BIOTECHNOLOGICAL APPROACHES IN AQUATIC ANIMAL HEALTH MANAGEMENT FOR IMPROVED AQUACULTURE PRODUCTION

1. Dr. T. Citarasu
Associate Professor
Tele-fax: + 91-4652-253078
Mobile: +91-9994273822
Email: citarasu@gmail.com
citarasu@msuniv.ac.in
Website: www.msuniv.ac.in
MANONMANIAM SUNDARANAR UNIVERSITY, TIRUNELVELI

2. BIOTECHNOLOGICAL APPROACHES IN
AQUATIC ANIMAL HEALTH MANAGEMENT
FOR IMPROVED PRODUCTION

3. CURRENT STATUS OF FISHERIES

4. CAPTURE FISHERIES
Fisheries play an important role in the economic development of our
country
It is recognized as a powerful source of income and employment creation
Also seen as a good foreign exchange earner
Million tones

5. PROBLEMS IN CAPTURE FISHERIES
Decline in catch rate due to overfishing and overdependence on
trawlers
Harmful methods and Negligent fishing – Dolphin, Turtle etc
Destructive fishing: Cyanide fishing; Dynamite fishing; long line
fishing; Ghost fishing; Purse- seine fishing and shark fining
Ecosystem destruction
Fish reduction
Rupture in food web

6. CULTURE FISHERIES (AQUACULTURE)
Lucrative food production industry
Fresh, brackish & Marine based culture
Increasing annual growth rate
Global sea food demand-70% in the next 33 years (7 fold)
Attractive Job opportunities

7. SHRIMP FARMING
Asia is the largest producer- 80 %
India is the second place of Asia &
fifth place in globally
>350 shrimp hatcheries 13 billion seed
production/ annum
Top 5 Countries L. Met. tones
China 7.5
India 7.0
Vietnam 5.5
Thailand 4.5
Indonesia 1.5
Aquaculture production (2018)

8. CULTURE TYPES
Extensive
Semi- Intensive
Intensive
Super- Intensive
 No external diet
 Low stocking densities (0-5 m3)
 Yield: 500 kg/Ha/yr
 External diet
 Intermediate stocking (10-30 m3)
 Yield: 1-2 tonnes/Ha/yr
 Formulated complete feeds
 High density stocking (30-60 m3)
 Yield: above 2 tonnes/Ha/yr
 Closed system with raceway + aeration
 Very high density stocking (>60 m3)
 Yield: above 60 tonnes/Ha/crop

9. 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, MrNV
Stress Induced diseases

10. Important Shrimp Diseases in India
Vibriosis White Patch Disease
White Spot Syndrome Virus (WSSV) Enterocytozoon hepatopenaei (EHP)

11. Finfish Diseases
Freshwater Fishes
Motile Aeromonad septicemia
(MAS)
Hemorrhagic septicemia
A. hydrophila, A. sobria
A. caviae, A. veronii,
A. salmonicida
Marine Fishes
Vibriosis
Pathogenic Vibrios
Photobacterium sp
Micrococcus sp
Flavobacterium- Gill disease
Enterobacter- Edwardsiella
Pseduomonas
Salmonella

12. CURRENT PROBLEMS IN DISEASE CONTROL
Current disease treatment
protocols – difficult
Chemicals & Synthetic drugs
Antibiotics & other chemicals
MPEDA abandoned more than
20 antibiotics

13. Chloramphenicol Metronidazole
Nitrofurans Ronidazole
Neomycin Ipronidazole
Nalidixic acid nitroimidazoles
Sulphamethoxazole Clenbuterol
Chlorpromazine Diethylstilbestrol
Colchicine Sulonamide
Dapsone Floroquinolones
Dimetridazole Glycopeptides
Banned Antibiotics (MPEDA)

15. BIOTECHNOLOGY TOOLS IN AQUATIC HEALTH

16. Tools can certainly revolutionize aquaculture
Genetic modification holds tremendous potential to improve the quality
and quantity of fish reared in aquaculture
Reduce the importation of foreign fish and aquaculture products and
increased foreign earnings
Tools derived from genomics can also improve our under-standing of how
aquatic organisms adapt and respond to their environments
Tools to enhanced production : Diagnostic tools; in silico approach;
Vaccine development; transgenic fish; Edible vaccine; Nanotechnology;
RNAi ; Gene editing and Phage therapy etc

