Disease diagnosis Alternative approaches

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
www.msuniv.irins.org/profile/174278
CENTRE FOR MARINE SCIENCE AND TECHNOLOGY
MANONMANIAM SUNDARANAR UNIVERSITY, TIRUNELVELI

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

3. 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

4. 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
Aquaculture production (2022)
Top 5 Countries L. Met. tones
Ecuador 13.0
India 8.0
China 8.0
Vietnam 6.80
Indonesia 4.0

5. 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

7. Important Shrimp Diseases in India
Vibriosis Early Mortality Syndrome
White Spot Syndrome Virus (WSSV) Enterocytozoon hepatopenaei (EHP)

8. 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

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

11. BIOTECHNOLOGY TOOLS IN AQUATIC HEALTH

12. DIAGNOSTIC TOOLS

13. 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

14. 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

15. 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)

16. 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.

17. 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

18. 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.

19. Detection and quantification of RNA molecules for infectious diseases
Amplify RNA sequences in a highly specific and sensitive manner with the
isothermal conditions at 41°C.
This assay relies on the activity of three enzymes, namely, T7 RNA polymerase,
RNase H, and AMV reverse transcriptase
The steps involved primer annealing and cDNA synthesis, RNA transcription,
hybridization and stand displacement, continuous amplification and finally
detection and quantification.
Detection of RNA viruses, retrovirus & Vibrio cholerae, Salmonella sp.,
Escherichia coli etc
Nucleic acid sequence based amplification (NASBA)

20. 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

21. 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

22. 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

24. Genomics
NGS: transcriptional profile, translation, metabolic pathways, antibiotic
gene resistance and virulent gene expression etc.
Pathogenic detection with its whole genome profiles including
virulence factors and binding sites etc.
Development of effective therapeutics, vaccines and drugs, to control
diseases using immune-related molecular understanding (PRRs, TLRs)

25. Transcriptomics
Valuable insights into the gene expression
patterns and regulatory mechanisms @
specific point in time.
Understanding the disease mechanisms,
identifying potential drug targets, gene
regulation, proteomics and metabolomics
etc.
Key transcriptomes are mRNAs, non-
coding RNAs and small RNAs; to determine
the transcriptional structure of genes
Quantify transcriptomes: cDNA synthesis,
hybridization, qPCR, RNA Sequencing and
microarray etc

26. 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); Antibiotic Resistant Gene
Identify new latent pathogens in asymptomatic
carriers, uncharacterized pathogens causing a
new disease or multiple pathogens associated
with disease syndromes in shrimp farms

27. PROTEOMICS BASED DIAGNOSIS

28. 1 & 2 D PROTEIN GEL ELECTROPHORESIS
1-DE: identify proteins in complex samples, diagnostic applications,
including protein characterization, purification & quantification
2-DE: protein separation based on isoelectric focusing (IEF) followed by
molecular mass protein separation.
Protein identification and expression analysis of thousands of proteins
on a single gel.
2D gel quantitative proteomics-DIGE is more sensitive and accurate
method with labeling of fluorescent tags (CyDyes™; Cy5, Cy3, and Cy2)

29. MASS SPECTROMETRY
High-throughput proteomics that also allow the measurement of
multiple properties
Abundance, tissue distribution, sub-cellular localization and protein-
protein interactions etc.
Need purification (HPLC) separate peptides and coupled with MS.
Shotgun proteomics helps to digests protein and detection with MS

30. LC-MS/MS
Systematic characterization of proteomes-host-pathogen interactions
Digested to peptides by a sequence-specific enzyme, typically trypsin
Peptides separated and analyzed by LC-MS/MS
LC-MS/MS has many applications in aquatic disease diagnosis.
In the case of viruses, several proteins have been modified although
differences depend on the type of virus.
Proteins involved in the glycolytic pathway and cytoskeletons were modified
during viral haemorrhagic septicaemia rhabdovirus in zebrafish
Functional analyses of the virion transmembrane proteome analysed in
Cyprinid herpesvirus 3 (CyHV-3)

31. MALDI-TOF
Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)
microbial identification and diagnosis, as a rapid, sensitive and
economical
Label-free and has high accuracy and reliability, but it requires a bulky
instrument for analysis and relies on pre-stored databases.
Virulence of Vibrio tapetis isolates and Photobacterium damselae
Envelope protein characterization of Singapore grouper iridovirus (SGIV)

32. PROTEIN-PROTEIN INTERACTION
PPIs play a crucial role in the functioning of cells
Essential for processes like signal transduction, enzyme activity regulation,
cell cycle progression, immune response etc.
In immune responses, interactions between immune-related proteins enable
the recognition and elimination of harmful pathogens
Pathogens often hijack host protein-protein interactions to facilitate their
invasion and survival.

