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Dr. T. CitarasuDr. T. CitarasuAssistant ProfessorAssistant ProfessorTele-fax: + 91-4652-253078Tele-fax: + 91-4652-253078Mobile: +91-9994273822Mobile: +91-9994273822Email:Email: [email protected]@gmail.com [email protected]@msuniv.ac.inWebsite:Website: www.msuniv.ac.inwww.msuniv.ac.in www.drcitarasu.comwww.drcitarasu.com
IMPORTANCE OF AQUACULTUREIMPORTANCE OF AQUACULTURE
Lucrative food production industry
Fresh, brackish & Marine based culture
10 % annual growth rate
Global sea food demand-70% in the next 35 years (7 fold)
Capture and culture fisheries
Shrimp FarmingShrimp Farming
Asia is the largest producer- 80 %Asia is the largest producer- 80 %
India is the second place of Asia & India is the second place of Asia & fifth place in globallyfifth place in globally
>350 shrimp hatcheries 13 billion seed >350 shrimp hatcheries 13 billion seed production/ annum production/ annum
Top 5 Countries M. M tonesChina 34.43India 3.13Vietnam 1.65Thailand 1.39Indonesia 1.30
Aquaculture production (FAO, 2009)
CURRENT PROBLEMS IN AQUACULTURE CURRENT PROBLEMS IN AQUACULTURE Culture methods
• Extensive: 0-5 m3
• Moderate extensive : 5-15 m3
• Semi intensive : 15-30 m3
• Intensive : 30-60 m3
• Super Intensive : > 60 m3
Significant economic losses
Bacterial diseases - Vibriosis
Viral diseases – WSSV; MBV
Stress Induced diseases
Finfish DiseasesFinfish Diseases
Freshwater FishesFreshwater FishesMotile Aeromonad septicemia Motile Aeromonad septicemia (MAS)(MAS)
Hemorrhagic septicemiaHemorrhagic septicemiaA. hydrophila, A. sobriaA.caviae, A. veronii, A. salmonicida
Marine FishesMarine FishesVibriosisVibriosis
Pathogenic VibriosPhotobacterium spMicrococcus spFlavobacterium- Gill diseaseEnterobacter- EdwardsiellaPseduomonas Salmonella
Important Shrimp Diseases in IndiaImportant Shrimp Diseases in India
VibriosisVibriosis RMS or White Patch DiseaseRMS or White Patch Disease
White Spot Syndrome Virus (WSSV)White Spot Syndrome Virus (WSSV) Enterocytozoon hepatopenaei (EHP)
CURRENT PROBLEMS IN DISEASE CONTROL CURRENT PROBLEMS IN DISEASE CONTROL
Current disease treatment Current disease treatment protocols – difficultprotocols – difficult
Chemicals & Synthetic drugsChemicals & Synthetic drugs
Antibiotics & other chemicalsAntibiotics & other chemicals
MPEDA abandoned more than MPEDA abandoned more than 10 antibiotics10 antibiotics
Chloramphenicol Metronidazole
Nitrofurans Ronidazole
Neomycin Ipronidazole
Nalidixic acid nitroimidazoles
Sulphamethoxazole Clenbuterol
Chlorpromazine Diethylstilbestrol
Colchicine Sulonamide
Dapsone Floroquinolones
Dimetridazole Glycopeptides
Banned Antibiotics (MPEDA)Banned Antibiotics (MPEDA)
Herbal MedicinesHerbal Medicines
Storehouses of safer & cheaper compounds
Low cost, effective, no side effects & biodegradable
Various activities such as growth promotion, immunostimulants, anti stress, appetizing, aphrodisiac, antibacterial, antiviral and antifungal etc.