17. DIAGNOSTIC TOOLS

18. POLYMERASE CHAIN REACTION (PCR)
PCR is a technique used to make thousands of copies of a DNA used in
diagnosis including paternity testing, mutation detection for disease and
cloning genes for research
Steps including Denaturation, Annealing and extension
DNTPs, Buffer, MgCl2, Taq DNA polymerase, primers & DNA template,
In aquaculture, PCR is highly useful to detect the bacterial, viral infection at
very earlier stage of infection

19. TYPE OF PCR USED IN AQUATIC DISEASE DIAGNOSIS
Nested PCR
Modification of PCR that was designed to
improve sensitivity and specificity
Two sets of primers used, first set of
primers used to amplify a target
sequence and the second one used to
amplify a region within the first target
sequence
For the impossible templates where the
GC content might be high or chance of
non-specific banding is higher, nested
PCR offers the best results. It is also
useful in the amplification of genes with
the low abundance.
Highly useful to detect WSSV, EHP etc

20. Multiplex PCR
Simultaneous detection of multiple
targets in a single reaction well,
with a different pair of primers for
each target. This technique requires
two or more probes that can be
distinguished from each other and
detected simultaneously.
This is achieved by designing
primers, which can amplify different
regions of the same template DNA
(particular pathogen) or primers
that can be amplify two entirely
different DNA templates (two
distinct virus)

21. Semi quantitative PCR
Reverse transcriptase to make DNA
from the RNA in the sample, and there
for qualitatively detect gene expression
by use of the complimentary DNA.
If the target sequence to be detected is
RNA, the conventional PCR step would
precede a reverse transcription (RT)
step by which RNA is enzymatically
converted to complementary DNA
(cDNA).
An oligo deoxynucleotide primer
hybridizes to mRNA and is extended by
an RNA dependent DNA polymerase
The newly synthesized single standard
cDNA can be amplified using specific
primers in a conventional PCR.

22. Quantitative PCR (qRT PCR)
PCR-based technique that quantify gene expression
Amplification produces increasing amounts of double-stranded DNA, which binds
SYBR green or TaqMan probe resulting in an increase in fluorescence.
Quantification of the amount of target in unknown samples is accomplished by
measuring CT and using the standard curve to determine starting copy number.
In the diagnostic format the greatest advantage is no post-PCR manipulation involved
in the visualization of the result, cross- contamination and false positive result can be
minimized.
Increased sensitivity, reproducibility and quantitative accuracy, a part from the
decreased hands-on time and less chances of contamination is a very important tool in
aquatic health management

23. Loop mediated isothermal amplification (LAMP)
Nucleic acid amplification method that amplifies DNA with high specificity (109-
1010 times in 15-60 mins), efficiency and rapidity under isothermal conditions
Simple, rapid, specific and cost-effective nucleic acid amplification
DNA polymerase (Bst polymerase) and a set of four specially designed primers to
recognize six distinct regions of the target DNA
Unlike PCR, LAMP is carried out in constant temperature (60–65°C) using an auto-
cycling strand displacement DNA synthesis and does not require thermal cycler.
The amplified product can be detected as white precipitate or yellow green color
solution after addition of SYBR Green.

24. IMMUNODIAGNOSIS TECHNIQUES
Polyclonal Antibodies (pAbs)
Polyclonal antibodies (pAbs) are mixture of heterogeneous which are
usually produced by different B cell clones in the body.
They can recognize and bind to many different epitopes of a single antigen.
Substances foreign to the host (bacteria or virus) known as antigens are
recognized by the host immune system as invaders.
Used to develop different kits for pathogen detection

25. Monoclonal Antibodies (mAbs)
Introducing an antigen to a mouse and then fusing polyclonal B cells from the
mouse's spleen to myeloma cells
Hybridoma cells are cultured and continue to produce antibodies to the
antigen
Hybridoma technology allows us to produce large amounts of pure antibodies
Used to develop different kits for pathogen detection we can obtain cells that
produce antibodies naturally & can grow continually in cell culture for mAbs
production
High specificity

