Correspondence: José Luis Balcázar, Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Científicas (CSIC), c/.Eduardo Cabello 6, 36208 Vigo, Spain. Tel.: +34 986 214 457; fax: +34 986 292 762; e-mail address: email@example.com
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 106 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.
Aeromonas salmonicida ssp. salmonicida is the causal agent of furunculosis, a disease of major importance in the culture of salmonid fish. The disease is characterized by the presence of hemorrhagic and necrotic lesions in the gills, intestine, and muscle (Hiney & Olivier, 1999). Owing to the high mortality and contagious nature of the disease, large amounts of antibiotics are often used for prevention and control of furunculosis (Munro & Hastings, 1993). The use of antibiotics as a preventive measure has been questioned because they can alter the intestinal microbiota and induce resistant populations of bacteria, with unpredictable long-term effects on public health (Kruse & Sørum, 1995). Vaccination has become an important strategy in the control of furunculosis among farmed fish (Ellis, 1997); however, the process is often ineffective when applied to immature fish because they are not fully immunocompetent and do not always respond to vaccination.
It is well known that the normal indigenous microbiota plays an important role in the health of animals (Balcázar et al., 2006a). A number of mechanisms may be responsible for the protective role of the microbiota, for example the production of antimicrobial substances (Bernet-Camard et al., 1997), specific competition for pathogen receptor sites on the mucosal surface (Coconnier et al., 1992), a source of nutrients and of enzymes contributing to digestion (Alm, 1982), and enhancement of the immune response against pathogenic microorganisms (Perdigón et al., 1998).
Probiotics, defined as live microorganisms that when administered in adequate amounts confer a health benefit on the host (Reid et al., 2003), are usually members of the healthy indigenous microbiota, and their addition can assist in returning a disturbed microbiota to its normal beneficial composition (Salminen et al., 2005).
There are several reports of disease prevention or enhancement of immune function resulting from the oral administration of probiotics such as Lactobacillus species in endothermic animals and humans (Alander et al., 1999; Marteau et al., 2001), while only a few studies have tested in vivo the protection conferred by indigenous lactic acid bacteria in fish experimentally infected with pathogens.
Studies by our research group have recently demonstrated that Lactococcus lactis ssp. lactis CLFP 100, Leuconostoc mesenteroides CLFP 196, and Lactobacillus sakei CLFP 202 isolated from the intestines of healthy salmonids exhibit high adhesion to intestinal mucus, a competitive exclusion of fish pathogens, and a high degree of resistance to pH 3.0 and 10% fish bile under in vitro conditions (Balcázar, 2006; Balcázar et al., 2007a). The aims of the current study were, therefore, to investigate whether Lactococcus lactis ssp. lactis CLFP 100, Leuconostoc mesenteroides CLFP 196, and Lactobacillus sakei CLFP 202 could stimulate the humoral and cellular immune responses of rainbow trout (Oncorhynchus mykiss), and to determine their capacity to protect against challenge with A. salmonicida ssp. salmonicida.
Materials and methods
Three bacterial strains, Lactococcus lactis ssp. lactis CLFP 100, Leuconostoc mesenteroides CLFP 196 and Lactobacillus sakei CLFP 202, isolated from salmonids and genetically identified by 16S rRNA gene sequencing, were selected from a pool of 246 strains obtained from the intestinal content of healthy salmonids, because their in vitro characteristics suggested that they could be considered as potential fish probiotics (Balcázar et al., 2006b, 2007a, b). They were grown aerobically in de Man, Rogosa and Sharpe (MRS; de Man et al., 1960) broth at 22°C. Stock cultures stored at −80°C were prepared from overnight cultures to which 20% (v/v) glycerol (Prolabo, Fontenay, France) was added just prior to freezing.
