Ø Aquaculture is one of the fastest growing
food-producing sectors.
The Use of Probiotics in
Aquaculture
Ø Need to reduce the use of antimicrobials.
Ø Emphasis needs to be on prevention.
Ø Antibiotic use in Norway dropped from 50
metric tons pa in 1987 to 746.5 kg in 1997.
Ø Production of farmed fish in Norway increased
from 5 x 104 to 3.5 x 10 5 metric tons.
Ø Vibrio alginolyticus has been used as a
probiotic in Ecuadoran shrimp hatcheries
since 1992.
Ø Hatchery down time reduced from 7 days /
month to < 21 days / year.
Ø Production ↑ by 35%, while antibiotic use ↓ by
94% between 1991 and 1994.
Definition of probiotics
Ø Fuller - a live microbial feed supplement
which beneficially affects the host animal
by improving its intestinal balance.
Ø Not adequate for aquatic organisms.
Ø Terrestrial - gut represents a moist habitat in a
water-limiting environment.
Ø Aquatic - hosts and microorganisms share the
ecosystem. Probiotic bacteria could also be
active on the gills and skin of the host, as well
as in its ambient environment.
Ø 10% annual growth between 1984 - 1995
Ø 3 % livestock meat
Ø 1.6% capture fisheries
Ø Disease outbreaks are a significant
constraint on aquaculture.
Ø The use of disinfectants and antimicrobials
have had limited success
Ø growing concern about the use of antibiotics
which encourage the emergence of bacterial
resistance.
Probiotics
Ø Probiotics are considered to be one of the
most significant technologies to have
evolved in response to disease control.
Ø FAO defined the development of affordable,
efficient vaccines, the use of
immunostimulants and non-specific
immune enhancers, and the use of
probiotics and bioaugmentation for the
improvement of aquatic environmental
quality as major areas for further research
in disease control in aquaculture.
Definition of probiotics
A probiotic is a live microbial adjunct which
has a beneficial effect on the host by
modifying the host-associated or ambient
microbial community, by ensuring
improved use of feed or enhancing its
nutritional value, by enhancing the host
response towards disease, or by improving
the quality of its ambient environment.
Verschuere et al. (2000) MMBR 64: 655-671
1
Can microbial communities be
manipulated?
Ø Aquaculture practices do not provide
appropriate environments for the
establishment of stable microbial
communities.
Ø Discontinuous culture cycles.
Ø Disinfection or cleaning of ponds or tanks
prior to stocking.
Ø sudden increases in nutrients due to
exogenous feeding.
Ø Instead of allowing spontaneous primary
colonisation of the rearing water by bacteria
accidentally present, the water could be pre-
emptively colonised by the addition of
probiotic bacteria.
- recognised that pre-emptive colonisation
extends the reign of pioneer organisms.
Ø A single addition would suffice if the probiotic
bacteria are well adapted to the prevailing
environmental conditions.
Ø If the host / environment already carries a
stable microbial community, repeated
inoculations will be necessary.
Ø Accepted that fish contain a specific
intestinal microbiota that is established at
the juvenile stage or after metamorphosis.
Ø Unlikely that a single inoculum of a probiotic
bacteria will result in long term dominant
colonisation.
Ø It will be necessary to supply the probiotic on
a regular basis if a continuous colonisation at
high densities is required.
- especially if the bacterial species used do
not belong to the normal dominant intestinal
microbiota of the cultured species or its
particular development stage.
Ø Microbial communities influenced by
deterministic and stochastic factors.
Ø Deterministic factors include:
Ø salinity
Ø temperature
Ø oxygen concentration
Ø quantity and quality of feed
Ø Stochastic factors:
Ø chance favours organisms which happen to
be in the right place at the right time to enter
the habitat and to proliferate if the conditions
are suitable.
Ø Lactic acid bacteria
Ø Not dominant in the normal intestinal
microbiota of fish.
Ø Addition of high doses in juvenile fish resulted
in a temporary change of the intestinal
microbial community.
Ø Lost within a few days after inoculations
halted.
Application
Ø Probiotics are applied in the feed or added
to the culture tank / pond as preventative
agents against infection by pathogenic
bacteria.
Ø Nutritional effects are also often attributed
to probiotics (especially for filter feeders).
2
Application
Ø Most probiotics used in aquaculture are:
Ø Lactobacillus; Carnobacterium (lactic acid
bacteria).
Ø Vibrio s p. (V. alginolyticus)
Ø Bacillus
Ø Pseudomonas
Fish eggs and larvae
Ø Improved the survival rate of larval turbot
(Scophthalmus maximus) by daily addition
of lactic acid bacteria to the enrichment
medium of the rotifers used as live food for
the turbot larvae (Gatesoupe, 1994).
Ø The added lactic acid bacteria could be
retrieved in large amounts from the turbot
larvae, and a significant reduction of larval
mortality was observed when the larvae were
challenged with a pathogenic Vibrio on day 9.
Fish juveniles and adults
Ø A good correlation was found between the
production of siderophores and the protective
action of P. fluorescens, suggesting that
competition for free iron is involved in the
mode of action.
Ø P. fluorescens exerted its effect from the
host’s exterior, since the strain did not
significantly invade the fish following bath
treatment.
Fish eggs and larvae
Ø It is assumed that uncontrolled microbial
development in hatcheries is one of the
main reasons for the unpredictable results.
Ø Probiotics may be beneficial as control.
