Lactococcus garvieae ( L. garvieae ) es un patogeno emergente tanto en acuicultura marina como en continental y que puede llegar a provocar perdidas economicas importantes sobre todo en verano, periodo en el cual el agua alcanza temperaturas elevadas (Vendrell y cols., 2006). Por este motivo, una vacunacion adecuada y eficaz es la clave para minimizar el impacto de esta bacteria en aquellas especies de cultivo que son mas susceptibles a sus efectos. En el presente estudio, se valoro la eficacia de tres vacunas frente a L. garvieae en trucha arcoiris, mediante el metodo de cohabitacion 28 dias post-vacunacion. La que presento una mayor proteccion fue la vacuna comercial (Icthiovac LG) frente a L. garvieae (RPS=87,50%), seguida de una vacuna experimental trivalente para L. garvieae y dos cepas de Yersinia ruckeri (RPS=81,25%) y por ultimo una vacuna experimental tetravalente para L. garvieae , Aeromonas salmonicida subsp. salmonicida y dos cepas de Y. ruckeri (RPS=70,93%). Los resultados de supervivencia de cada grupo se compararon con los del grupo control, indicando un incremento significativo de la resistencia frente al patogeno en todos los grupos vacunados (p<0,05) Eficacy’s evaluation through cohabitation challenge of three Lactococcus garvieae vaccines in rainbow trout (Oncorhynchus mykiss) Lactococcus garvieae (L. garvieae) is considered an emergent pathogen because of its zoonotic potential, an emergent pathogen (Meyburgh y cols., 2017) both in marine and freshwater aquaculture, and it causes important economic losses in summer, when water temperature rises (Vendrell y cols., 2006). Because of this, an adequate vaccination is the key to minimize the impact of that bacterium in those cultivated species that are more susceptible to his effects. In the present study, the efficacy of three L. garvieae vaccines was evaluated with a cohabitation infection 28 days post-vaccination. A commercial vaccine (Icthiovac LG) showed the best results with a RPS=87.50%, followed by an experimental trivalent vaccine (L. garvieae and two Y. ruckeri strains) with a RPS=81.25% and finally an experimental tetravalent vaccine (L. garvieae, two Y. ruckeri strains and Aeromonas salmonicida subsp. salmonicida) with a RPS=70.93%. The mortality results of each group was compared with those to the control group, indicating an increase of the immunity to this pathogen in all vaccinated groups (p<0.05).

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 study was conducted to evaluate the probiotic properties of endogenous rainbow trout microbiota against pathogenic Lactococcus garvieae. A total of 335 bacterial strains were isolated from rainbow trout and screened for antagonistic activity against L. garvieae using an agar spot assay. Antagonistic strains were grouped by PCR amplification of repetitive bacterial DNA elements (rep-PCR) and identified by 16S rRNA gene sequence analysis. The results revealed that the antagonistic strains belonged to the genera Lactobacillus, Lactococcus and Leuconostoc. Further probiotic characteristics, such as specific growth rate, doubling time, resistance to biological barriers, antibiotic resistance, hydrophobicity and production of antimicrobial substances, were also studied. These strains were able to survive low pH and high bile concentrations, showed good adherence characteristics and a broad spectrum of antibiotic resistance. The antagonistic efficacy was maintained after sterile filtration and was sensitive to proteinase K, indicating that proteinaceous extracellular inhibitory compounds were at least partially responsible for pathogen antagonism. Based on these results, these strains should be further studied to explore their probiotic effects in challenge experiments in vivo. This study shows clear evidence that the indigenous trout-associated microbiota may provide a defensive barrier against L. garvieae.

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.

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.

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.

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.

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.

The minimum inhibitory concentrations (MICs) of erythromycin against 31 strains of Lactococcits garvicae isolated from diseased rainbow trout were determined. All isolates were sensitive to the antimicrobial agent with MIC values between 0.06 and 0.125 pg ml-'. This study suggests that erythromycin could be used as an effective treatment to control lactococcosis Outbreaks in rainbow trout, although more information on toxicity and efficacy are still needed.