26. APPLICATIONS OF IMMUNO DIAGNOSIS
Western Blot Fluorescent Abs
ELISA
Immunoperoxidase
Lateral Flow Dot Blot

27. DNA BASED BLOTTING TECHNIQUES
Southern Blot
Detection of a specific DNA sequence in
DNA samples
Combines transfer of electrophoresis-
separated DNA fragments to a filter
membrane and subsequent fragment
detection by probe hybridization
Northern Blot
Detect specific RNA molecules among a
mixture of RNA
Can be used to analyze a sample of RNA
from a particular tissue or cell type in
order to measure the RNA expression of
particular genes

28. HYBRIDIZATION TECHNIQUES
In Situ Hybridization (ISH)
Labeled complementary DNA, RNA or modified nucleic
acids strand to localize a specific DNA or RNA
sequence in a portion or section of tissue or if the
tissue
RNA ISH is used to measure and localize RNAs
(mRNAs, lncRNAs, and miRNAs) within tissue
sections, cells, whole mounts, and circulating tumor
cells
Fluorescence In Situ Hybridization
Molecular cytogenetic technique that
uses fluorescent probes that bind to only those parts of
a nucleic acid sequence
Fluorescence microscopy can be used to find out the
signal
Detect bacterial and viral DNA in an infected cell (~ 300
bp)

29. METAGENOMICS
Modern genomics techniques to the study of
communities of microbial organisms directly in
their natural environments
Sampling and nucleic acids extraction, Library
construction and Analysis of metagenomics
libraries.
Nodavirus (Farfantepenaeus duorarum
nodavirus, FdNV) and a new DNA virus
possessing a circular genome designated
shrimp hepatopancreas-associated circular DNA
virus (Shrimp CDV)
Identify new latent pathogens in asymptomatic
carriers, uncharacterized pathogens causing a
new disease or multiple pathogens associated
with disease syndromes in shrimp farms

30. DNA MICROARRAY
Expression rate of thousands of genes and
identify wide range of pathogens from complex
samples in one single reaction
Involves hybridization of DNA with large
number of probes and can overcome the
shortcomings of multiplex PCR, which can
detect only a maximum of six pathogens at a
time
Fluorescent labeled DNA sequences that are
hybridized to the microarray slide help to
identify the pathogens
Fluorescent microarray detector and computer
program will analyze the fluorescent array for
the presence or absence of the species/strain
specific DNA sequence

31. BIOTECHNOLOGICAL TOOLS TO IMPROVE PRODUCTION

32. Immunostimulants
A substances that stimulate the immune system by inducing activation
or increasing activity of any of its components.
Improve immunological & haemotological parameters
Secretion of antioxidant enzymes & AMP
Resist or eliminate pathogens
CHITIN
LACTOFERIN
FUCOIDON
BETA GLUCAN
LPS
HERBALS

33. Herbal Immunostimulants
Phyllanthus niruri Leucus aspara Ocimum basilicum
Boost immune system
Suppress the multiplication of WSSV
Activate PO system and secretion of antioxidant enzymes
Psidium guajava
Phenol, 2,5- bis
(1,1-dimethylethyl)

34. Agathi grandiflora, Aegle marmelos, Cyperus rotundus, Cynodon dactlylon,
Eclipta alba, Picrorhiza kurroa, Tinospora cordifolia, Withania somnifera and
Zingiber officinalis

35. CRUSTACEAN IMMUNE SYSTEM
Three main types of haemocytes involve in immunity
Fixed phagocytes in the hepatopancreas and nephrocytes in the gills also
augment the activities of the hemocytes
Cellular defense reactions are phagocytosis, nodule formation and encapsulation
Both are responsible for invading microbes and macroparasites.
Humoral factors including AMP and proteins such as lysozyme, lectins, and the
products of the prophenoloxidase-activating system.

36. Identification of important genes involved in
humoral immune pathways
Pattern recognition receptors (PRRs)
PRRs are proteins expressed by cells of the
innate immune system to identify pathogen-
associated molecular patterns (PAMPs), which
are associated with microbial pathogens or
cellular stress
PRRs play very important roles in innate
immunity. They are lectin, LGBP and tetraspanin
which are key factors to recognizing different
pathogens and triggering a series of immune
reactions
Toll-like receptors (TLRs) are a class of proteins
that play a key role in the innate immune system.
They are single, membrane-spanning, non-
catalytic receptors that recognize structurally
conserved molecules derived from microbes.
Once these microbes have breached physical
barriers such as the skin or intestinal tract
mucosa, they are recognized by TLRs and
immune cell responses are activated.

