Introduction

Intensive culture of fish entails loss of a large number of animals due to bacterial infections, despite the fact that care is taken to ensure optimal nutritional and environmental factors (Trust 1986; Alderman 1988; Rigos and Troisi 2005). In euryaline fish farming the most prominent pathogenic bacteria are Vibrio (Listonella) anguillarum and Photobacterium damsella subsp. piscicida (Zorilla et al. 2003; Toranzo et al. 2005). The treatment of the bacterial diseases of fish especially in larval stages consists in the application of antibiotics either by bath (Samuelsen 2003), or using the live feed Artemia as a carrier of the antibiotics (Duis et al. 1995; Touraki et al. 1996, 1999). Although the application of antimicrobials has well documented beneficial effects to the infected animals, it might present an environmental hazard due to the development of microbial resistance of pathogens (Spangaard et al. 1993; Samuelsen et al. 1994; Rigos and Troisi 2005). Since strict food safety and quality requirements are required to increase food safety, the use of antibiotics is restricted and preventative measures are recommended against the introduction of pathogens to aquatic animal health, also being considered more cost effective than cure (FAO 2010). The most promising preventive method to control potential pathogens in fish culture is the use of probiotics (Kesarcodi-Watson et al. 2008) although the direct use of a probiotic by addition in the water column, presents great environmental consideration, due to the risk of microorganism pollution (Lara-Flores 2011). According to the broadened definition of Gram et al. (1999), probiotics are “live microbial supplements that beneficially affect the host animal by improving its microbial balance”. Various probiotics have been extensively used over the recent years to improve growth or survival of fish (Carnevali et al. 2006; Makridis et al. 2008; Fjellheim et al. 2010), as immunomodulators (Salinas et al. 2005; Picchietti et al. 2007; Tapia-Paniagua et al. 2011), or to protect fish from vibriosis (Gildberg et al. 1997; Planas et al. 2006; Sorroza et al. 2012). However data on the effect of Bacillus subtilis and Lactobacillus plantarum on V. anguillarum are scarce and their suitability as probiotics to be used to protect fish larvae requires clarification. The administration of probiotics to target animals, namely fish larvae, using the technique of bioencapsulation of the probiotic in the live fish feed Artemia has been reported to be an interesting but scarcely studied approach (Gomez-Gil et al. 2000; Hai et al. 2010). Selected bacterial strains have been reported to protect Artemia from pathogens (Verschuere et al. 2000), while recently the incorporation of the probiotics B. subtilis and B. licheniformis in adults of Artemia urniana led to modulation of their gut microbiota (Motlagh et al. 2012).

The present study focused on the effect of two potential probiotics B. subtilis and L. plantarum to Artemia nauplii in terms of survival and protection from a challenge with V. anguillarum, and the efficacy of Artemia nauplii enriched with the selected probiotic to protect European sea bass larvae from vibriosis.

Materials and methods

Bacterial strains and culture conditions

Microbial strains originated from the Collection Espanola de Cultivos Tipo (CECT) and the National Collection of Industrial Bacteria (NCIMB, Aberdeen, Scotland). The strains that were used were Bacillus subtilis NCIMB 3610 and L. plantarum CECT 220. Stock cultures were maintained at −80 °C in 20 % glycerol, for long term storage. Bacteria were grown in MRS or NB broth at 30 °C, for 24 h (Papagianni et al. 2006). Optical density of cultures was monitored at 600 nm where maximum absorbance was observed. The Greek strain of the pathogen Vibrio (Listonella) anguillarum, 332A serotype O2, used for challenge experiments was grown in Brain Heart Infusion agar containing 2 % NaCl, at 22–24 °C, for 24 h (Magarinos et al. 1992).

Growth inhibition in co-culture assays

Both candidate probiotic strains were tested for antagonistic effects against the fish pathogen using broth co-culture assays. Two series were performed for each probiotic, one adding 104 cells of either B. subtilis or L. plantarum to 1 ml of BHI broth containing 2 % NaCl and a second by adding 107 probiotic cells. The original cell count of the pathogen V. anguillarum 332A was the same amounting to 104 cells. Mono cultures of each probiotic and of the pathogen acted as controls. An average CFU ml−1 was calculated by counting in each case and plotted against OD600. Growth of probiotics and the pathogen was daily monitored at 600 nm and by spreading the co-culture on Brain Heart Infusion agar containing 2 % NaCl, morphological identification of each bacterium and counting. All cultures were performed in triplicate.