Preparation of the feed
The three selected strains (Lactococcus lactis ssp. lactis CLFP 100, Leuconostoc mesenteroides CLFP 196, and Lactobacillus sakei CLFP 202) were grown in MRS broth at 22°C overnight with agitation in a shaking incubator. After incubation, the cells were harvested by centrifugation (2000 g), washed twice with phosphate-buffered saline (PBS; 10 mM sodium phosphate, 150 mM sodium chloride [pH 7.2]), and resuspended in the same buffer. The A600 nm was adjusted to 0.25±0.05 in order to standardize the number of bacteria (107–108 CFU mL−1). Dilution plating in MRS was used to verify the relationship between A600 nm and CFU per milliliter.
Commercial feed (ProAqua, Palencia, Spain) was used as the basal diet for supplementation with the three selected strains. The required amount of bacterial suspension was applied into the feed slowly, mixing part by part in a drum mixer. The amount of lactic acid bacteria in each feed was determined by plate counting on MRS agar.
Fish and experimental conditions
Rainbow trout were obtained from a commercial fish farm. The fish were fed with a standard commercial feed at a rate of 1.5% of biomass per day. The fish had not been vaccinated nor had they been exposed to fish diseases, and were deemed pathogen-free by standard microbiological techniques and by a previously described multiplex PCR for simultaneous detection of Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri (Del Cerro et al., 2002). The fish were acclimated for 1 week in tanks before the start of the trial. After the acclimation period, the average weight of the fish was 40 g and the fish were divided into four 500 L tanks, each containing 100 fish. All fish were maintained in static aerated fresh water at 14±1°C with a 25% water change every day and a 12 h dark/12 h light photoperiod for 2 weeks. On day 15, the challenge was started by cohabitation (see below) in the three probiotic-fed groups and the control group. During the challenge trial, the water temperature was raised to 16±1°C and kept at this temperature for the rest of the study period.
One group served as the control and was fed un-supplemented feed during the entire trial period. The other three groups were fed with feed containing different viable lactic acid bacteria until the end of the experiment. The first group was fed a diet supplemented with 106 CFU g−1Lactococcus lactis ssp. Lactis; the second group was fed a diet supplemented with 106 CFU g−1Leuconostoc mesenteroides; and the third group was fed a diet supplemented with 106 CFU g−1Lactobacillus sakei. Fish in all groups were fed twice daily at 1.0% of biomass.
Bacterial counts and specific identification of the probiotics
The microbial analyses were performed twice before the trial and at the end of the second week. The fish were sacrificed by immersion for 15 min in a tank containing tricaine methanesulfonate (MS-222; Syndel Laboratories Ltd, Canada) at a concentration of 150 mg L−1 of water. They were then opened aseptically and the whole intestine was removed. The intestine was dissected, and the contents were collected by carefully scraping with a rubber spatula, and then weighed. The microbial analyses were performed by spreading appropriate dilutions in PBS from 10−1 to 10−6 on MRS agar, a selective media for lactic acid bacteria. The plates were incubated aerobically at 22°C for 2 days.
Specific identification through random amplification of polymorphic DNA (RAPD) was carried out by amplification of 20–30 colonies randomly selected from the MRS plates containing 30–300 colonies. Bacterial cultures (1.0 mL) were homogenized in 200 μL of Tris-EDTA buffer (pH 8.0; 10 mM Tris, 1 mM EDTA), and centrifuged at 12 000 g for 1 min. Pellets were then extracted with InstaGene Matrix (Bio-Rad Laboratories, Hercules, CA) following the manufacturer's instructions. DNA yield and purity were determined spectrophotometrically by measuring 260 nm/280 nm absorbance ratios (Gene Quant pro RNA/DNA Calculator, Amersham Pharmacia Biotec, Cambridge, UK).
The primer used for RAPD analysis of bacterial DNA has been previously described (Johansson et al., 1995). A total volume of 50 μL of reaction mixture contained 0.2 mM of each dNTP, 2.5 mM MgCl2, 2.0 μM primer 5′-ACGCGCCCT-3′, PCR buffer [10 mM Tris-HCl (pH 8.3), 50 mM KCl], 1.0 U of Taq polymerase (Invitrogen Corp.), and 1.0 μL of DNA template. Amplification of DNA was performed in a GeneAmp PCR System 2400 thermocycler (Perkin-Elmer Corp., Wellesley, MA) with the following conditions: an initial denaturation of 10 min at 94°C; four cycles of 45 s at 94°C, 2 min at 30°C, and 45 s at 72°C; 36 cycles of 15 s at 94°C, 30 s at 36°C, and 45 s at 72°C; and 10 min at 72°C. The DNA amplicons were separated on a 4% acrylamide gel and compared with a 1-kb ladder (Bio-Rad Laboratories). The mixture was stored at −20°C until use.