37. Antimicrobial peptides(AMP)
AMP (host defense peptides) are an
evolutionarily conserved component of the
innate immune response
AMP are penaeidin, anti-lipopolysaccharride
factor (ALF), crustin and lysozyme were found in
shrimp
Play crucial roles in a shrimp’s immunity to
bacterial infection.
Antioxidant enzymes
They are neutralizing free radicals
During pathogen encounters NADPHoxidase will
activate, in turn enhancing the glycolytic
reactions that will increase the consumption of
oxygen and induce the production of a mass of
reactive oxygen species (ROS) such as
superoxide anion (O2-), hydrogen peroxide (H2O2)
and hydroxyl radical (OH-)
In F. chinensis, oxidant enzymes including
mMnSOD, peroxiredoxin and catalase that are
closely related to the immune response of shrimp
to pathogens

38. In silico Drug designing
COMPUTATIONAL DRUG SCREENING
Antiviral, immunostimulant, anti
apoptotic inhibitors and PO negative
regulator inhibitor compounds
-Terrestrial
- Marine origin – NRPS/PKS
- Solar salt works origin – NRPS/PKS
Ligand databases like PubChem
Compounds, Drug Bank, Zinc
Database etc.
3D structure of WSSV download from
PDB database or homology modeling
or threading method with I-TASSER
Active site prediction by PDBSUM
database

39. MOLECULAR DOCKING –Antiviral
Computational simulation of a candidate ligand binding to a
receptor and form a stable complex
Predict the suitable ligand compounds which bind the WSSV
proteins and form a complex.
The complex forming will help to arrest the multiplication of
WSSV

40. MOLECULAR DYNAMICS (MD) SIMULATION
In order to the study the activity or stability of the ligand and
target protein at in vivo level MD simulation or water dynamic
analysis will be useful for the hydrogen band interaction of
particular amino acid from protein and the drug.
Physical movement of atom and molecules
Interact with water molecules in body system for more stability

41. MODE OF ACTION (Immunostimulation)
Immunostimulant 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
Possibility to activate the PRPs in SPC to activate proPO system and
immunity developed against pathogens
IMM
SPC
PRPs
PPAE
Activation
PO ActivationTLR Activation
AMP synthesis
& Phagocytosis
Immune gene Exp
& ROI, RNI

42. Inhibition of anti apoptotic protein
WSSV hijack protein AAP1 (Anti
Apoptotic Protein)
Designing drugs for inhibiting the
expression of AAP1
Help to up regulation of Caspase
expression & leading to
immunostimulation - q & semi qPCR
Inhibition of PO negative regulation
Proteinase inhibitors as negative
regulation for PO affect the
stimulation - SERPIN
By inhibit/ down regulate SERPIN PO
doesn't affect leading to good
immunostimulation
Expression of SERPIN by qRT PCR
SPC
PPAETLR
SERPIN
Ligand
PO positive

43. RECOMBINANT VACCINE DEVELOPMENT

44. Vaccine Generations
First Generation of vaccine
Live, attenuated and killed forms
killer
Cellular immune responses
Second Generation of vaccine
Subunit vaccines
T Helper cells immunity
Third Generation of vaccine
DNA vaccines
Humoral & cellular immunity
Strong & long lasting

45. Vaccines in Aquaculture
Whole Cell Vaccine
Bacterial Cells
Viral Particles
Inactivated forms
Virus- Grown in cell lines/ eggs
Demerit- revert
ECP Vaccine
ECP – haemolysin, virulent factors
Need adjuvant
Improved survival
Partial purified forms
52 kDa

46. SUBUNIT VACCINE
Proteins
Envelop / OMPs (37kDa)
Strong Immunity
Need adjuvant
Hematological & immunological improvement
BIO FILM VACCINE
Matrix of Protein, carbohydrate & DNA
90 % Bacteria secretes
Difficult to treat by antimicrobials
Modulate cytokine synthesis and
interrupt production of antibodies

48. Demerits of Conventional Vaccines
Gives positive effects and have some demerits
such as week and shorter immunity, reversion of
virulence, high cost, some times ineffective, heat
liable and need of high cost adjuvant etc.
Recombinant DNA vaccine technology is an
attractive alternative to traditional vaccines
because of certain advantages, which includes
straightforward design and construction, heat
stability, low production costs, long-term storage
capabilities and no risk of reversion
This approach can elicit very strong and long-
lasting immune responses, also offers economic
benefit, environmental and safety advantages,
which are particularly attractive for the farmers.