The aim of this study was to evaluate the ability of three lactic acid bacteria (LAB) isolated from fish, Lactococcus lactis CLFP 101, Lactobacillus plantarum CLFP 238, and Lactobacillus fermentum CLFP 242 to inhibit adhesion of several fish pathogens (Aeromonas hydrophila, Aeromonas salmonicida, Yersinia ruckeri and Vibrio anguillarum) to host intestinal mucus under in vitro conditions. The results showed that only Lc. lactis CLFP 101 reduced the adhesion of all fish pathogens, whereas L. plantarum CLFP 238 reduced the adhesion of A. hydrophila and A. salmonicida. With the exception of V. anguillarum, the adhesion of all fish pathogens to intestinal mucus was reduced by L. fermentum CLFP 242 and by mixture of the three LAB strains. In addition, only Lc. lactis CLFP 101 showed antibacterial activities against all fish pathogens as measured in spent culture liquid. All LAB strains were able to survive relatively low pH and high fish bile concentrations. Based on the positive results of this study, Lc. lactis CLFP 101, L. plantarum CLFP 238 and L. fermentum CLFP 242 should be further studied in challenge experiments in fish to explore their probiotic effects.

We analysed the effect of probiotic strains on the cellular and humoral immune responses of rainbow trout (Oncorhynchus mykiss), and their capacity to prevent furunculosis during a challenge trial. Probiotic strains (Lactococcus lactis ssp. lactis CLFP 100, Leuconostoc mesenteroides CLFP 196, and Lactobacillus sakei CLFP 202) were administered orally to fish for 2 weeks at 10(6) CFU g(-1) of feed. In comparison to untreated control fish, the phagocytic activity of head kidney leukocytes and the alternative complement activity in serum were significantly greater in all probiotic groups at the end of the second week. With the exception of the group fed with Lactobacillus sakei, superoxide anion production was also significantly increased in the probiotic groups. Analysis of lysozyme activity did not exhibit any significant difference in the probiotic and control groups. Fifteen days after the start of the probiotic feeding, fish were challenged with Aeromonas salmonicida ssp. salmonicida. The fish supplemented with probiotics exhibited survival rates ranging from 97.8% to 100%, whereas survival was 65.6% in fish not treated with the probiotics. These results demonstrate that probiotic supplementation to fish can reduce the severity of furunculosis, and suggest that this reduction may be associated with enhanced humoral and cellular immune response.

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 106 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 × 102 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.

We analysed the effect of probiotic supplementation on the control of lactococcosis in rainbow trout. Probiotic strains Leuconostoc mesenteroides CLFP 196 and Lactobacillus plantarum CLFP 238 were administered orally to fish for 30 days at 107 CFU g(-1) feed. Thirty days after the start of the probiotic feeding, fish were challenged with Lactococcus garvieae. Probiotic supplementation reduced fish mortality significantly, from 78% in the control group to 46-54% in the probiotic groups. (C) 2007 Elsevier Ltd. All rights reserved.

The aim of this study was to identify lactic acid bacteria (LAB) using polymerase chain reaction (PCR) amplification of variable regions of the 16S rRNA gene. Thirteen LAB strains were isolated from the intestinal microbiota of healthy salmonids. A similar to 500-bp region of the highly conserved 16S rRNA gene was PCR-amplified and following this, a portion of the amplicon (272-bp) including the V1 and V2 variable regions was sequenced. The sequence containing both the V1 and V2 region provided strong evidence for the identification of LAB. The LAB strains were identified as Camobacteriton maltaroinaticum, Lactobacillus curvatus, Lactobacillus sakei, Lactobacillits plantarum, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, and Leuconostoc mesenteroides. The method described was found to be a very simple, rapid, specific, and low-cost tool for the identification of unknown strains of LAB. (c) 2007 Elsevier Ltd. All rights reserved.