EcliptineEcliptine Tinosporone
Ocemine Aegilin
Percolation ExtractionPercolation ExtractionHerbal powder+ non polar solvent -Herbal powder+ non polar solvent -
Hexane (1:1) at 3 daysHexane (1:1) at 3 days
Filter & centrifugeFilter & centrifuge
Filtrate Filtrate SlurrySlurry
Slurry + Mid polar solvent Slurry + Mid polar solvent (1:1) at 3 days(1:1) at 3 days
Filter & centrifugeFilter & centrifuge
Filtrate Filtrate SlurrySlurry
Slurry + polar solvent (1:1) Slurry + polar solvent (1:1) at 3 daysat 3 days
Filtrate Filtrate
CondenseCondense
Rotary EvaporatorRotary Evaporator
LyophilizeLyophilize
ScreeningScreening
StorageStorage
Soxhlet ExtractionSoxhlet Extraction
Herbal powderHerbal powder
Particular solventParticular solvent
16 h extraction16 h extraction
LyophilizeLyophilize
ScreeningScreening
StorageStorage
condensecondense
Column PurificationColumn Purification
Herbal ExtractsHerbal Extracts
Loaded with silica column with Loaded with silica column with the mobile phase is hexanethe mobile phase is hexane
Elute with mixture of non polar + Elute with mixture of non polar + mid polar solvent 100 hexane; mid polar solvent 100 hexane; 80H + 20EA; 60H+40EA.…… 100 80H + 20EA; 60H+40EA.…… 100 % EA% EA
Elute with mixture of MP + P Elute with mixture of MP + P solvent 80EA+20 solvent 80EA+20 methanol………………100% methanol………………100% methanolmethanol
Collect elution & screeningCollect elution & screening
ELUTION STEPSELUTION STEPS
HEXANEHEXANE
100100
9090
8080
7070
6060
5050
4040
3030
2020
1010
00
ETHYL ACETATE ETHYL ACETATE
0 0
10 10
20 20
30 30
40 40
50 50
60 60
70 70
80 80
90 90
100 100
ETHYL ACETATE ETHYL ACETATE
100100
90 90
8080
7070
6060
50 50
40 40
30 30
20 20
10 10
00
METHANOL METHANOL
00
10 10
2020
3030
4040
50 50
60 60
70 70
80 80
90 90
100100
Non
Pol
arM
id P
olar
Mid
Pol
arM
id P
olar
Pola
rPo
lar
Thin Layer Chromatography (TLC)Thin Layer Chromatography (TLC)
Small amount of ExtractsSmall amount of Extracts
Spotted on TLCSpotted on TLC
Placed in a beaker having the Placed in a beaker having the mobile phase of 1:1 Hexane & mobile phase of 1:1 Hexane &
Ethyl acetate Ethyl acetate
Run by capillary actionRun by capillary action
Rf valueRf value
distance from origin to component spotdistance from origin to component spot
Rf value = ------------------------------------------- Rf value = -------------------------------------------
distance from origin to solvent frontdistance from origin to solvent front
Further purificationFurther purification Lengthy column Lengthy column
100-200 100-200 μm of silica particle sizeμm of silica particle size
Fine separationFine separation
Partial Characterization of Active compoundsPartial Characterization of Active compounds
UV SpectrometryUV Spectrometry
FT-IR spectroscopy FT-IR spectroscopy
HPLC-MSHPLC-MS
GC-MSGC-MS
LCMSLCMS
NMRNMR
Antibacterial ScreeningAntibacterial Screening
Piper longumPiper longumMurraya koeniji Murraya koeniji Quercus Quercus infectoriainfectoria Psoralea Psoralea
corylifoliacorylifolia
More than 50 herbals screened against More than 50 herbals screened against aquatic important pathogensaquatic important pathogens
In vitro In vitro antibacterial activityantibacterial activity
Some of them characterizedSome of them characterized
Beta Ocemine
Quercucin PsoralinPiperine
Mode of Herbal Medicines DeliveryMode of Herbal Medicines Delivery
Artificial Diet PreparationArtificial Diet PreparationIngredients
Diet composition (g.kg -1)C HAD1 HAD2 HAD3 HAD4
Brown fish meal 280 280 280 280 280Shrimp head meal 100 100 100 100 100Squid meal 20 20 20 20 20Squid liver powder 30 30 30 30 30Wheat gluten 60 60 60 60 60Wheat flour 200 200 200 200 200Soy bean meal 100 100 100 100 100Broken rice 102 102 102 102 102Fish oil 20 20 20 20 20Vitamin premix 7.8 7.8 7.8 7.8 7.8Mineral mix 40 40 40 40 40Mixtures of Herbal antibacterial extracts 0 0.2 0.4 0.6 0.8
The basal diet contained 45.1 % protein; 7.2 lipid; 14.6% ash; 7.1 % moisture and 3% fibre.