Production of antibacterial substances by the probiotics

Two series of experiments were performed one for each probiotic. Aliquots of fresh cultures of each candidate probiotic were inoculated in Nutrient broth at an initial cell density of 107 CFU ml−1 and incubated at 30 °C. Samples of 1 ml were withdrawn at 0, 24, 48 and 96 h, centrifuged at 200 × g and the supernatants sterilized through 0.45 μm pore -size filters and stored at 4 °C. The inhibitory activity of each supernatant was tested by the method of Gram et al. (1999). Briefly 450 μl of supernatant were added in tubes containing 500 μl Nutrient broth with 2 % NaCl and these were inoculated with 50 μl of V. anguillarum, yielding approximately 104 CFU ml−1 of the pathogen. As controls served samples in which the pathogen was inoculated in 950 μl of BHI broth. The three replicates used for each combination were incubated in a shaker (220 rotations/min, 30 °C) and the pathogen growth was monitored by optical density recordings of 100 μl aliquots at 600 nm in a Microplate Autoreader photometer (Bio-Tek Instruments), at 0, 24, 48, 72 and 120 h.

Administration of probiotics to Artemia nauplii

Experiments were performed on A. franciscana (Kellog) cysts (E.G. grade, Great Salt Lake strain, batch 11.119.03) kindly provided by INVE (Aquaculture Artemia Systems, Baasrode, Belgium) and stored at 4 °C. Hydration and decapsulation of the cysts in hypochlorite solution were performed as previously described (Sorgeloos et al. 1986), resulting in bacteria-free cysts. The cysts were re-suspended in a vessel containing 500 ml of filtered and autoclaved artificial seawater (35 g l−1, pH 8.7) and they were allowed to hatch for 24 h under continuous filtered aeration and illumination (2,000 lux) at 28 ± 1 °C (Soltanian et al. 2007). At the end of the incubation period nauplii instar I were collected aseptically and used for enrichment according to the method of Patra and Mohamed (2003). Briefly, nauplii were thoroughly rinsed with filtered and autoclaved artificial seawater, transferred to vessels containing 500 ml sterile seawater at a density of 15 nauplii ml−1 and the appropriate probiotic or its culture cell free supernatant was administered to the nauplii.

Three treatments were performed for each candidate probiotic. In the first treatment with B. subtilis, a probiotic culture of 3 × 109 CFU ml−1 was used and 75 μl were administered per dose to the nauplii daily, 2 doses per day for a total of 2 days corresponding to the administration of a total of 1.8 × 106 CFU ml−1 of Artemia culture medium, after four doses to the nauplii over a period of 48 h. In the second treatment 150 μl of a B. subtilis culture of 7 × 109 CFU ml−1 were administered to the nauplii corresponding to 8.4 × 106 CFU ml−1 of Artemia culture medium after the total of four administered doses. In the third treatment the cell-free B. subtilis culture supernatant, after sterilization, was added in the sterile artificial seawater used for the culture of the nauplii at a concentration of 50 % (v/v).

In the first treatment with L. plantarum a probiotic culture of 2.6 × 109 CFU ml−1 was used and 75 μl were administered per dose to the nauplii daily, 2 doses per day for a total of 2 days corresponding to the administration of 1.5 × 106 CFU ml−1 of Artemia culture medium after four doses to the nauplii over a period of 48 h. In the second treatment 150 μl of a L. plantarum culture of 7.1 × 109 CFU ml−1 were administered to the nauplii corresponding to 8.4  × 106 CFU ml−1 of Artemia culture medium after the total of four doses. In the third treatment the cell-free L. plantarum culture supernatant, after sterilization, was added in the sterile artificial seawater used for the culture of the nauplii at a concentration of 50 % (v/v).

All experiments were performed in triplicate an as control served nauplii that did not receive any treatment with either candidate probiotic. Survival of Artemia nauplii was monitored for 48 h after the addition of the probiotics. The presence of the probiotic in the nauplii was confirmed at the end of each experiment after thorough washing of the nauplii with sterile seawater, homogenization and inoculation on plates with Nutrient agar, BHI agar containing 2 % NaCl and McConkey agar and incubation at 25 °C for 24–72 h.

