Probiotics and Aquaculture

1. Probiotics and Aquaculture Professor B. Austin

2. Fish diseases Biological information • Description of new pathogens (e.g. Yersinia ruckeri biotype 1; organism related to ultramicrobacterium) • Improved taxonomy (vibrios; aeromonads) • Ecology (role of Aeromonas in the aquatic environment) • Pathogenicity (role of bacteriophage in the pathogenicity of Vibrio harveyi) Disease diagnosis • Serology (ELISA’s developed for field use) • Molecular methods (sequencing)

3. Disease control • Improved husbandry/management • Movement restrictions • Genetic improvement of stock • Antimicrobial compounds (possibly will be banned in future) • Non-specific immunostimulants (e.g. ß, 1-3 glucan; LPS) • Dietary supplements (vitamins, etc) • Water treatments • Vaccines (greater range of antigens needed; improved adjuvants and oralisers • Probiotics (biological control?)

4. Probiotics in aquaculture • See: Health and Nutritional Properties of Probiotics in Food including Powder Milk with Live Lactic Acid Bacteria - FAO. • The term probiotic is a relatively new word meaning “for life” and is currently used to name bacteria associated with beneficial effects for humans and animals. • The original observation of the positive role played by some selected bacteria is attributed to Eli Metchnikoff, the Russian born Nobel Prize recipient working at the Institut Pasteur at the beginning of the last century, who suggested that "The dependence of the intestinal microbes on the food makes it possible to adopt measures to modify the flora in our bodies and to replace the harmful microbes by useful microbes" (Metchnikoff, 1907).

5. Probiotics in aquaculture • The word "probiotic" was not coined until 1960, to name substances produced by micro-organisms which promoted the growth of other microorganisms. • In order to be termed a probiotic, the micro-organism must be able to confer defined health benefits on the host, in the actual product vehicle that will be made available to [humans].

6. Definition • Fuller (1987) considered that a probiotic is a cultured product or live microbial feed supplement, which beneficially affects the host by improving its intestinal (microbial) balance. The probiotic should provide actual benefit to the host, be able to survive in the digestive tract, be capable of commercialisation, i.e. grown on an industrial scale, and should be stable and viable for prolonged storage conditions, and in the field. • FAO/WHO – alive micro-organism, which when consumed in adequate amounts, confers a health effect on the host” • Other workers have broadened the definition, for example removing any association with feed (use in water?), or the need for live cells.

7. It is clear that: • Probiotics must not be harmful to the host • They need to be effective over a range of temperature extremes and variations in salinity Confusion: • There could be confusion between a probiotic applied by the oral route, injection or immersion, and a vaccine? • Confusion could have legal implications, for example, are they feed additives or oral vaccines? Is the effect genuine or could the probiotic really be a placebo?

8. Bacillus spp. Bacillus toyoi Entrecoccus faecium Micrococcus luteus Weissella hellenica Probiotics evaluated in aquaculture Gram-positive bacteria lactic-acid producing bacteria (Carnobacterium divergens, Carnobacterium inhibens, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus rhamnosus, Lactococcus lactis)

9. Aeromonas hydrophila Aeromonas media Alteromonas Photorhodobacterium Pseudomonas fluorescens Pseudomonas spp. Roseobacter Vibrio alginolyticus Vibrio fluvialis Vibrio proteolyticus Probiotics evaluated in aquaculture Gram-negative bacteria

10. Debaryomyces hansenii Phaffia rhodozyma Saccharomyces cerevisiae S. exiguous Tetraselmis suecica Probiotics evaluated in aquaculture Bacteriophages Yeasts Microalgae

11. Immunostimulants • Lipopolysaccharides (LPS) • Vibrio vaccines • Clostridium butyricum spores • Glucan from yeast cell walls (increase phagocytosis, antibody production, increase the chemiluminescent response, and superoxide anion production).

12. Reduction in mortalities • Lead to reduction in the levels of antimicrobial compounds • Lead to improved appetite and/or growth performance Benefits of probiotics:

13. Source of probiotics • Seawater • Beach sand • Fish guts • Commercial preparations

14. Mode of action of probiotics • Improved nutrition - enzymes (biotin, B12) • Stimulation of humoral and/or cellular immune response (phagocytic and lysozyme activities) • Alteration of microbial metabolism by the increase or decrease of relevant (digestive) enzyme levels • Competitive exclusion by which the probiotic antagonises the potential pathogen by colonising the gut, producing inhibitory compounds or competing for nutrients, space (= adhesion sites in the digestive tract) or oxygen

15. Vibrio alginolyticus • Used extensively in Ecuador • Claims to have reduced mortalities and the need for antibiotics by >90% • Weakly inhibitory to fish pathogens • Protected salmonids against subsequent challenge by Aeromonas salmonicida and Vibrio anguillarum

16. Carnobacterium inhibens K • Recovered from the digestive tract of salmonids and turbot • Weakly inhibitory to fish pathogens • Protected salmonids against subsequent challenge by Aeromonas salmonicida and Vibrio anguillarum

17. Aeromonas salmonicida, A. sobria, Brochothrix thermosphacta, Micrococcus • Recovered from the digestive tract of salmonids and turbot • Weakly inhibitory to Aeromonas salmonicida • Harmless following injection ofsalmonids • Stimulated appetite (aggressively) • 14-day feeding regime was effective at resisting challenge by A. salmonicida

18. Aeromonas salmonicida, A. sobria, Brochothrix thermosphacta, Micrococcus • Dead cells were as effective as live preparations • Field evidence - less none-specific mortalities and tail/fin erosion • Mechanism of action - stimulation of innate immune response, i.e. increased number of erythrocytes, macrophages, lymphocytes and leucocytes, and enhanced lysozyme activity [no effect on antibody levels; digestive enzymes]

19. Control of Yersina ruckeri biotype 1 • This biotype is not controlled by the currently available vaccines • An isolate of Micrococcus luteus was effective when fed to the fish as a probiotic • Appetite was stimulated • The isolate was also effective against Lactococcus garvieae and Streptococcus iniae

20. Conclusions • What is the difference between probiotics, immunostimulants, and oral vaccines? • Health benefits are clearly demonstrated • Indirect benefit: reduction in use of antimicrobial compounds • Must ensure that probiotic is harmless and stays harmless to the host • However, there are market opportunities