Bioenrichment or BioencapsulationBioenrichment or Bioencapsulation
Artemia naupli/ Pre adutArtemia naupli/ Pre adut
Gut EvacuationGut Evacuation
Enrichment 800mg/lit of herbal Enrichment 800mg/lit of herbal extracts emulsified with Egg extracts emulsified with Egg
YolkYolk
1 hr enrichment1 hr enrichment
Feeding to fishFeeding to fish
Herbal Antiviral
Phyllanthus niruri Leucus aspara
Ocimum basilicum
Effectively controlled WSSV at earlier stage of infection
Inhibit viral replication
Effective against other shrimp virus such as MBV and YHV
Psidium guajava
Phenol, 2,5- bis Phenol, 2,5- bis (1,1-dimethylethyl)(1,1-dimethylethyl)
MethodsMethods
5050µg extract+5 µl WSSV suspension (30µg µg extract+5 µl WSSV suspension (30µg protein)protein))- incubate 29)- incubate 290 0 C at 3 hr C at 3 hr
Centrifuged, filtered & LyophilizedCentrifuged, filtered & Lyophilized
Antiviral Herbal ExtractsAntiviral Herbal Extracts
Inject to ShrimpsInject to Shrimps
Monitor survival & external symptoms Monitor survival & external symptoms
PCR amplification PCR amplification
If positive ??If positive ?? If negative ??If negative ??
++
ImmunostimulantsAegle marmelosAegle marmelos
Picrorhiza kurooaPicrorhiza kurooa
Cynodon dactylonCynodon dactylon
Tinospora Tinospora cordifoliacordifolia
Eclipta albaEclipta alba
A substances that A substances that stimulate the immune stimulate the immune system by inducing system by inducing activation or increasing activation or increasing activity of any of its activity of any of its components. components. Improvement of…… • Hematology• Biochemical• Phagocytosis• Prophenol oxidase activity• Lysozyme activity• Bacterial & viral clearance
Types of Immunostimulants Types of Immunostimulants
Chitin, Chitoson & Oilgomers Chitin, Chitoson & Oilgomers Shell wasteShell waste
Fucoidon Fucoidon Sulfated polysaccharides –SeaweedsSulfated polysaccharides –Seaweeds
GlucansGlucansYeastYeast
LactoferinLactoferinMilkMilk
LipopolysaccharidesBacterial Cell wallBacterial Cell wall
HerbalsHerbalsW. somniferaW. somnifera, , A. calamos, T. cordifoliaA. calamos, T. cordifolia
Immunostimulant DrugsImmunostimulant DrugsImmunostimulant molecules recognize PRPs (PPAE) & culminate proteolytic cleavage of proPO to PO
Immunostimulant molecules interaction leading to PO activation
Melanin synthesis ROI, RNI Expression of immune genes& TLR TLR AMP & Phagocytosis
PPAE expression in haemocytes
TLR (DSCAM), MYD88, TRAF6
Immune genes (Caspase, crustin)
Enzyme assay- proPO, Intracellular Superoxide anion & Lysozyme
IMMIMM
SPCSPCPRPsPRPs
PPAEPPAE
ActivationActivation
PO ActivationPO ActivationTLR ActivationTLR Activation
AMP synthesis AMP synthesis & Phagocytosis & Phagocytosis
Immune gene ExpImmune gene Exp& ROI, RNI & ROI, RNI
““Quasi-Immune Response” Quasi-Immune Response” proved with herbal Immunostimulants proved with herbal Immunostimulants
Algal Drugs SeaweedsSeaweeds
Broad spectrum of activities
antiviral, anthelmintic, antifungal
and antibacterial
Brominated, aromatics, sterols, dibutanoids, proteins, peptides, sulphated polysaccharides, terpenoids, phlorotannins, acrylic acid, phenolic compounds and steroids.
