François-Joël Gatesoupe

The use of probiotics in aquaculture

The research of probiotics for aquatic animals is increasing with the demand for environmentfriendly aquaculture. The probiotics were defined as live microbial feed supplements that improve health of man and terrestrial livestock. The gastrointestinal microbiota of fish and shellfish are peculiarly dependent on the external environment, due to the water flow passing through the digestive tract. Most bacterial cells are transient in the gut, with continuous intrusion of microbes coming from water and food. Some commercial products are referred to as probiotics, though they were designed to treat the rearing medium, not to supplement the diet. This extension of the probiotic concept is pertinent when the administered microbes survive in the gastrointestinal tract. Otherwise, more general terms are suggested, like biocontrol when the treatment is antagonistic to pathogens, or bioremediation when water quality is improved. However, the first probiotics tested in fish were commercial preparations devised for land animals. Though some effects were observed with such preparations, the survival of these bacteria was uncertain in aquatic environment. Most attempts to propose probiotics have been undertaken by isolating and selecting strains from aquatic environment. These microbes were Vibrionaceae, pseudomonads, lactic acid bacteria, Bacillus spp. and yeasts. Three main characteristics have been searched in microbes as candidates to improve the health of their host. 1 . The antagonism to pathogens was shown in vitro in most cases. 2 . The colonization potential of some candidate probionts was also studied. 3 . Challenge tests confirmed that some strains could increase the resistance to disease of their host. Many other beneficial effects may be expected from probiotics, e.g., competition with pathogens for nutrients or for adhesion sites, and stimulation of the immune system. The most promising prospects are sketched out, but considerable efforts of research will be necessary to develop the applications to aquaculture.

Lactic acid bacteria in fish: a review

Fish are continuously exposed to a wide range of microorganisms present in the environment, and the microbiota of fish have been the subject of several reviews. This review evaluates lactic acid bacteria in fish, and focuses on the several investigations that have demonstrated that Streptococcus, Leuconostoc, Lactobacillus, and Carnobacterium belong to the normal microbiota of the gastrointestinal tract in healthy fish. However, it is well known that the population level of lactic acid bacteria associated with the digestive tract is affected by nutritional and environmental factors like dietary polyunsaturated fatty acids, chromic oxide, stress and salinity. Pathogenic lactic acid bacteria such as Streptococcus, Enterococcus, Lactobacillus, Carnobacterium and Lactococcus have been detected from ascites, kidney, liver, heart and spleen. Some antibiotic treatments and vaccinations have been proposed to cure or prevent these diseases that seem, however, to spread with the development of fish culture. It has also been reported that some lactic acid bacteria isolated from the gastrointestinal tract of fish can act as probiotics. These candidates are able to colonise the gut, and act antagonistic against Gram-negative fish pathogens. These harmless bacteriocin-producing strains may reduce the need to use antibiotics in future aquaculture.

Updating the importance of lactic acid bacteria in fish farming: natural occurrence and probiotic treatments.

Many recent papers have deepened the state of knowledge about lactic acid bacteria (LAB) in fish gut. In spite of high variability in fish microbiota, LAB are sometimes abundant in the intestine, notably in freshwater fish. Several strains of Streptococcus are pathogenic to fish. Streptococcus iniae and Lactococcus garvieae are major fish pathogens, against which commercial vaccines are available. Fortunately, most LAB are harmless, and some strains have been reported for beneficial effects on fish health. A major step forward in recent years was the converging evidence that LAB can stimulate the immune system in fish. An open question is whether viability can affect immunostimulation. The issue is crucial to commercialize live probiotics rather than inactivated preparations or extracts. There has been a regain of interest in allochthonous strains used as probiotics for terrestrial animals or humans, due to economical and regulatory constraints, but the short survival in sea water may limit application to marine fish. If viability is required, alternative treatments may include the incorporation of prebiotics in feed, and other dietary manipulations that could promote intestinal LAB. Antagonism to pathogens is the other main feature of candidate probiotics, and there are many reports concerning mainly carnobacteria and Enterococcus. Some bacteriocins were characterized which may be of interest not only for aquaculture, but also for food preservation.

Effect of live yeast incorporation in compound diet on digestive enzyme activity in sea bass (Dicentrarchus labrax) larvae

Yeasts produce polyamines, and some strains have a strong adhesion potential to intestinal mucus, an important condition for probiotic efficiency. The aim of this study was to explore an in situ production of polyamines by Debaryomyces hansenii HF1 (DH), a yeast strain isolated from fish gut, in comparison with Saccharomyces cerevisiae X2180 (SC) (Goteborg University Collection). The production of polyamines by DH was three times higher than that of SC. The main polyamines were spermine and spermidine, produced at a similar level. Both strains adhered to the gut of sea bass larvae. When the yeasts were introduced into a compound diet, the colonization was effective in the larvae (104 CFU g−1 on a body weight basis). The DH diet led to an increase in amylase secretion in 27-day-old larvae in comparison with the control diet. The secretion of amylase and trypsin was lower in the SC diet, and some delay in trypsin secretion was still observed in this group at day 42. At day 27, the activity of brush border membrane enzymes was stimulated by the DH diet, and delayed by the SC diet, in comparison with the control diet. The survival of the larvae was also increased in the DH diet, but the growth rate was lower than that in the control group. This may be due to the introduction of live yeast into the diet, which needs to be optimised.