Challenge of Artemia nauplii enriched with probiotics with V. anguillarum

Challenge of Artemia nauplii was performed according to the method used by Defoirdt et al. (2005). Briefly, cells from of a V. anguillarum culture (1 × 109 CFU ml−1) were washed twice with sterile saline and 200 μl were used to challenge the nauplii. This corresponds to a bacterial density of 2 × 105 CFU ml−1 of Artemia culture medium. Nauplii that were not treated with any probiotic nor challenged served as negative control while nauplii that were not fed with probiotics but were challenged with the pathogen were the positive control. All experiments were performed in triplicate and Artemia survival was monitored for a total of 96 h. The presence of the probiotic or the pathogen in the nauplii was confirmed at the end of each experiment after thorough washing of the nauplii with sterile seawater, homogenization and inoculation on plates with Nutrient agar, BHI agar containing 2 % NaCl and McConkey agar and incubation at 25 °C for 24–72 h.

Administration of Artemia nauplii enriched with the probiotic B. subtilis to D. labrax larvae to evaluate harmlessness of probiotic

Sea bass larvae, 50 days old, (of an average weight of 80 mg, length 1.3–1.9 cm approximately), kindly provided by a commercial local hatchery, were kept in filtered artificial seawater under continuous aeration at 20 °C, with a photoperiod of 12 h. Prior to any experiments a 10-day conditioning period was allowed. Fish were fed twice daily using A. franciscana nauplii at a level of 5–10 nauplii ml−1 seawater. Fish density never exceeded 5 individuals per liter and about one-third of the tank water was renewed daily. After the acclimatization period, groups of 20 fish were thoroughly rinsed and transferred in clean artificial seawater at separate aquaria of a capacity of 10 l each.

Since in the experiments with both probiotics on Artemia nauplii, B. subtilis resulted in better survival rates of the nauplii with or without challenge with the pathogen, this probiotic was chosen for further experiments. To examine the harmlessness of probiotic on sea bass larvae, fish were fed with nauplii enriched with the B. subtilis for 5 consecutive days and their survival was recorded for a total of 15 days. Artemia nauplii enriched with four doses of 150 μl of a B. subtilis culture of 7 × 109 CFU ml−1, as described above, after being thoroughly washed with autoclaved and filtered artificial seawater they were administered to fish larvae at 2 doses of nauplii daily (one every 12 h) at a density of 750 nauplii per individual per dose according to Touraki and Niopas (2012). Following the 5 days period, fish were fed with Artemia nauplii without any probiotic. As control served fish that were fed throughout the experiment with Artemia nauplii that received no probiotic treatment. Experiments were conducted in triplicate and survival was recorded daily for a total of 15 days. Fish samples were microbiologically tested on BHI agar for the presence of the inoculated strain.

Challenge of fish larvae with V. anguillarum and evaluation of treatment with the probiotic B. subtilis

Sea bass larvae of an average body weight of 90 mg were with Artemia nauplii enriched with B. subtilis for 5 consecutive days, twice a day, as described above. All experiments were conducted in triplicate, with 20 fish per experimental tank. A negative control group was used in which fish were neither fed with the probiotic nor challenged with the pathogen as well as a positive control that was challenged with the pathogen but did not receive any probiotic treatment. The challenge with V. anguillarum 332A (1.6 × 108 CFU ml−1) was performed in baths for 8 h at 21–24 °C as recommended by Sorroza et al. (2012). Survival of fish was recorded daily and each moribund or dead fish was removed from the tank and further analyzed for the presence of the pathogen.

Statistical analysis

Statistical analysis of the data was performed using one-way analysis of variance (ANOVA) followed by Tukey test in case the values showed significant differences. Statistical significance was set at a level of 0.05. The survival curves of sea bass larvae were estimated using survival analysis by the Kaplan–Meier method followed by curve comparison by long-rank test and Gehan–Breslow–Wilcoxon test.