33. IMMUNODIAGNOSIS TECHNIQUES

34. 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

35. 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 with High specificity
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

36. APPLICATIONS OF IMMUNO DIAGNOSIS
Enzyme Linked Immuno Sorbent Assay (ELISA)
Detection and quantification of specific proteins
Types include, direct, indirect, sandwich and competitive ELISA.
Coating the target antigen, blocking, Washing, primary antibody
treatment, incubation, washing, secondary antibody treatment with
incubation, washing and detection
Signal generated is quantified using a spectrophotometer

37. Western Blot
Immunoblot : study of protein expression; identify post-translational
modifications useful for disease diagnosis.
Proteins to be transferred NC or PVDF membrane by electro-transfer.
blocking, Washing, primary antibody treatment, incubation,
washing, secondary antibody treatment with incubation, washing
and detection

38. Agglutination
Can help identify the specific strain of
bacteria based on the presence of
specific surface antigens.
Latex beads are coated with antibodies
specific to a particular antigen will
cause the latex particles to agglutinate,
indicating a positive reaction
Dot Blot
Semi-quantitative technique, identifying
proteins for disease diagnosis
Like western blot no need for protein
purification
Lateral Flow
Diagnostic technique used for the rapid
detection virus and bacterial pathogens
Time consuming diagnosis, antibodies
coated in membrane, apply the antigen
then color develop

39. Visualize the presence, location,
and distribution of specific
proteins within tissue samples.
It combines principles of
immunology and histology to
provide insights into the cellular
and subcellular localization of
target antigens.
Localization of proteins in tissue
whole-mount or sections using
fluorescent labeled antibodies
that binds via antigen-antibody
interactions.
Immunohistochemistry & Fluorescent antibodies

40. APPLICATIONS OF IMMUNO DIAGNOSIS
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)
Immunoperoxidase
Visualize the presence and localization of
antigens in tissue samples using an
enzyme-linked detection system.
Enzyme, peroxidase, which catalyzes a
reaction that produces a colored or
visible precipitate at the site of the target
antigen, making it easily detectable
under a light microscope.

41. BIOTECHNOLOGICAL TOOLS TO IMPROVE
PRODUCTION

42. 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
IMMUNOSTIMULANTS

43. 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 Activation
TLR Activation
AMP synthesis
& Phagocytosis
Immune gene Exp
& ROI, RNI

44. 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

45. 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

46. 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
PPAE
TLR
SERPIN
Ligand
PO positive

47. RECOMBINANT VACCINE DEVELOPMENT

48. 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

49. 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.

50. RECOMBINANT VACCINES

51. 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

52. 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. 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

54. 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

55. 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.

56. 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.

57. EDIBLE ANTIBODY OR EDIBLE VACCINES

58. 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.

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

60. NANOTECHNOLOGY
DNA Nano-Vaccines: Use of nanoparticle carriers likes chitosan and poly-
lactide-co-glycolide acid (PLGA) for vaccine antigens may give a high level of
protection
Resistant to digestion and degradation and controlled release
Tripolyphosphate (TPP), a cross-linking agent, has been added to the aqueous
phase containing chitosan in a method for creating chitosan nanogels
These nanocapsules contain short strand DNA which when applied to water
containing fishes are absorbed into fish cells.
Chitosan-TPP : siRNA delivery vehicles

61. QUORUM SENSING CONTROL
Sophisticated cell-to-cell
communication mechanism within
a bacterial population.
Regulating gene expression,
virulence factor production and
biofilm formation
They continuously release small
signaling molecules called
autoinducers, acylated
homoserine lactones (AHLs)
They have specific receptors on
their surfaces that can detect the
autoinducers and transmitted and
leading to bloom.
Several herbal compounds able to
bind AHLs and molecular docking
approach solves this

62. Specific Pathogen Free (SPF)
Development of SPF doubling the production in Litopenaeus
vannamei in US aqua industry during 1990s
They cross bread between two traits
CPF and Oceana Institute develop the SPF by cross bread the
better characters such as salinity, low DO, pH and disease
tolerance traits and analyze the characters at 30 generation and
commercialization

63. 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

64. 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

65. TRASGENIC TELEOSTS FOR EPA AND DHA PRODUCTION

67. “A process in which the introduction of double-stranded
RNA into a cell inhibits the expression of genes”
RNA INTERFERNECE (RNAi)

68. 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

69. 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)

70. PHAGE THERAPY

71. 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.

72. 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

73. 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

74. Gene editing (CRISPR-Cas9)

75. 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. Small piece of RNA that is
designed to target a specific DNA sequence.
The Cas enzyme then acts as molecular
scissors, cutting the DNA at the targeted
location.
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
Edited fish successfully reproduce and give
rise to an F1 generation, the genetic changes to
be inherited by their offspring
CRISPR-Cas technology can also be used to
control the viral and bacterial diseases
particularly in shrimps and prawns

76. 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

77. CONCLUSIONS & FUTURE DIRECTIONS

78. Tools can certainly revolutionize
aquaculture & Improved health and
wealth
Reduce the importation of foreign fish
and aquaculture products and increased
foreign earnings & Improved economy to
the farmers
Genetic modification holds tremendous
potential to improve the quality and
quantity of fish reared in aquaculture
Environmental spoilage can be avoided &
Preventing emergence of resistant strains
Consignment rejection can be controlled
I am Healthy

79. THANK YOU