PROBIOTICS AND THEIR USE IN AQUACULTURE

April 2006 10th Aquaculture Insurance and Risk Management Conference

Presented by: William P. Long Chief Executive Officer Epicore BioNetworks Inc.

BASIC AQUACULTURE MARKET NEEDS

To survive and make money

To overcome problems of: Pond crashes Farm abandonment Low survival Poor quality animals Low weights Poor feed conversion ratio (FCR) Its not a hobby; its a business!

MURPHYS LAW

Whatever can go wrong, will go wrong! Mismanagement

Weak PL, bad feed, inadequate oxygen, poor harvest procedures

Natural events
Weather, predators

Disease Theft Crop market prices

THE SCIENCE OF SURVIVAL

Through microbiological science aquaculturists can fight Murphy's Law Probiotics are a natural, environmentally friendly application of microbiology

TRADITIONAL DISEASE CONTROL UNDER FIRE

Many

older antibiotics proven unsafe so are banned from all uses Shrimp with antibiotic residues cannot be sold in many major markets State of art antibiotics should be reserved for humans

ROLE OF ENVIRONMENTAL MICROBIOLOGY IN AQUACULTURE

Pollution control
Bioremediation of wastes Cleaner water & pond bottoms Disease resistance imparted by creating a lower stress growing environment

Probiotic action
Directly protects animals from disease

Pathogenic Vibrio parahemolyticus, Vibrio harveyi bacteria, Aeromonas

Indirectly protects from some viral diseases

POLLUTION CONTROL

Waste is major problem in aquaculture pond and hatchery waters Affects animal health and vigor Lowers harvest yield value Forces frequent water exchange

A biosecurity risk

Bacteria are natures chosen waste degraders End result of microbial action is biomass - a soil amendment

Harnessing the power of natures microbial workforce

BIOREMEDIATION MODE OF ACTION

Aquaculture wastes are organic in nature Microorganisms eat organic wastes

Bacteria + N + C + P + O2 = More Bacteria + H2O + CO2

Microorganisms digest pollutants

Ammonia: very toxic to crustaceans and fish Nitrite: very toxic to crustaceans and fish

And is food for disease causing Vibrio

Nitrate: too much causes algae crashes Pond bottom waste: release nutrients in older ponds to promote natural algae bloom without added fertilizer

WHAT IS A PROBIOTIC
A

live microbial supplement that benefits the host animal

From the Greek probios:

Pro = for

Bios = life

Opposite

of antibiotic

A microbial killer or suppressant

PROBIOTIC MODES OF ACTION

Competitive exclusion

Out-competes pathogenic bacteria for nutrients Displaces pathogens from gut wall sites Produces acids in gut to inhibit pathogenic bacteria Repels or kills pathogenic bacteria

Bacteriocin (antimicrobial) excretion

Stimulates immune response Aids digestion

Produces enzymes to help digest & absorb feed Reduces toxic amines

PROBIOTIC BENEFITS vs. ANTIBIOTICS

Probiotic use eliminates need for antibiotics Probiotics create no illegal residue Bacteria cannot develop resistance to probiotics but can to antibiotics Antibiotics lead to slow growth rate of larvae Probiotics boost immune system of shrimp Probiotics also digest pollutants Probiotics promote high survival so are costefficient to use Cheaper to use probiotics than antibiotics

PROBIOTICS AND VIRUSES

Anecdotal evidence that probiotics mitigate pathogenicity of WSSV virus

Pond in Vietnam saved by double does of probiotic after WSSV outbreak Pond in Mexico with same experience after PCR testing and mortality confirmed WSSV presence

Some researchers believe probiotics stimulate shrimp immune response Some researchers believe that probiotics mitigate WSSV by controlling secondary pathogens

PROBIOTIC COMPOSITION

Single strain of live microorganism

Usually bacterial or yeast e.g. Lactobacillus acidophilus, Saccharomyces cerevisae, Vibrio alginolyticus
Lactic bacteria typical of human probiotics

Microbial ecosystems
Mixtures of specially selected, live but dormant microorganisms Synergistic mixtures High strength for economy of use

Usually are dry powders

Excellent shelf life if kept dry

WHY MICROBIAL ECOSYSTEMS?