CarrageenanCarrageenanPowerful antioxidantsPowerful antioxidants
AlginateAlginate polysaccharaidepolysaccharaide
Laminaria and FucusLaminaria and Fucus
Fucoidon Fucoidon
Powerful antiviral against WSSVPowerful antiviral against WSSV2-(2-hydroxyphenoxy)-1-2-(2-hydroxyphenoxy)-1-
phenylethanolphenylethanol
2-(2-hydroxyphenoxy)-1-phenylethanol
Micro AlgaeMicro Algae
Polyketides, amides, alkaloids, peptides and beta carotenoids
antiviral and antibacterial
Antioxidants
Euglena viridisEuglena viridisAgainst fish & Shellfish pathogensAgainst fish & Shellfish pathogens
Chlorella Chlorella AntibacterialAntibacterial
Dunaliella spDunaliella sp
From Halophilic origin From Halophilic origin
Control shellfish virusControl shellfish virus
Powerful antioxidants & Immunostimulants Powerful antioxidants & Immunostimulants
Marine & Halophilic secondary metabolites
Metabolite Production & CharacterizationMetabolite Production & Characterization
Bacterial culture @2000 RPM/25Bacterial culture @2000 RPM/2500C/ 7 C/ 7 DaysDays
Spin the Broth with 5000 RPMSpin the Broth with 5000 RPM
Remove the brothRemove the broth
3 times extraction with ethyl 3 times extraction with ethyl acetateacetate
Column purification Column purification
Filter and lyophilizeFilter and lyophilize
Structural elucidationStructural elucidation
1,10-Biphenyl-3-amine
Pharmacological Important BiosurfactantsPharmacological Important Biosurfactants
Extracellular Amphiphilic compoundsExtracellular Amphiphilic compounds
Glycolipids, lipopeptides, fatty acids, Glycolipids, lipopeptides, fatty acids,
polymeric surfactants & particulate polymeric surfactants & particulate
naturesnatures
Broad range of applications Broad range of applications
Antibacterial, antiviral, antifungal, anti-Antibacterial, antiviral, antifungal, anti-
adhesive, anti-cancer, antioxidants, adhesive, anti-cancer, antioxidants,
adjuvant etcadjuvant etc
9-OctadecenamideFatty acid
Mannosamine- Polymeric natureMannosamine- Polymeric nature
ProbioticsProbiotics
Probiotics have been defined as “live microbial food supplements which beneficially affect the host by improving the intestinal microflora balance”
Gut & Water ProbioticsGut & Water Probiotics
Competitive exclusion principleCompetitive exclusion principle
Used in Shrimp farms and polluted areasUsed in Shrimp farms and polluted areas
Improved strains of ProbioticsImproved strains of Probiotics
Lactobacillus and Bifidobacterium spLactobacillus and Bifidobacterium sp
Probiotic Diet making ProtocolProbiotic Diet making Protocol
Mixed substrates Mixed substrates [Rice bran-300 g;[Rice bran-300 g; Wheat bran-300 g; Rice flour-200g &Wheat bran-300 g; Rice flour-200g &
Wheat flour-200 g]Wheat flour-200 g]
600 ml bacterial culture broth600 ml bacterial culture broth
Make small balls & dried @ 40 ºCMake small balls & dried @ 40 ºC
Powdered Powdered
Bacterial countBacterial count
Vacuum packed Vacuum packed
Apply to pondApply to pond
SPECIFIC PATHOGEN FREE (SPF) SHRIMPSSPECIFIC PATHOGEN FREE (SPF) SHRIMPS
Specific Pathogen Free (SPF)Specific Pathogen Free (SPF)Development of SPF doubling the production in Development of SPF doubling the production in Litopenaeus Litopenaeus vannamei vannamei in US aqua industry during 1990sin US aqua industry during 1990s
They cross bread between two traitsThey cross bread between two traits
CPF and Oceana Institute develop the SPF by cross bread the CPF and Oceana Institute develop the SPF by cross bread the better characters such as better characters such as salinity, low DO, pH tolerance & disease salinity, low DO, pH tolerance & disease tolerence traits tolerence traits and analyze the characters at 30 generation and and analyze the characters at 30 generation and commercializationcommercialization
EDIBLE ANTIBODY OR EDIBLE VACCINESEDIBLE ANTIBODY OR EDIBLE VACCINES
Production of Yolk antibodyProduction of Yolk antibody
Principle
When chickens are faced with a foreign virus or bacterium, they produce antibodies to fight the invader. They pass that immunity on to their offspring, and antibodies wind up in the eggs.