49. RECOMBINANT VACCINES

50. Immunization with a circular piece of
DNA that code for an antigen
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
DNA VACCINE

51. 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/ keep the mammary
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?

53. 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

54. Fast production of proteins with large quantities
Short generation times, as bacteria grow and multiply rapidly
The expressed proteins often do not fold properly and so are biologically
inactive
The synthesized proteins are often toxic to bacteria
Lack of enzymes responsible for post-translational modifications
Subunit vaccines through Bacterial Expression

55. Subunit vaccines through Baculovirus Expression
Viral recombinant proteins from baculovirus infected cells - Fast production
of proteins with large quantities
Advantages including improved solubility, ability to incorporate post-
translational modifications, and higher yields for secreted proteins
90 % efficiency with 500 mg of protein per liter of culture
Proper protein folding & biologically active proteins
Eukaryotic posttranslational modification

57. Subunit Vaccines through Yeast Expression System
The galactose induction system in Saccharomyces cerevisiae
GAL1 promoter used to conditionally over express genes
Advantages: growth speed, easy genetic manipulation, low cost media,
post translational modifications & secretory expression
Saccharomyces, Pichia, Kluyveromyces, Hansenula and Yarrowia.

58. Subunit Vaccine –WSSV VP28-pESC-URA
Benefits
Recombinant subunit vaccine developed against White Spot Syndrome
Virus (WSSV) using the immunogenic gene through yeast expression
vector pESC-URA
VP28-pESC-URA subunit vaccine administrated pacific white leg shrimp
Litopenaeus vannamei had survived more than 80 % against WSSV
infection
The vaccine helps also helped to improve the immune system by
expression of more immune genes and Toll Like Receptors (TRL) and
reduction of viral load after WSSV challenge

59. Micro algal Recombinant vaccines
Antigens expressed in the chloroplast or anchored to the surface of plasma
membrane
Safe and inexpensive to immunize fishes.
Foreign antigens can be expressed in the chloroplast or the cytoplasm with
high yields
Algae are a potential food source for larval fish (10 µm)
Chlamydomonas is innocuous, nontoxic and nonpathogenic.

60. EDIBLE ANTIBODY OR EDIBLE VACCINES

61. PRODUCTION 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.

62. IgY production & purification
Yolk
DDH2O Wash
Cut Open
Isoproponanl wash 1: 3 – 2 times
Acetone wash- 1 time
Fitered & store
Complete removal of
Lipids

63. Anti WSSV IgY (37 k Da)
M C IgY IgY- Adj

65. Vaccine Delivery Methods
Injection
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
Bacterial Transformants
DNA vaccine transformant (Bacterial
mode)

66. Through Diets
DNA and subunit vaccine transformants /
proteins will be coated to artificial fed
Bioencapsulation
Bioencapsulation may be a preferred method
of oral delivery (rotifers, brine shrimp)
Nano delivery
Conjugate with Chitoson / sodium alginate
complex
Subunit vaccines with nanoparticles,
Poly(lactic-co-glycolic acid) (PLGA)
Micro algal delivery
Recombibnant vaccine producing micro
algae

67. TRANSGENIC DEVELOPMENT

68. TRANSGENIC FISH
Transgenesis:
Foreign DNA is introduced into the animal, using rDNA technology, then
transmitted through the germ line so that, the animal gets the same
modified genetic material
Organisms into which heterologous DNA (transgene) has been artificially
introduced and integrated in their genomes are called transgenics
A transgenic animal is one whose genome has been changed to carry genes
from other species.
The transgenic fish may change their change their characters

69. Need for Transgenic fish
Growth enhancement
Adaptation for environments
Increase disease resistance
Sexual maturation
Enhance nutritional quality
Improve food utilization
Transgenic manipulation of antimicrobial
peptide genes may lead to the production
of fish strains with elevated resistance to
bacterial, viral and protozoan pathogens

70. TRASGENIC TELEOSTS FOR EPA AND DHA PRODUCTION

72. RNA INTERFERNECE (RNAi)

73. RNA Interference (RNAi) against Viral disease
“A process in which the introduction of double-stranded
RNA into a cell inhibits the expression of genes”