The effect of three strains of lactic bacteria on the production rate of rotifers, Brachionus plicatilis, and their dietary value for larval turbot, Scophthalmus maximus

Abstract Rotifers were cultured with three food additives differing only in their strain of live lactic bacteria. Two Lactobacillus strains significantly increased the population density of rotifers, but L. plantarum was more efficient than L. helveticus , while the effect of Streptococcus thermophilus was not significant. The amount of aerobic bacteria in rotifer cultures was significantly decreased with L. plantarum . The growth of Aeromonas salmonicida in rotifers was particularly inhibited by L. plantarum . However, the dietary value of the rotifers was improved by the three bacterial additives, since the mean weight of turbot at day 20 was significantly increased when the rotifers had been fed with any of the three additives. No further improvement of the growth rate was observed when Artemia nauplii were also fed with the additives.

Probiotic and formaldehyde treatments of Artemia nauplii as food for larval pollack, Pollachius pollachius

Abstract Formaldehyde was used to disinfect Artemia cysts and nauplii, while introducing two probiotics in the enrichment process: Bactocell (Pediococcus acidilactici) and Levucell (Saccharomyces cerevisiae). The disinfectant was selected due to its potential compatibility with probiotics, since it was more effective against Gram-negative bacteria than against lactic acid bacteria and yeast. However, the presence of formaldehyde reduced the intake of P. acidilactici in Artemia. Consequently, the disinfection was stopped before Bactocell supplementation to the nauplii fed to pollack (Pollachius pollachius) larvae. The mean weight of pollack was higher with this probiotic treatment. Growth was even better with the combination of Levucell and Bactocell, but the yeast should be introduced circumspectly. A high bacterial load was found in the nauplii enriched with Levucell, but not treated with formaldehyde. In the absence of Bactocell, the discontinuation of disinfecting Artemia after Levucell enrichment caused poor growth of pollack. Resistant strains had emerged after 3 months of daily cyst incubation with 50 mg l−1 formaldehyde, rising from 1.4 CFU per newly hatched nauplius before the experiments, up to 3.4×104 CFU nauplius−1 by the end of 3 months. The phenotypes and genotypes of these opportunistic resistant strains were quite different from the initial resistant strains. Particular attention was paid to Vibrio alginolyticus-like isolates that were not detected before the experiments. These isolates were compared by amplified ribosomal DNA-restriction analysis (ARDRA) and RAPD to two reference strains previously isolated in turbot larvae, indicating some genotypic distance between the new isolates and the reference strains. Thus, the formaldehyde treatment cannot be recommended due to the risk of resistance spread. It was concluded that P. acidilactici is a promising probiotic for fish larvae. Its combination with S. cerevisiae may be valuable, but on the condition that a concentrated form that would not jeopardize the bacterial balance in the absence of disinfectant should be prepared.

Pathogenicity of Vibrio splendidus strains associated with turbot larvae, Scophthalmus maximus

Turbot larvae were challenged with eight strains of Vibrio splendidus isolated from diseased larvae, plus a ninth strain pathogenic to scallop larvae (A515; Nicolas et al. 1996 ). Six strains caused heavy mortality but the scallop pathogen and the other two strains did not. All the strains shared a large number of phenotypic traits, and an attempt was made to relate virulence to genotype and phenotype. Five of the six pathogenic strains were very similar, as shown by RAPD fingerprinting and phenotypic characteristics. The relatedness of the other strains was intermediate between the main pathogenic group and V. splendidus ATCC 33125, but the DNA–DNA homology between the pathogenic group and the reference strain was still high (78% of reassociation rate). The non‐pathogenic isolates may be a useful tool for determining the possible virulence factors, as all the isolates differed by few characteristics.

The continuous feeding of turbot larvae, Scophthalmus maximus, and control of the bacterial environment of rotifers

Turbot larvae were fed continuously with rotifers pumped from the culture tanks and rinsed before distribution. This technique improved the growth and survival rates of turbot, in comparison with the results obtained with a single daily distribution of rotifers enriched for 6 h. Special attention was paid to associated bacteria. Most bacteria detected in healthy rotifers and turbot were identified as Vibrio alginolyticus. An opportunistic strain of Aeromonas sp. was observed in large numbers, but only in tanks where the larvae were on the verge of high mortality. A food additive containing live lactic bacteria improved the growth rate of turbot when it was put into the rotifer medium. A similar improvement was observed when the pH of this medium was lowered to 5.26–6, while the temperature was increased from 19 to 26°C. The combination of the bacterial additive and the pH-temperature changes was not efficient and might have led to the proliferation of Aeromonas.

The effect of bacterial additives on the production rate and dietary value of rotifers as food for Japanese flounder, Paralichthys olivaceus

Two food additives containing live lactic bacteria were given to S-type rotifers fed on live algae and bakers yeast. One of them improved the production rate of rotifers (59 rotifers ml−1 day−1) in comparison with the control group fed without any additive (48 rotifers ml−1 day−1). The second additive did not improve the production rate (42 rotifers ml−1 day−1), but it improved the dietary value of the rotifers. Indeed, at day 18, the mean length of Japanese flounder fed on these rotifers (7.5 mm) was significantly higher than that of the control group (7.3 mm). The total amount of aerobic bacteria in rotifers was also affected by this additive. Three thousand bacteria per rotifer were recorded in the production tanks. Bacterial growth was particularly high during the enrichment phase. After 17 h of enrichment with an emulsion of cuttlefish liver oil, 99 000 bacteria were counted per control rotifer. The second bacterial additive limited the bacteria to 54 000 per rotifer after 17 h of enrichment. This may be a reason why this additive improved the dietary value of the rotifers.

Bacteria associated with cultured rotifers and artemia are detrimental to larval turbot, Scophthalmus maximus L.

Abstract The survival rate of turbot is improved significantly by disinfecting rotifers, Brachionus plicatilis for 24 h with antibiotics. During the second step of the food sequence, the same effect is observed with the disinfection of Artemia . It is concluded that both food organisms convey detrimental bacteria and that they should be cultured as cleanly as possible.