1. Chickens are more apt than mammals to make high-avidity antibodies
2. A single chicken can produce an enormous amount of antibody, upto 3 grams of IgY per month, which is 10-20 times the amount of a rabbit
3. chickens produce antibody much quicker-high-titre antibody is available from eggs as early as day 25
4. Storing of eggs (antibodies) is very easy
5. It is cheaper to feed and house chickens than rabbits
6. Effective against drug-resistant bacteria.
IgY IgY production & production & purificationpurification
YolkYolk
DDHDDH22O WashO Wash
Cut Open Cut Open
Isoproponanl wash 1: 3 – 2 times Isoproponanl wash 1: 3 – 2 times
Acetone wash- 1 timeAcetone wash- 1 time
Fitered & storeFitered & store
Complete removal of Complete removal of LipidsLipids
Anti WSSV IgY (37 k Da)
M C IgY IgY- Adj
VACCINE DEVELOPMENTVACCINE DEVELOPMENT
Vaccine Generations Vaccine Generations
First Generation of vaccineLive, attenuated and killed forms
killerCellular immune responses
Second Generation of vaccineSubunit vaccines T Helper cells immunity
Third Generation of vaccineDNA vaccinesHumoral & cellular immunityStrong & long lasting
Vaccines in AquacultureVaccines in Aquaculture
Whole Cell VaccineWhole Cell Vaccine
Bacterial CellsBacterial Cells
Viral ParticlesViral Particles
Inactivated formsInactivated forms
Virus- Grown in cell lines/ eggsVirus- Grown in cell lines/ eggs
Demerit- revert Demerit- revert
ECP VaccineECP Vaccine
ECP – haemolysin, virulent factorsECP – haemolysin, virulent factors
Need adjuvantNeed adjuvant
Improved survivalImproved survival
Partial purified forms Partial purified forms 52 kDa52 kDa
Subunit VaccineSubunit Vaccine
Proteins Proteins
Envelop / OMPs (37kDa) Envelop / OMPs (37kDa)
Strong ImmunityStrong Immunity
Need adjuvantNeed adjuvant
Hematological & immunological improvementHematological & immunological improvement
Bio Film Vaccine Bio Film Vaccine
Matrix of Protein, carbohydrate & DNA Matrix of Protein, carbohydrate & DNA
90 % Bacteria secretes 90 % Bacteria secretes
Difficult to treat by antimicrobialsDifficult to treat by antimicrobials
Modulate cytokine synthesis and interrupt production of antibodies
DNA VACCINEDNA VACCINE
DNA VaccineDNA Vaccine
Immunization with a circular piece of DNA that code for an antigen
Plasmids consist of strong viral promoter to drive the in vivo transcription and translation
When injected into an individual, the plasmid is taken up by the host cells and its genetic information is translated into the immunizing protein.
Multicistronic vectors are sometimes constructed to express more than one immunogen, or to express an immunogen and an immunostimulatory protein.
Plasmids consist of strong viral promoter (SV-40/CMV ) to drive the in vivo transcription and translation of gene interest.
Intron A may sometimes be included to improve mRNA stability and hence increase protein expression.
Plasmids also include a strong polyadenylation/transcriptional termination signal, such as bovine growth hormone or rabbit beta-globulin polyadenylation sequences.
Multicistronic vectors are sometimes constructed to express more than one immunogen, or to express an immunogen and an immunostimulatory protein.
Vector DesignVector Design
Recombinant plasmids enter to the host cell
Gene of interest is transcribed by the RNA polymerase II & synthesis of messenger RNA (mRNA)
Translated into the corresponding protein in the cytoplasm of the host cells
T- Cell as well as B-Cell immunity will developed & produced antibody against the proteins
When pathogen (antigen) enters to the host cell, the T-Cells as well as B cells recognize and killed.
How DNA vaccine plasmids Stimulate immune responses?
Immune system Aquatic species
Shell Fishes
Innate immune system
No clear responses
No antibody production
Pathogen persists
Infectious to others
Humoral immunity AMP, PO & Coagulation
Cellular immunity Encap, nodule & pago..
Adaptive immune system
Specific responses
Antibody produced
Pathogen eliminated
May or may not
Humoral immunity B cells
Cellular immunity T cells
Fin Fishes
DNA VACCINE DEVELOPMENT PROTOCOL DNA Vaccine ConstructDNA Vaccine Construct
PCR amplification of desired gene
Design & amplify the gene by specific primers
5’-GGATCCAATCATGGATCTTTCTTTCAC-3’-BamHI
5’- GAATTCTTACTCGGTCTCAGTGCC-3’- EcoRI Cloning to suitable vectors
Purify the PCR products Clone to “T” Easy vectors Ligation @ 4 or16° C for 16 hrs/ Over Night Transformation to DH5 α Sequencing by using M13+ and M13- primers
DNA Vaccine Construct by cloning
Plasmid extraction from positive clonesRestriction of REC plasmid by respective enzymesLigate to suitable DNA vaccine vector such as DNAVAX or pCDNA 3.1 @ ratio of 1: 3
Transformation
Recombinant plasmids & comp. cells will be mix and keep on ice 30 mts
The mixture will be put in water bath at 42°C for 90 seconds
Snap chill the and add 400 µl LB broths
10 µl of this culture will be plated on LB agar in presence of antibiotics.