Blood samples and isolation of head kidney phagocytes
Blood samples were taken from 10 fish of each group at the end of the second week. The fish were sacrificed as described above. Blood was drawn from the caudal vein of individual fish. The sera were then separated by centrifugation at 1500 g for 25 min and stored at −80°C prior to analysis.
Following blood collection, head kidney cells were removed aseptically, dissected, and suspended in L-15 medium (Sigma Chemical Corp., St Louis, MO) containing 2% fetal calf serum (FCS). The cell suspension was then filtered thought a 100 μm nylon mesh (Becton Dickinson Labware, Bedford, MA), and the leukocyte fractions were separated using the Histopaque-1119 method (Sigma). In order to obtain leukocyte fractions, 1.0 mL of supernatant was layered onto 0.5 mL of the Histopaque-1119 solution in a sterile centrifuge tube. The tube was capped and then centrifuged at 700 g for 30 min at room temperature. A diffuse band of leukocytes occurred above the erythrocyte pellet. This layer of cells was aseptically removed with a pipette and resuspended in L-15 medium. The viability of the cells was confirmed by staining with 0.1% trypan blue, and the cell number was calculated and adjusted to 6 × 107cells mL−1 with L-15 medium.
Radiolabelling of bacteria for phagocytosis assay
The A. salmonicida ssp. salmonicida CLFP 501 strain used was isolated at the Laboratory of Fish Pathology, University of Zaragoza, Spain, during a natural outbreak of furunculosis in rainbow trout. Aeromonas salmonicida ssp. salmonicida was grown in brain heart infusion broth (BHI; Scharlau Chemie, Barcelona, Spain) overnight at 22°C. Tritiated thymidine ([methyl-1,2-3H]thymidine; 10 μL mL−1, 117 Ci mmol−1) was added to the medium to label the bacteria metabolically. After incubation, bacteria were harvested by centrifugation (2000 g), washed twice with PBS, and resuspended in the same buffer. Bacterial suspensions were adjusted to an absorbance (600 nm) of 0.25±0.05, which corresponded to 6 × 107 CFU mL−1. Dilution plating was used to verify the relationship between A600 nm and CFU per milliliter.
Phagocytic activity of leucocytes was determined according to Balcázar et al. (2006c). The head kidney leucocytes (see above) were adjusted to a final concentration of 1.67 × 105 mL−1 with L-15 medium supplemented with 0.1% FCS. A volume of 900 μL of the leukocyte suspension per well was pipetted into 96 flat-bottomed well plates. Phagocytosis of bacterial cells was assayed by adding 100 μL of radioactively labelled bacteria (see above) adjusted to 6 × 107 mL−1 (leukocyte-to-bacteria ratio, c. 1/40 well−1) and incubating for 30 min at room temperature with gentle mixing at frequent intervals. After incubation, the homogenates were transferred to sterile centrifuge tubes, and the nonphagocytized bacteria were removed by density-gradient centrifugation using the Histopaque-1119 method (see above). Phagocytized bacteria were lysed by incubation at 60°C for 1 h with 1.0% sodium dodecyl sulphate (SDS) in 0.1 M NaOH. Phagocytosis was assessed by quantifying the amount of radioactivity by liquid scintillation and was expressed as the percentage of radioactivity recovered after phagocytosis relative to the radioactivity in the bacterial suspension added to the plates.