Results

Growth inhibition in co-culture assays

The two potential probiotic strains were tested for antagonistic effects on the growth of V. anguillarum in broth co-culture assays with the pathogen. After 96 h in co-culture B. subtilis inhibited the growth of the pathogen by 33 % when added at the low concentration of 8.2 × 104 CFU ml−1 and this inhibition increased to 42 % when the concentration of the probiotic was 1.1 × 107 CFU ml−1 (Fig. 1a, b). In the case of L. plantarum, the inhibition of growth of the pathogen amounted to 16 % at the low concentration of the probiotic and to 21 % at the higher concentration of the probiotic. (Fig. 1c, d). It should be noted that in the case of B. subtilis the inhibition of growth was gradual and the growth of the pathogen declined from 0 to 96 h of co-culture, without however complete inhibition of its growth. To the contrary when L. plantarum was used, the growth of the pathogen was completely inhibited at 24 and 48 h of co-culture of the pathogen with the high concentration of the probiotic, rising again after 96 h of co-culture at levels significantly lower from control (P < 0.001).

Fig. 1
figure 1

Growth inhibition in co-culture assays of V. anguillarum with: a B. subtilis at 104 CFU ml−1, b B. subtilis at 107 CFU ml−1, c L. plantarum at 104 CFU ml−1 and d L. plantarum at 107 CFU ml−1. Open triangle growth of V. anguillarum alone, open square growth of probiotic alone, filled triangle growth of V. anguillarum in the presence of the probiotic, filled square growth of the probiotic in the presence of Vibrio

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Production of antibacterial substances by the probiotics

In order to screen whether the inhibition of the pathogen was due to antagonism or to antibacterial substances produced by the potential probiotics, the pathogen was cultured in cell-free supernatants of the two probiotics. Growth inhibition of the pathogen was significant in the 48 and 72 h supernatants of B. subtilis (P < 0.001), while in the other supernatants of the probiotic tested there was no significant reduction of the growth of the pathogen (P > 0.05) (Fig. 2a). The inhibition of growth was however more evident in the case of culture of the pathogen in the 48 and 72 h supernatants of L. plantarum and complete inhibition of the pathogen was observed after 24 h of culture (Fig. 2b).

Fig. 2
figure 2

Growth curves of V. anguillarum cultured in the presence of cell free supernatants of a B. subtilis and b L. plantarum to screen for the production of antibacterial substances by the probiotics. Open triangle control Vibrio with no S/N, open square culture of pathogen in S/N 0 h, filled triangle culture of pathogen in S/N 24 h, open circle culture of pathogen in S/N 48 h, filled square culture of pathogen in S/N 72 h and filled circle culture of pathogen in S/N 96 h

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Administration of probiotics to Artemia nauplii

Artemia survival was reduced due to the administration of probiotics (Fig. 3a, b). In the case of B. subtilis, although survival rates were slightly lower at both concentrations of the probiotic (Fig. 3a), they were not significantly different compared to the control values (P > 0.05). However, in the L. plantarum series, survival appeared significantly reduced compared to control (P < 0.05) reaching 58 % in the higher concentrations of 109 CFU ml−1 of the probiotic (Fig. 3b). The cell free supernatant of both probiotics was highly toxic to Artemia nauplii, leading to zero survival rates at 48 h after the administration.

Fig. 3
figure 3

Effect of the administration of B. subtilis (a) and L. plantarum (b) on the survival of Artemia nauplii. Diamond control survival of Artemia nauplii without any probiotic, filled square survival of Artemia following administration of 108 CFU ml−1 probiotics, open triangle survival of Artemia following administration of 109 CFU ml−1 probiotics, open square survival of Artemia following administration of cell free supernatant of probiotic culture

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Challenge of Artemia nauplii enriched with probiotics with V. anguillarum

In order to evaluate any possible protection offered to Artemia by the probiotics, the nauplii were challenged following the administration of the probiotics (Fig. 4). Our results showed a marked protection of the nauplii due to probiotics, which was higher when the higher concentrations of probiotics were used. In terms of survival, nauplii that were not treated with either probiotic showed survival rates of 33 %. Treatment with the high concentration of 109 CFU ml−1 of L. plantarum resulted in improved survival rates of 84 %, a value however significantly different from the survival of 95 % of control nauplii that were not challenged (P < 0.001). A better protection of Artemia nauplii from the pathogen appeared to be offered by B. subtilis, with survival amounting to 58 % at the low dose of the probiotic and to 99 % at the high dose of the probiotic, a value not significantly different (P > 0.05) from the survival of the control nauplii that received no challenge.