Plasmid extraction
Plasmid will be purified from the positive clones using standard methods
Steps 1. Alkaline lysis2. Phenol chloroform Extraction3. Ethanol precipitation
Plasmid will be suspended to PBS or sterile saline and store to -20° C
Diluted to sterile saline for delivery
Vaccine Delivery
Small volume of DNA vaccines is enough 0.1 or 0.2 ml volume is enough 10 µg DNA is enough (5+ 5= 2 doses or 10= 1 dose) Disadvantage: Cant’ apply for small fishes
Methods of DeliveryGenerally injectionLarvae- injection impossibleAlternative methods are
1. The DNA vaccine transformants will be coated to artificial fed using fish oil and feed them.
2. Bioencapsulation may be a preferred method of oral delivery (rotifers, brine shrimp)
3. DNA vaccine transformant (Bacterial mode)4. Conjugate with Chitoson / sodium alginate
complex
Tissue Distribution studies
RNA extracted from different tissues
2µg of total RNA will be primed with 1µg oligodT(18) and reverse-transcribed into cDNA with 200U MMLV reverse transcriptase
PCR amplification with specific gene primers & product confirmation
q-RT PCR for gene copy
RNA INTERFERENCE (RNAi)RNA INTERFERENCE (RNAi)
RNA Interference (RNAi) against Viral diseaseRNA Interference (RNAi) against Viral disease
““A process in which the introduction of double-stranded A process in which the introduction of double-stranded RNA into a cell RNA into a cell inhibits the expressioninhibits the expression of genes” of genes”
Post Translational Gene Silencing Post Translational Gene Silencing (PTGS) Pathway(PTGS) Pathway
ds RNA in the cytoplasm triggers the ds RNA in the cytoplasm triggers the multi domain multi domain ribonuclease II enzyme ribonuclease II enzyme DICER which cleaves the ds RNA in to si DICER which cleaves the ds RNA in to si RNA which are RNA which are 21 to 23 21 to 23 nucleotide nucleotide fragments.fragments.
These si RNA is recognized by the These si RNA is recognized by the RNA RNA induced Silencing complex (RISC)induced Silencing complex (RISC), a , a multi enzyme unit that brings about multi enzyme unit that brings about separation of two si RNA stands.separation of two si RNA stands.
The antisense Si RNA stand remains The antisense Si RNA stand remains bound to RISC while the sense strand is bound to RISC while the sense strand is released. Finally the antisense and released. Finally the antisense and RISC complex bind the target mRNA RISC complex bind the target mRNA allowing the nuclease activity and allowing the nuclease activity and degraded the target gene. degraded the target gene.
Crustaceans such as penaeid shrimp, which can be infected by more than twenty different viruses.
Bunyaviridae, Herpesviridae, Picornaviridae, Parvoviridae, Reoviridae, Rhabdoviridae, Togaviridae, Iridoviridae or a new virus family, the Nimaviridae
Culture practices leading to stress induced diseases, bacterial and viral infections leading to severe economic losses
White spot syndrome virus (WSSV), Yellow head virus (YHV), and Taura syndrome virus (TSV)
Role of RNA Interference in aquatic disease control Role of RNA Interference in aquatic disease control
CONCLUSIONS & FUTURE DIRECTIONS CONCLUSIONS & FUTURE DIRECTIONS
Improved health and wealthImproved health and wealth
Environmental spoilage can be avoidedEnvironmental spoilage can be avoided
Preventing emergence of resistant Preventing emergence of resistant
strainsstrains
Biomagnifications can be controlledBiomagnifications can be controlled
I am I am HealthyHealthy
Consumption of antibiotic treated Consumption of antibiotic treated
shrimp/ fish in humans can be prevented shrimp/ fish in humans can be prevented
Consignment rejection can be controlledConsignment rejection can be controlled
The product will be of great benefit for The product will be of great benefit for
the farmersthe farmers
Improved economy to the farmers Improved economy to the farmers
Affected farms due to indiscriminate Affected farms due to indiscriminate
usage of antibiotics can be reutilized.usage of antibiotics can be reutilized.