Assessment of Sample Size Calculations Used in Aquaculture by Simulation Techniques.

An adequate sampling methodology is the key to knowing the health status of aquatic populations. Usually, the aims of epidemiological surveys in aquaculture are to detect an infection and estimate the disease prevalence, and different formulas are used to calculate the sample size. The main objective of this study was to assess if the sample sizes calculated using classical epidemiological formulas are valid considering the sampling methodology, the population size, and the spatial distribution of diseased animals in the population (non-clustered or clustered). However, the use of sample sizes of 30, 60, and 150 fish is widely accepted in aquaculture, due to the requirements of the World Organization for Animal Health (OIE) for epidemiological surveillance. We have developed a specific software using ASP (Active Server Pages) language and MySQL database in order to generate aquatic populations from 100 to 10 000 brown trouts infected by Aeromonas salmonicida with different levels of prevalence: 2, 5, 10, and 50%. Then we implemented several Monte Carlo simulations to estimate empirically the sample sizes corresponding to the different scenarios. Furthermore, we compared these results with the values calculated by classical formulas. We determined that simple random sampling was more accurate in detecting an infection, because it is independent of the distribution of infected animals in the population. However, if diseased animals are non-clustered it is more efficient to use systematic methods, even in the case of small populations. Finally, the formula to calculate sample size to estimate disease prevalence is not valid when the expected prevalence is far from 50%, and it is necessary to increase the sample size to reach the desired precision.

Nisin Z Production by Lactococcus lactis subsp. cremoris WA2-67 of Aquatic Origin as a Defense Mechanism to Protect Rainbow Trout (Oncorhynchus mykiss, Walbaum) Against Lactococcus garvieae.

Probiotics represent an alternative to chemotherapy and vaccination to control fish diseases, including lactococcosis caused by Lactococcus garvieae. The aims of this study were (i) to determine the in vitro probiotic properties of three bacteriocinogenic Lactococcus lactis subsp. cremoris of aquatic origin, (ii) to evaluate in vivo the ability of L. cremoris WA2-67 to protect rainbow trout (Oncorhynchus mykiss, Walbaum) against infection by L. garvieae, and (iii) to demonstrate the role of nisin Z (NisZ) production as an anti-infective mechanism. The three L. cremoris strains survived in freshwater at 18 °C for 7 days, withstood exposure to pH 3.0 and 10 % (v/v) rainbow trout bile, and showed different cell surface hydrophobicity (37.93-58.52 %). The wild-type NisZ-producer L. cremoris WA2-67 and its non-bacteriocinogenic mutant L. cremoris WA2-67 ∆nisZ were administered orally (10(6) CFU/g) to rainbow trout for 21 days and, subsequently, fish were challenged with L. garvieae CLG4 by the cohabitation method. The fish fed with the bacteriocinogenic strain L. cremoris WA2-67 reduced significantly (p < 0.01) the mortality (20 %) compared to the fish treated with its non-bacteriocinogenic knockout isogenic mutant (50 %) and the control (72.5 %). We demonstrated the effectiveness of L. cremoris WA2-67 to protect rainbow trout against infection with the invasive pathogen L. garvieae and the relevance of NisZ production as an anti-infective mechanism. This is the first report demonstrating the effective in vivo role of LAB bacteriocin (NisZ) production as a mechanism to protect fish against bacterial infection. Our results suggest that the wild-type NisZ-producer strain L. cremoris WA2-67 could be used in fish farming to prevent lactococcosis in rainbow trout.

Uptake and retention of Vibrio parahaemolyticus in a cohabitating population of Ruditapes decussatus and Ruditapes philippinarum under experimental conditions.

The presence of Vibrio parahaemolyticus in bivalve mollusc is an important cause of foodborne illnesses, and their levels are influenced by environmental changes, such as temperature and salinity. Clams are common species in estuaries and are used in environmental monitoring programmes. Present study compared the uptake and retention of nonpathogenic V. parahaemolyticus by two species of clam (Ruditapes decussatus and R. philippinarum), cohabitating in a closed system. Results showed no significant differences were found between both species of clams. Bacterial levels are following a similar trend with values between 3.48 and 3.70 log CFU/g for R. decussatus and between 3.15 and 3.49 log CFU/g for R. philippinarum. So, in the absence of water renewal, high and stable levels of V. parahaemolyticus were observed in cultured clams after exposure. Changes in physical parameters should be taken into account to design surveillance programmes in bivalves, and sampling should focus on species that have faster filtration rates at that water temperature since they potentially represent the worst-case scenario.

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.

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-1ß, 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-1ß, 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)).

Detection and identification of tdh- and trh-positive Vibrio parahaemolyticus strains from four species of cultured bivalve molluscs on the Spanish Mediterranean Coast.

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.

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.

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.

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.

Protection of rainbow trout (Oncorhynchus mykiss) from lactococcosis by probiotic bacteria.

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 10(7) 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.

Enhancement of the immune response and protection induced by probiotic lactic acid bacteria against furunculosis in rainbow trout (Oncorhynchus mykiss).

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.

In vitro competitive adhesion and production of antagonistic compounds by lactic acid bacteria against fish pathogens.

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.

Safety and efficacy of an inactivated vaccine against Lactococcus garvieae in rainbow trout (Oncorhynchus mykiss).

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 10(6) 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.

Quantitative detection of Aeromonas salmonicida in fish tissue by real-time PCR using self-quenched, fluorogenic primers.

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.

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.

Sequencing of variable regions of the 16S rRNA gene for identification of lactic acid bacteria isolated from the intestinal microbiota of healthy salmonids.

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 approximately 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 Carnobacterium maltaromaticum, Lactobacillus curvatus, Lactobacillus sakei, Lactobacillus 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.

Immune modulation by probiotic strains: quantification of phagocytosis of Aeromonas salmonicida by leukocytes isolated from gut of rainbow trout (Oncorhynchus mykiss) using a radiolabelling assay.

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 in fish: a review.

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.

The role of probiotics in aquaculture.

The increase of productivity in aquaculture has been accompanied by ecological impacts including emergence of a large variety of pathogens and bacterial resistance. These impacts are in part due to the indiscriminate use of chemotherapeutic agents as a result of management practices in production cycles. This review provides a summary of the use of probiotics for prevention of bacterial diseases in aquaculture, with a critical evaluation of results obtained to date.