Teams work better than individuals

Ecosystems work better than single strains

What makes a good ecosystem?

Enough players on the team

High cell count (cfu = colony forming units) More than one type (i.e. strain) Too many strains is bad (poor QC, low individual count) Best strain for each intended purpose (e.g. enzyme production or specific waste digestion) Safe microorganisms

Enough of the right types of players

High quality players of each type

TYPICAL AQUACULTURE PROBIOTIC

Selected live microorganisms like:

Bacillus spp., Lactobacillus spp., Yeast Soil-based, non-toxic, not genetically modified Not close relative of pathogens

Minimum strength = 2E+09 CFU/gm

i.e. 2,000,000,000 colony forming units per gram

Fortified with a broad spectrum of free enzymes to start waste digestion Contains proprietary growth stimulants Free of pathogens and toxins Biodegradable carrier

TYPICAL PROBIOTIC PRODUCT

PROBIOTIC SUCCESS FACTORS

Existence of water quality or disease problem Use proper dosage & treatment frequency
Follow literature or ask local distributor

Time to work
Bacteria, unlike chemicals, take time to work For curative application allow 24-48 hours

Organic matter present

Add biological growth media to new or lined ponds

Proprietary media or home-made (e.g. sucrose-based)

Salinity
Good probiotics are tolerant but best in salinity <40 ppt

Pond pH tolerant but best at 6-10 Temperature: Slower at low temperatures

AMMONIA DIGESTION
Ammonia (TAN) Concentration Over Time at Various Dose Rates of EPICIN EPICIN (mg/l)
4.0 error bars represent +/- sd 3.0 TAN (mg/l)

10
2.0

1.0

50

0.0 0 1 2
Time (Days)

Probiotics take time to work Ammonia digestion is dose sensitive

SALINITY TOLERANCE
4 3.5 3 TAN (ppm) 2.5 2 1.5 1 0.5 0 0 1 Time (days) 2 3 48 37 24 12 0

ORGANIC CARBON IMPORTANCE

4 3.5 TAN (mg/l) 3 2.5 2 1.5 1 0.5 0

22

43

64

86

Total Organic Carbon (mg/l)

Works best with organic carbon present

PROBIOTIC HYDRATION

Best to hydrate (or wake up) dormant bacteria Success factors

Water quality: use clean or disinfected pond water

Neutralize disinfectants before adding PROBIOTICS

Aeration: use air stone or stir frequently Time: follow literature for hydration time

Growth media
Specially designed media offers control and optimum growth Pond-side grow-up lowers treatment costs

PROBIOTIC CELL GROWTH

HYDRATION IN POND WATER
Cell Count (billion cfu/ml) 600 500 400 300 200 100 0 0 2 4 6 8 10 12 Grow-up time (hours)

Growth reverses unless special growth media is used

PROBIOTIC GROW-UP WITH MEDIA In FRESH and SEA WATER

4.0E+08 3.5E+08 3.0E+08 cfu/ml 2.5E+08 2.0E+08 1.5E+08 1.0E+08 5.0E+07 0.0E+00 0.0 hrs 4.0 hrs 8.0 hrs Fresh Water Sea Water

Slightly more growth in fresh water; BGM media allows more growth

PROBIOTICS BENEFIT THROUGHOUT THE LIFECYCLE

In In In

maturation hatcheries grow-out

To produce the eggs To produce the juveniles To produce the adults

PROBIOTICS IN GROW-OUT PONDS

TYPICAL GROW-OUT POND

PROBIOTIC APPLICATION TO POND

GROW-OUT SHRIMP

BENEFITS OF POND PROBIOTICS

Better water quality Less toxic environment for culturing animals Higher stocking densities Reduced animal stress Better growth & survival Larger animals or shorter cycles Higher yield Probiotic, beneficial microbial environment Better growth & survival Reduced water exchange Greater biosecurity Less pollution output Lower fertilizer costs in older ponds