Superoxide anion production by the head kidney leucocytes was determined based on the reduction of nitroblue tetrazolium (NBT) as described by Puangkaew et al. (2004). In brief, 15 μL of 2 × 107 cell mL−1 isolated leucocytes was mixed with an equal volume of L-15 containing NBT (solution of NBT in L-15 at 1.0 mg mL−1). After incubation for 1 h, 400 μL of N,N-dimethylformamide was added and centrifuged at 3000 g. The OD of the supernatant was measured at 540 nm.
The complement activity (alternative pathway) was assayed following the procedure of Yano (1992) using rabbit red blood cells (RaRBC). Briefly, the RaRBC were washed and adjusted to 2 × 108 cell mL−1 in 0.01 M ethylene glycol tetraacetic acid–magnesium–gelatin veronal buffer (EGTA–Mg–GVB). The 100% lysis value was obtained by lysing 100 μL of RaRBC with 3.4 mL of distilled water and measuring the OD of haemolysate at 414 nm against distilled water. The test serum was appropriately diluted and volumes ranging from 0.1 to 0.25 mL were made up to 0.25 mL total volume before being allowed to react with 0.1 mL of RaRBC in test tubes. After incubation at 20°C for 90 min with occasional shaking, 3.15 mL of a saline solution was added to each tube, and tubes were centrifuged at 1600 g for 10 min at 4°C. The OD of the supernatant was measured at 414 nm. A lysis curve was obtained by plotting the percentage of haemolysis against the volume of serum added. The volume yielding 50% haemolysis was determined and in turn used for assaying the complement activity of the sample (ACH50 value=units mL−1).
Serum lysozyme activity was measured using the turbidimetric method, originally described by Parry et al. (1965) and modified for microtitre assay by Demers & Bayne (1997). Serum (25 μL well−1) was placed in triplicate in a 96-well plate, and 175 μL of a suspension of Micrococcus lysodeikticus (75 mg mL−1 in 0.1 M phosphate buffer with 0.09% NaCl, pH 5.8) was added. After the plate had been shaken, the decrease in absorbance at 450 nm was recorded for 5 min. Lysozyme activities were converted to lysozyme concentration using hen egg white lysozyme as a standard.
Experimental infection was carried out with permission from the Ethical Committee for Animal Experiments, University of Zaragoza, Spain. Aeromonas salmonicida ssp. salmonicida CLFP 501, previously isolated during a natural outbreak of furunculosis in rainbow trout, was grown for 12 h at 22°C in BHI broth. After incubation, the cells were harvested by centrifugation (2000 g), washed twice with PBS, and resuspended in the same buffer. The A600 nm was adjusted to 0.25±0.05 in order to standardize the number of bacteria (6 × 107 CFU mL−1). The bacterial suspension was diluted to a density of 1.7 × 106 CFU mL−1, and 0.1 mL of this suspension was injected intraperitonealy into cohabitation fish. The fish were anaesthetized with MS-222 before injection. All cohabitants (16 for each group) were marked by clipping the adipose fin after injection, and were replaced into the appropriate tank with experimental fish. All fish were monitored closely postexposure. Moribund and dead fish were collected and recorded daily; moribund animals were killed with an overdose of MS-222. Aeromonas salmonicida ssp. salmonicida was isolated from tissue samples of freshly dead fish on tryptic soy agar plates (TSA; Scharlau Chemie) at 22°C for 72 h, and its identity was verified by a previously described real-time PCR method (Balcázar et al., 2007c).
anova and Duncan's multiple range test were used to determine the significant variation (P<0.05) in the immune response between the control and experimental groups. For the results from the challenge trial, differences in cumulative survival were calculated according to the Kaplan–Meier method. All statistics were calculated using spss for Windows version 11.5 (SPSS, Chicago, IL).