Fig. 4
figure 4

Effect of the administration of probiotics to Artemia nauplii following challenge with V. anguillarum. Filled square positive control group challenged with pathogen without any probiotic treatment, open square group receiving low dose of 108 CFU ml−1 B. subtilis prior to challenge, filled triangle group receiving high dose of 109 CFU ml−1 B. subtilis prior to challenge, open triangle group receiving low dose of 108 CFU ml−1 L. plantarum prior to challenge, filled circle group receiving high dose of 109 CFU ml−1 L. plantarum prior to challenge and open circle negative control group, no challenge, no probiotic treatment

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Administration of Artemia nauplii enriched with the probiotic B. subtilis to D. labrax larvae to evaluate harmlessness of probiotic

Since best survival rates of Artemia nauplii and best protection of them against a V. anguillarum infection was observed using B. subtilis, this probiotic was chosen to be administrated to fish larvae. Evaluation of the possible harmful effects of the probiotic to fish by the administration of Artemia nauplii enriched with the probiotic showed no harmful effects (Fig. 5), since survival (86 % ± 5.7) was not significantly different (P > 0.05) from control (95 % ± 8.6). Comparison of the survival curves showed absence of any significant difference between control and B. subtilis treated sea bass larvae (χ 2 3.38, P value 0.066 at Log-Rank test).

Fig. 5
figure 5

Survival of D. labrax larvae fed with B. subtilis enriched Artemia nauplii and challenged with V. anguillarum. Open circle negative control group of fish that did not receive probiotic treatment or challenge, filled square fish that were fed with Artemia nauplii enriched with B. subtilis but were not challenged, filled triangle positive control group of fish that did not receive probiotic treatment and were challenged with V. anguillarum, (open triangle) group of fish that was fed with Artemia nauplii enriched with B. subtilis prior to challenge with the pathogen

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Challenge of fish larvae with V. anguillarum and evaluation of treatment with the probiotic B. subtilis

In the challenge experiment, the mortality of the positive control, that is untreated challenged fish, amounted to 54 %, the mortality of the negative control to 5 %. The mortality of the probiotic treated sea bass larvae after challenge amounted to 14 % and was significantly reduced in comparison to the negative control group (Fig. 5). Comparison of the survival curves showed significant difference among the B. subtilis treated—challenged fish and the positive control group (χ 2 59.22, P value <0.001). Fish that were affected showed signs of vibriosis, mainly consisting of hemorrhagic septicaemia and the pathogen was recovered from the dead fish. Therefore fish mortalities were attributed to the pathogen.

Discussion

The extensive use of antibiotics to treat fish infections in aquaculture has led to the necessity of finding novel alternative methods, rather prophylactic treatments instead of therapeutic. As such, the use of probiotics has been suggested only a few years ago (Gomez-Gil et al. 2000; Burr and Gatlin 2005). Recently probiotics have been successfully used either to promote growth of fish (Carnevali et al. 2006), or to induce immunological responses (Picchietti et al. 2007, Makridis et al. 2008, Tapia-Paniagua et al. 2011). It has recently been reported (Swain et al. 2009) that the probiotics Streptococcus phocae and Enterococcus faecium control vibriosis in the shrimp Penaeus monodon. Protection of fish from infections has been reported using a variety of probiotics such as Pseudomonas fluorescens (Gram et al. 1999), Roseobacter (Planas et al. 2006), Vagococcus fluvialis (Sorroza et al. 2012), Salvenilus fontinalis (Boutin et al. 2011) and mixtures of Lactobacillus bacteria (Talpur et al. 2011). However, the risk of microbiological pollution of the environment when the probiotics are added in the water (Lara-Flores 2011) and the short survival of probiotics in seawater (Gatesoupe 2008) have been reported as limiting factors in their use in aquaculture. In the present study an alternative approach of the incorporation of probiotics in the live fish feed Artemia nauplii is used. Our results showed that B. subtilis and L. plantarum protect Artemia nauplii against V. anguillarum and in addition the probiotic B. subtilis protects sea bass larvae from V. anguillarum infection when administered through the nauplii of Artemia.