In this study a real-time PCR assay using self-quenched primers labelled with a single fluorophore for the detection of Aeromonas salmonicida was developed. Probe specificity was confirmed by amplification of 16 A. salmonicida strain templates and by the lack of a PCR product with 26 non-A. salmonicida strains. With a pure culture of A. salmonicida, the assay was linear over a range of 0.5 pg to 50 ng and was able to detect 16 c.f.u. per reaction. A similar sensitivity was observed in DNA extracted from a mixture of A. salmonicida and fish tissue. Results using artificially inoculated tissues and diseased fish from outbreaks indicated that the assay can provide sensitive species-specific detection and quantification of A. salmonicida in fish tissue.

We studied the safety and efficacy of an inactivated vaccine (Ichtiovac-Lg) against Lactococcus garvieae in rainbow trout (Oncorhynchus mykiss). In an initial dose-response experiment to test safety, we injected 50 rainbow trout weighing 30-40 g with a double dose of vaccine (0.2 ml) intraperitoneally. We observed these fish three times a day until day 50 post-vaccination when they were killed to evaluate visceral reactions, adhesions and intraperitoneal absorption. Survival was 100% in both the treatment and control groups and no significant differences were found in percentage of severe adhesions and pigmentation of peritonea and viscera.In a second trial, we injected 50 rainbow trout weighing 30-40 g with 0.1 ml of vaccine and a control group was injected with 0.1 ml of PBS intraperitoneally. On day 29 post-vaccination, both groups were challenged by intraperitoneal injection with 0. 1 ml of a virulent heterologous strain of L. garvieae at 3 x 106 Cfu ml(-1) and fish were observed for a further 21 days. At the end of the experiment, the survivals of the vaccinated fish and control group were 94 and 4%, respectively. (C) 2007 Elsevier B.V. All rights reserved.

The present study describes the screening of five lactic acid bacteria (LAB) for use as probiotics based on their competitive adhesion and production of antagonistic substances against some fish pathogens. A reduction of adhesion of all pathogenic strains tested was obtained with three of the LAB strains (Lactococcus lactis subsp. lactis CLFP100, Lactococcus lactis subsp. cremoris CLFP102 and Lactobacillus curvatus CLFP150). With the exception of fish pathogens Flavobacterium psychrophilum and Renibacterium salmoninarum that were not inhibited by LAB strains, production of antagonistic compounds by all tested LAB was observed against at least one of the indicator strains. Based on mucus adhesion, competitive exclusion, and suppression of fish pathogen growth, the selected LAB strains can be considered for future challenge experiments in fish as a very promising alternative to the use of chemotherapeutic agents.

A method is described for the rapid and sensitive assay of phagocytosis that utilizes radioactively labelled bacteria. With this method, we observed that phagocytosis of heat-inactivated Aeromonas salmonicida by leukocytes isolated from gut of rainbow trout fed with different viable probiotics (Lactococcus lactis subsp. lactis, Lactobacillus sakei, and Leuconostoc mesenteroides) was significantly higher (P<0.05) after 2 weeks of probiotic-feeding than the control group. However, only phagocytosis of live A. salmonicida by the leukocytes isolated from gut of rainbow trout fed with L. lactis subsp. lactis was significantly higher (P<0.05) than the control group.

Lactococcus garvieae is the etiological agent of Lactococcosis, an emergent disease which affects many fish species and causes important economic losses both in marine and freshwater aquaculture when water temperature increases over 16 degrees C in summer months. Normally, it causes a hyperacute and haemorrhagic septicemia. This paper presents a state of the art review of fish Lactococcosis including aspects such as pathogen characterization, pathogenesis, epidemiology, diagnosis and control measures of the disease in farmed fish. (c) 2006 Elsevier Ltd. All rights reserved.

Six lactic acid bacteria isolated from salmonids and identified as Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactobacillus curvatus, Leuconostoc mesenteroides, Lactobacillus sakei and Carnobacterium maltaromaticum were found to inhibit the growth of several Aeromonas species. These results suggest that these strains could be suitable as biological control agents in aquaculture.