To examine the role of the probiotics colonizing the intestine of the fish, we used rainbow trout receiving the probiotic strains at a dose equivalent to 106 CFU g−1 of feed for 2 weeks. Before the trial the fish had no detectable Lactococcus lactis ssp. lactis, Lactobacillus sakei, or Leuconostoc mesenteroides in the intestine. The number of viable lactic acid bacteria increased rapidly in all probiotic groups and ranged from 7.5 × 106 to 2.3 × 107 CFU g−1 at the end of the second week (Fig. 1). Specific identification through RAPD profiles of colonies randomly selected from the MRS plates confirmed the capacity of probiotic strains to survive and colonize the gastrointestinal tract. In addition, it is important to point out that the control group had a low number of lactic acid bacteria (2.4 × 102 CFU g−1), especially of Carnobacterium maltaromaticum, at the end of the second week.
Cellular immunological response
The phagocytic activity of the head kidney leucocytes of rainbow trout fed with the various viable probiotics was significantly higher (P<0.05) after 2 weeks of probiotic feeding than that of the control group (Fig. 2).
At the end of the second week of probiotic feeding, a significant increase in the superoxide anion production was observed in the groups fed with Lactococcus lactis ssp. lactis and Leuconostoc mesenteroides (P<0.05) compared with the control (Fig. 3). The superoxide anion production in the group fed with Lactobacillus sakei also increased, but was not statistically significant (P>0.05).
Humoral immunological response
The alternative complement activity (ACH50) in the serum was found to be significantly (P<0.05) greater in all probiotic groups than in the control group at the end of the second week (Fig. 4). The analysis of lysozyme activity did not demonstrate any significant difference in the probiotic groups compared with the control (Fig. 5).
To investigate whether the probiotics are able to protect rainbow trout against furunculosis infection, fish were infected with A. salmonicida ssp. salmonicida by the cohabitation method. The fish that had been supplemented with the probiotics showed a cumulative survival ranging from 97.8% to 100%, whereas survival was 65.6% in fish not treated with the probiotics (Fig. 6). Statistical analysis demonstrated significant differences (P<0.001) in the survival rate of fish between the probiotic and control groups. External examination and bacteriological analysis of fish that died during the study revealed the presence of furunculosis in all cases.
Probiotic supplementation can enhance nonspecific host resistance to microbial pathogens and thereby facilitate the exclusion of potential pathogens in the digestive tract. In this study, we were able to detect high amounts of lactic acid bacteria in the intestine after 2 week of probiotic supplementation, which demonstrates that Lactococcus lactis ssp. lactis CLFP 100, Leuconostoc mesenteroides CLFP 196, and Lactobacillus sakei CLFP 202, previously isolated from the endogenous microbiota of salmonids, have a strong ability to adhere to and survive in the intestinal mucus. At the beginning of the study, we observed that all fish were colonized with several Aeromonas species, particularly Aeromonas hydrophila, which constitutes a large component of the resident microbiota of freshwater fish (Gonzalez et al., 1999). However, probiotic supplementation demonstrated an ability to antagonize the resident microbiota, as the increase of lactic acid bacteria observed is possibly a result of the decrease in the intestinal pH induced by lactic acid or other fermented products produced by lactic acid bacteria.
In addition, we demonstrated that administration of the viable lactic acid bacteria Lactococcus lactis ssp. lactis CLFP 100, Leuconostoc mesenteroides CLFP 196, and Lactobacillus sakei CLFP 202 enhanced both cellular and humoral immune functions in rainbow trout by increasing the proportion of phagocytically active cells from head kidneys and activating the complement receptor expression. Enhanced phagocytosis has also been reported in gilthead seabream (Sparus aurata L.) following administration of Lactobacillus delbrüeckii spp. lactis (CECT 287) in the feed for 14 days at a dose equivalent to 107 CFU g−1 of feed (Salinas et al., 2005). Phagocytosis is responsible for early activation of the inflammatory response before antibody production, and is mediated by phagocytic cells such as neutrophils, monocytes and macrophages in fish. The in vivo activation of phagocytic cells by immunomodulators may have also led to the secretion of a wide range of biologically active molecules such as enzyme inhibitors, cationic peptides, and complement components, and to the production of reactive oxygen and nitrogen species that are involved in bactericidal activity (Adams & Hamilton, 1984; Kwak et al., 2003). The administration of Lactococcus lactis ssp. lactis CLFP 100 and Leuconostoc mesenteroides CLFP 196 for 2 weeks in feed stimulated phagocytic activity and superoxide anion production in fish. Superoxide anion production in the group fed with Lactobacillus sakei CLFP 202 also increased, but did not result in a significant difference compared with the control group. The differences observed in superoxide anion production between the probiotic groups can be related to colonization capacity on the intestinal mucus.