The growth of the pathogen was successfully inhibited by both probiotics in co-culture as well as when the pathogen was incubated in cell free supernatants of the probiotics. Similar inhibition of growth has been reported for V. harveyi in co-culture experiments with B. subtilis BT23 (Vaseeharan and Ramasamy 2003; Banerjee et al. 2007) as well as in co-culture with L. plantarum (Kongnum and Hongpattarakere 2012) and these probiotics have been suggested respectively for use as probiotic treatments of the black tiger shrimp Penaeus monodon and the white shrimp Litopenaeus vanamei. The recovery of V. anguillarum after 96 h of culture with the high dose of L. plantarum in combination to the slight decrease in growth of the probiotic observed in our study, might be attributed to competition for nutrients. Growth of lactic acid bacteria is inhibited in nutrient exhaustion conditions (Leroy and De Vuyst 2001) while V. anguillarum has been reported to survive long term starvation (Nelson et al. 1997). However, the fact that inhibition of growth of the pathogen was evident in our study when the pathogen was cultured in the 48 and 72 h cell free supernatants of the probiotics, suggests that inhibition might be attributed to the production of bacteriocins by the probiotics.

The administration of probiotics to Artemia nauplii led to slightly decreased survival rates when high concentrations of the probiotics were used and this decrease in survival was significant (P < 0.05) in the L. plantarum treated group. The higher survival rates of Artemia nauplii observed by other investigators (Patra and Mohamed 2003; Motlagh et al. 2012) are possibly due to the lower concentrations of probiotics used in these studies. The lower survival rates observed in our study are in agreement with the adverse effects of the administration of increased levels of Bacillus sp to angelfish (Farahi et al. 2011). In a study conducted by Dehghan et al. (2011) on the enrichment of Artemia urniana nauplii with B. subtilis, although survival of nauplii was not recorded, the authors concluded that Artemia nauplii are suitable to be used for bio-vaccination for the control and treatment of diseases, with time of enrichment being of crucial importance since it affects the levels of probiotics. The potency of the antibacterial substances produced by B. subtilis and L. plantarum is evident from the fact that zero survival rates were observed for nauplii in cell free supernatants of both probiotics. It appears that an optimal concentration of the appropriate probiotic is necessary to offer protection to Artemia and possibly fish against pathogens, without however showing any toxicity effects to the means of delivery, namely the nauplii. Selected bacterial strains have been reported to protect Artemia against the pathogen V. proteolyticus only when living cells or the probiotics were present (Verschuere et al. 2000). In our study B. subtilis administered at 109 CFU ml−1 of culture, offered the best protection to Artemia in a challenge with V. anguillarum, since the survival of the nauplii after challenge was not significantly different than the negative control. Hence B. subtilis was selected for further experiments with sea bass larvae. This strain was harmless to sea bass larvae since no mortality was observed 10 days after the administration of Artemia nauplii enriched with B. subtilis for a 5 day period and the comparison of the survival curves of the probiotic treated and the control group of fish showed no significant difference. In the experimental challenge however performed to evaluate the protection offered by this probiotic against infection by V. anguillarum, mortality appeared greatly reduced in the B. subtilis treated fish compared to the positive control group. A similar reduction in mortality was reported for rainbow trout after the administration of the probiotic Pseudomonas fluorescens in the water and following a challenge with V. anguillarum (Gram et al. 1999). In addition Sorroza et al. (2012) observed protection of the European sea bass following the administration of the probiotic Vagococcus fluvialis by means of a commercial dry fish feed. However there is no report on the ability of probiotics to offer protection to fish larvae against pathogens, or studies employing the administration of the probiotics through the live fish feed Artemia in challenge tests. The positive immunostimulatory effect of probiotics administered to sea bream larvae through the live fish feed Artemia was reported by Picchietti et al. (2007), although no challenge test was performed. Since an environmentally friendly method is to deliver the probiotics to fish is necessary to avoid microbial pollution (Lara-Flores 2011) and fish larvae feed on Artemia nauplii, the live fish feed as route of administration was considered an interesting but scarcely studied approach (Gomez-Gil et al. 2000). Recently Vine et al. (2006) suggested that an optimization of the loading of probiotic bacteria to live food is required. The results in our study present evidence for the ability of Artemia nauplii to act as carriers of the probiotic B. subtilis to sea bass larvae, offering protection against vibriosis.

Conclusion

In conclusion, our data provide strong evidence that B. subtilis could be used as probiotic bacteria administered through the live fish feed Artemia nauplii, to protect sea bass larvae against infection by V. anguillarum providing an environmentally friendly and antibiotic-free alternative method for the prophylaxis of marine fish larvae of vibriosis.