagnosing Renibacterium salmoninarum in rainbow trout (Oncorhynchus mykiss) and comparison with other techniques

Bacterial Kidney Disease of salmonid is caused by a slow-growing gram-positive bacterium, Renibacterium salmoninarum. This bacterium lives both extra-cellular and intra-cellular in the host. Serological and molecular diagnostic methods to detect the bacterium major surface protein antigen p57 have been developed. In the present work, a newly developed quantitative Reverse Transcriptase-PCR (RT-QPCR), using self-quenched fluorescent primer (Lux), a nested PCR (NPCR), a commercial ELISA and recently commercially available Immune-chromatographic strip test(IC-Strip) were compared for their ability to detect BKD in kidney tissue samples obtained from experimentally infected fish. ELISA test resulted to be rapid, simple and indicative for the bacterial load. The IC-Strip test had similar characteristics for bacterial detection. Both tests are a good option for rapid and relatively inexpensive screening studies, despite the one and two log decrease in bacterial detection limits compared to NPCR and RT-QPCR, respectively. The use of Lux primers in the newly developed RT-QPCR revealed to be a cost-effective alternative to other fluorescence-based PCR techniques. The option of generating a melting temperature curve with the real time PCR instrument confirmed the specificity of the PCR product. The RT-QPCR technique had the advantage of detecting low numbers of viable bacterial mRNA which implied a higher capacity of detecting chronically infected animals. For instance, some fish in the group infected by cohabitation had very low bacterial load and were only detected by this technique.