PROOF OF EFFICACY
In

vitro tests

Lab ammonia digestion test Microbiological competitive growth tests vs. pathogenic bacteria
In

vivo tests

Shrimp Vibrio challenge test Field tests

COMPETITIVE GROWTH

24 hour probiotic pre-treatment of five PL30 P. vannamei Vibrio parahemolyticus challenge (approx. 2E+04 cfu/ml) Record survival after 24 hours
120 Survival Increase (%) 100 80 60 40 20 0 -20 0 -40 EPICIN:Vibrio Cell ratio 5 10 15 20 25

EPICORE SHRIMP CHALLENGE TESTS

Probiotic protection is dose sensitive

SEMI-INTENSIVE VANNAMEI POND RESULTS Ecuador 1995-1998

Parameter Ponds (#) Avg. Size (ha) Avg. S.D. (PL/sq m) Avg. Cycle (days) Avg. weight (gm) Avg. survival rate Annual yield (kg/ha)

Probiotic 20 3.25 18.2 117 10.6 39.5% 1720

Control 26 8.7 16.9 121 10.4 34.2% 1524

Probiotic gave 12.9% average higher yield

Microbiological Assay of 1995 Ecuadorian Shrimp Ponds

Pond 51 - CONTROL

POND 58 Probiotic + BGM TREATED

INTENSIVE VANNAMEI POND RESULTS Brazil 2001

Parameter Pond Cycles Avg. size (ha) Avg. S.D. (PL/sq m) Avg. cycle (days) Avg. weight (gm) Avg. S.R. Cycle yield (kg/ha) Probiotic 3 2.25 95.4 114 12.0 73.5% 8,470 Control 4 2.25 64.3 95 11.6 69.5% 4,750

Probiotic allowed higher stocking and gave 78% yield increase

PROBIOTIC ADDITION TO FEED

Ecuadorian Pond Survival Results

60.0% 50.0% Survival 40.0% 30.0% 20.0% 10.0% 0.0% Control 2.2 g/kg feed EPICIN Treatment Rate 4.4 g/kg feed

Very efficient for low SD ponds

TYPICAL SHRIMP HATCHERY

TYPICAL SHRIMP HATCHERY TANKS

POSTLARVAL SHRIMP

PROBIOTICS IN HATCHERIES

Prevention better than curative approach

Use throughout cycle to avoid problems

Product grade selection

Special grades for hatcheries & for zoea stage

Product hydration important

Pass mixture through a 100 micron mesh and discard the remaining solid material

Use extra when conditions dictate Follow supplier literature DO NOT USE with antibiotics or microbiocides

PROBIOTIC HYDRATION AND APPLICATION

Larvipac 3 Year Data Comparison

500,000 Production '000 400,000 300,000 200,000 100,000 33.42 2000 2001 2002 47.46 62.21 70 60 50 40 30 Survival %

EPICORE SYSTEM TRIAL

NORAMAC HATCHERY - ECUADOR
70% 60%

Average Survival %

50%

40%

AVERAGE 9 TANKS USING PROBIOTIC

30%

AVERAGE 3 TANKS WITHOUT PROBIOTIC

20%

10%

0% 1

Probiotics improve all liquid feed results.

NON-SHRIMP SPECIES
Probiotics

work in fish aquaculture as well as in shrimp

In hatcheries In grow-out

Research

on several species

Fish Crabs Oysters

FLORIDA KEYS FISH HATCHERY

U. OF MIAMI SEA TROUT STUDY

Mean sea trout lengths 21 days after hatching using lumped trial data for analysis
6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1
Lum ped Probiotics Lumped Control

length (mm)

p = 0.08 (NS)

Treatment

U. OF MIAMI SEA TROUT STUDY

Mean Sea Trout Survival 21 days after hatching
1200

1000

800

Count

600

p = 0.21

400

200

Probiotics
Treatment

Control

PROBIOTICS MAKE A DIFFERENCE

Probiotics always come out on top!

PROBIOTICS FOR AQUACULTURE

The science of survival

Help to improve aquaculture yield and profits Thanks for your interest.
2006 Epicore BioNetworks Inc.