Ø 2 week old halibut larvae is affected by
incubation with indigenous fish bacteria
Ø survival in presence of V. salmonicida-like
strains and Lactobacillus plantarum = 95%.
Fish juveniles and adults
Ø Several strains of Pseudomonas fluorescens
were able to exclude a pathogenic A.
salmonicida strain from Atlantic salmon
presmolts with stress-inducible furunculosis
infection and to limit the mortality of rainbow
trout (40 g) infected with V. anguillarum.
Ø Short-term bathing of the fishes in a bacterial
suspension of the probiotic, long-term
exposure in the rearing water, or a
combination of the two treatments led to a
significant decrease in mortality after the
challenge trial.
Fish juveniles and adults
Ø A V. alginolyticus strain was applied in a bath
treatment to Atlantic salmon (21 g) maintained
in freshwater.
Ø V. alginoiyticus was found in the intestine up
to 21 days after the initial probiotic
application.
Ø Challenge experiments revealed that the
probiont led to a reduction in mortality after
exposure to A. salmonicida and to a lesser
extent after exposure to V. anguillarum and V.
ordalii.
3
Penaeid shrimps
Ø A non-pathogenic V. alginolyticus strain was
inoculated daily into 25-and 60-ton larval
rearing tanks containing Litopenaeus
vannamei postlarvae.
Ø Survival and wet wt. were higher in the
probiotic-treated shrimps compared to
shrimps receiving prophylactic doses of
oxytetracycline and the control group.
Ø V. parahaemolyticus was not detected in any
of the probiotic treated shrimps, while control
tanks and those containing shrimps receiving
antibiotics had V. parahaemolyticus in
approximately 10% of the samples.
Penaeid shrimps
Ø Strain BY-9 was produced on large scale and
inoculated at 10‘ CFU/ml into an 18-ton larval
rearing tank for mass production of P.
monodon larvae.
Ø The strain is known to inhibit the in vitro
growth of V. harveyi.
Ø BY-9 inoculation gave a lower Vibrio density
(46.1%) and a higher survival rate (10.6%) than
control larvae cultured up to the tenth
postlarval stage.
Bivalve molluscs
Ø Alteromonas holoplanktis was isolated
from the gonads of Chilean scallop
(Argopecten purpuratus) broodstock.
Ø Displayed in vitro inhibitory activity against
the known pathogens V. ordalii, V.
parahaemolyticus, V. anguillarum V.
alginolyticus, and Aeromanas hydrophila.
Ø Protected the scallop larvae against V.
anguillarum in an experimental infection.
Penaeid shrimps
Ø The use of several Bacillus cultures in
penaeid culture ponds in Indonesia allowed
culture of the shrimps for over 160 days
without problems.
Ø Farms that did not use the Bacillus cultures
experienced almost complete failure in all
ponds, with luminescent Vibrio disease killing
the shrimps before 80 days of culture was
reached.
Ø A cost-benefit analysis of the use of Bacillus
cultures on a particular farm in Thailand
showed a clear benefit to the producer.
Crabs
Ø After an inoculation of diatoms and rotifers,
the bacterial strain PM-4 was introduced daily
for 7 days in 200-m3 tanks containing crab
(Portunus trituberculatus) larvae and was also
inoculated with diatoms and rotifers.
Ø There was a negative correlation between the
presence of PM-4 and the densities of Vibrio
spp.
Ø In 7 trials, the ave. survival of crab larvae +
strain PM-4 was 27.2%. In 6/9 trials without
PM-4, no larvae grew into adults, resulting in
an ave. survival of only 6.8%.
Bivalve molluscs
Ø Aeromonas media A199 inhibited 89 strains of
aeromonads and Vibrio in vitro.
Ø Prevented mortality in oyster (Crassostrea
gigas) larvae challenged with Vibrio tubiashii.
Ø Since A. media A199 could no longer be
detected on the host only 4 days after the
probiotic treatment, it would be necessary to
introduce the probiotic at regular intervals if a
prolonged protective effect is required.
4
Live food
Ø Unicellular algae are often given as a first
food or are included in the culture system
as a food for rotifers and Artemia. Bacteria
increase the growth rate and yield of algae.
Ø Since bacteria may also inhibit algal
growth, careful screening is necessary
when bacteria are to be used as probiotics
in larval rearing or in the green-water
technique.
Artemia
Ø Verschuere et al. selected 9 bacterial strains
that positively influenced the growth and/or
survival of juveniles of the brine shrimp
Artemia cultured as a live food for other
species.
Ø All 9 strains delayed the death of the Artemia
when experimentally infected with pathogenic
V. proteolyticus CWST2.
Ø While all Artemia in the axenic control died within 2
days after infection, the survival rates of the
Artemia cultures inoculated with strain LVSS or a
mixture of the 9 strains showed > 80% survival
after 4 days.
Unicellular algae
Ø Strain SK-05 was selected for its active growth
in organic-poor substrates and inoculated it
into a Skeletonema costatum culture.
Ø V. alginolyticus was inoculated as a typical
contaminant.
Ø Strain SK-05 prevented V. alginolyticus
growth.
Ø Since it exerted no in vitro inhibitory action
against V. alginolyticus, it seems that the
protection was due to competitive exclusion
as only strain SK-05 was able to utilise S.
costatum exudates.
Nutritional effects
Ø Addition of bacteria to the rearing water of
filter feeders such as rotifers, bivalve larvae
or adults, and crustacean larvae may result
in massive uptake of these bacteria
Ø Bacteria may act as a (complementary)
food source or contribute to the digestion
of the food.