74. Experimentally, this technology has been proved to be very useful for the
control of several pathogens including WSSV
It has been particularly very effective for the control of many viral diseases
where treatments through medicines are not possible
Specific virulence genes of the pathogens are targeted to develop short RNA
fragments This brings degradation of the pathogen through post translational
modification
This method is particularly look promising for the treatment of viral diseases
of shrimp where neither any treatment methods nor any vaccines are
available

75. Post Translational Gene Silencing
(PTGS) Pathway
ds RNA in the cytoplasm triggers the multi
domain ribonuclease II enzyme DICER which
cleaves the ds RNA in to si RNA which are 21
to 23 nucleotide fragments
These si RNA is recognized by the RNA
induced Silencing complex (RISC), a multi
enzyme unit that brings about separation of
two si RNA stands.
The antisense Si RNA stand remains bound to
RISC while the sense strand is released.
Finally the antisense and RISC complex bind
the target mRNA allowing the nuclease activity
and degraded the target gene

76. ROLE OF RNA INTERFERENCE IN AQUATIC DISEASE CONTROL
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)

77. PHAGE THERAPY

78. Phage Therapy is the therapeutic use
of lytic bacteriophages to treat
pathogenic bacterial infections.
Bacteriophages, the viruses that
infect and kill their specific hosts,
have been reported to offer scope as
an alternative to antibiotics as
therapeutic agents in controlling
bacterial infections
The purpose of phage and antibiotic
therapy are same in controlling
bacterial infections but antibiotics
are banned in many countries due to
multidrug resistant strain
development.

79. Phage activity is very specific, attacking
only host bacterial cells without affecting
other (normal) micro flora
The capability of phages to not only target
and destroy a specific bacterium, but also
replicate exponentially, underscores their
potential role in treating infectious
diseases
Phages also have several advantages over
antibiotics: they are ecologically safe
(i.e.harmless to humans, plants and
animals), and phage preparations are
readily producible, and easy to apply.
In aquaculture phages effectively
controlled Aeromonas salmonicida and
Vibrio harveyi

80. Advantages in Phage therapy
Multiply at exponential growth rates but antibiotics suppress the host immune
system during their therapeutic use
Phages infect a relatively limited range of bacterial hosts but antibiotic have the
broad spectrum in controlling bacterial populations
Ability to self-mutate themselves against phage- resistant mutants.
However, a single phage may be insufficient to control an infection. Cocktail
phages can enhance their effects, a viable alternative to control opportunistic
or antibiotic-resistant bacteria
Best alternatives against vaccines

82. Gene editing (CRISPR-Cas9)

83. CRISPR gene editing is a method by which the
genomes of living organisms may be edited.
It is based on a simplified version of the
bacterial CRISPR/Cas (CRISPR-Cas9) antiviral
defense system. By delivering the Cas9
nuclease complexed with a synthetic guide
RNA (gRNA) into a cell, the cell's genome can
be cut at a desired location, allowing existing
genes to be removed and/or new ones added
Genome editing can rapidly introduce favorable
changes to the genome, such as fixing alleles
at existing trait loci, creating de novo alleles, or
introducing alleles from other strains or
species
Infectious diseases are one of the primary
threats to sustainable aquaculture, with an
estimated 40% of the total potential production
lost per annum
CRISPR-Cas technology can also be used to
control the viral and bacterial diseases
particularly in shrimps and prawns

85. Applications in aquaculture
Combining in vivo and in vitro screening
approaches has the potential to identify functional
disease resistance alleles for disease resistance
Salmonidae, Cyprinidae, Siluridae , Pacific oyster,
Nile tilapia, and gilthead sea bream etc
Immunity and disease resistance have already been
investigated using genome editing in Rohu carp
and Grass carp
Can also be applied to develop models for studying
fundamental immunology, such as the targeted
disruption of the TLR22 gene in carp
improved cell lines for fish species, by enabling
more efficient production of viruses for future
vaccine development by knocking out key
components of the interferon pathway

86. CONCLUSIONS & FUTURE DIRECTIONS

87. Improved health and wealth
Environmental spoilage can be avoided
Preventing emergence of resistant
strains
Biomagnifications can be controlled
I am Healthy

88. Consumption of antibiotic treated
shrimp/ fish in humans can be prevented
Consignment rejection can be controlled
The product will be of great benefit for
the farmers
Improved economy to the farmers
Affected farms due to indiscriminate
usage of antibiotics can be reutilized.

89. THANK YOU