The complement system is composed of more than 35 soluble plasma proteins that play key roles in innate and adaptive immunity (Boshra et al., 2006). Complement is initiated by one or a combination of three pathways, namely the alternative, lectin and classical. The alternative complement pathway, which is antibody independent, can be directly activated by the lipopolysaccharide of Gram-negative bacteria, and can result in lysis of the bacterial cell (Sakai, 1992; Boshra et al., 2006). In the present study, the alternative complement activity in the serum was found to be significantly greater in all probiotic groups than in the control group at the end of the second week.
Lysozyme is a cationic enzyme that attacks the β-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the peptidoglycan of bacterial cell walls. This enables lysozyme to lyse certain Gram-positive bacteria, and, in conjunction with complement, even some Gram-negative bacteria (Alexander & Ingram, 1992). Previous studies suggest that lysosyme production in fish may be upregulated in response to microbial polysaccharides. Panigrahi et al. (2004) showed significantly higher serum lysozyme activity in rainbow trout fed with Lactobacillus rhamnosus JCM 1136 in the feed for 30 days at a dose equivalent to 109 CFU g−1 of feed. However, the results obtained in the present study did not exhibit any significant difference in terms of lysozyme activity in the serum in the probiotic groups compared with the control at the end of the second week. The differences between these studies can be explained by a different dose in the feed and the length of the studies. Direct comparisons between these studies are also difficult because these strains are functionally different and may be affected differently by genetic, nutritional and environmental factors, and by the origin of the probiotic strain.
The results reinforce the currently held view that viable microbial food supplements have a beneficial effect on the health of the host by enhancing the immune response and modifying the composition of the intestinal microbiota, thereby providing protection against pathogenic microorganims (Balcázar et al., 2007d). These data support other studies that have demonstrated the ability of probiotics to reduce significantly the impact of furunculosis in rainbow trout (Nikoskelainen et al., 2001; Irianto & Austin, 2002).
To our knowledge this is the first report demonstrating the probiotic effect of endogenous Lactococcus lactis ssp. lactis, Leuconostoc mesenteroides, and Lactobacillus sakei against furunculosis in rainbow trout. To date, Lactococcus lactis is the most extensively studied lactic acid bacterium, from which a variety of genetic tools have been developed, leading to the successful production of enzymes (Kuipers et al., 1997), therapeutic proteins (Steidler et al., 2003), and nutraceuticals (Hugenholtz et al., 2002). For example, an engineered strain of interleukin-10-secreting Lactococcus lactis has been used to treat chemically induced inflammatory bowel disease in mice (Steidler et al., 2000).
In conclusion, the three endogenous lactic acid bacteria used here were clearly beneficial for rainbow trout when administered as food additives in terms of colonizing the gastrointestinal tract, enhancing cellular and humoral immune responses, and resisting challenge from A. salmonicida ssp. salmonicida. These findings, together with evidence from previous studies of the immunity-enhancing and protective effects of lactic acid bacteria against microbial pathogens (Nikoskelainen et al., 2001; Kim & Austin, 2006; Vendrell et al., 2007), suggest that dietary supplementation with defined probiotics may represent an effective prophylactic means of countering infections in fish. Moreover, probiotic supplementation offers an alternative to antibiotics and chemotherapeutic agents routinely used as feed supplements in fish farming (Balcázar et al., 2006d).
This study was supported by a grant from the National Adviser Body of Continental Cultures (JACUCON). J.L.B. was supported by a fellowship from the Spanish International Cooperation Agency (AECI). We thank M.C. Uriel, J. Orós, and R. Claver for their skilled technical assistance; and C. Rumbaitis del Rio and M.S. Devall for their assistance and critical reading of the manuscript.