ALTERNATIVE APPROACH IN AQUATIC HEALTH MANAGEMENT

1. Dr. T. Citarasu
Assistant Professor
Tele-fax: + 91-4652-253078
Mobile: +91-9994273822
Email: citarasu@gmail.com
citarasu@msuniv.ac.in
Website: www.msuniv.ac.in

2. CMST
Beach
Back Water Nursery ground
Mangroves
Our Institute, Centre for Marine Science & Technology
Courses Offered
M. Sc Marine Science
M. Sc Marine Biotechnology
M. Sc Microbiology
M. Phil programmes
PhD in various discipline
Rajakkamangalam

3. IMPORTANCE OF AQUACULTURE
Lucrative food production industry
Fresh, brackish & Marine based culture
10 % annual growth rate
Global sea food demand-70% in the next
33 years (7 fold)
Capture and culture fisheries
Attractive Job oppertunities

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
Top 5 Countries L. Met. tones
China 7.5
India 7.0
Vietnam 5.5
Thailand 4.5
Indonesia 1.5
Aquaculture production (2018)

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

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

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

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

11. Herbal 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.
Ecliptine Tinosporone
Ocemine Aegilin

12. Antibacterial Screening
Piper longumMurraya koeniji
Quercus infectoria
Psoralea corylifolia
Several herbals screened against aquatic important
pathogens
In vitro antibacterial activity
Some of them characterized
Beta Ocemine
Quercucin Psoralin
Piperine

13. 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
(1,1-dimethylethyl)

14. ANTIVIRAL COMPOUNDS
Aegelin
Andrographolide
Cyclohexane, 1,1'-dode
cylidenebis 4-methyl-
Phenol 2 5-bis(1 1-
dimethylethyl)-
1,10- biphenyl-3-amine
9-octadecenamide
Mannosamine
3-beta-hydroxy-5-cholen-2-4-
oic acid
Surfactin A synthetase C

15. Anti EHP

17. Immunostimulants
Aegle marmelos
Picrorhiza kurooa
Cynodon dactylon
Tinospora cordifoliaEclipta alba
A substances that stimulate the immune system
by inducing activation or increasing activity of
anyofits components.
Improvement of……
• Hematology
Immunological parameters
• Phagocytosis
• Prophenol oxidase activity
• Lysozyme activity
• Bacterial & viral clearance

19. Types of Immunostimulants
Chitin, Chitoson & Oilgomers
Shell waste
Fucoidon
Sulfated polysaccharides –Seaweeds
Glucans
Yeast
Lactoferin
Milk
Lipopolysaccharides
Bacterial Cell wall
Herbals
W. somnifera, A. calamos, T. cordifolia

20. MODE OF ACTION
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

21. Inhibition of anti apoptotic protein
WSSV hijack protein AAP1
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 negetive
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

22. ALGAL DRUGS
SEAWEEDS
Broad spectrum of activities
antiviral, anthelmintic, antifungal & antibacterial
Brominated, aromatics, sterols, dibutanoids,
proteins, peptides, sulphated polysaccharides,
terpenoids, phlorotannins, acrylic acid, phenolic
compounds and steroids.
Carrageenan
Powerful antioxidants
Alginate
polysaccharaide
Laminaria and Fucus
Fucoidon
Powerful antiviral against WSSV
2-(2-hydroxyphenoxy)-1-
phenylethanol

23. MICRO ALGAE
Polyketides, amides, alkaloids, peptides and
beta carotenoids
antiviral and antibacterial
Antioxidants
Euglena viridis
Against fish & Shellfish pathogens
Chlorella
Antibacterial
Dunaliella sp
From Halophilic origin
Control shellfish virus
Powerful antioxidants & Immunostimulants

24. Pharmacological Important Biosurfactants
Extracellular Amphiphilic compounds
Glycolipids, lipopeptides, fatty acids,
polymeric surfactants & particulate
natures
Broad range of applications
Antibacterial, antiviral, antifungal, anti-
adhesive, anti-cancer, antioxidants,
adjuvant etc

27. PROBIOTICS
Probiotics have been defined as “live microbial
food supplements which beneficially affect the
host by improving the intestinal microflora
balance”
Gut & Water Probiotics
Competitive exclusion principle
Used in Shrimp farms and polluted areas
Improved strains of Probiotics
Lactobacillus and Bifidobacterium sp

28. WATER PROBIOTICS
Mixed substrates [Rice bran-300 g;
Wheat bran-300 g; Rice flour-200g &
Wheat flour-200 g]
600 ml bacterial culture broth
Make small balls & dried @ 40 ºC
Powdered
Bacterial count
Vacuum packed
Apply to pond

30. EDIBLE ANTIBODY OR EDIBLE VACCINES

31. 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.

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

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

35. VACCINE DEVELOPMENT

36. 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

37. 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

38. 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

39. RECOMBINANT VACCINES

40. 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

42. 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?

43. 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

44. 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

45. 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

47. 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.

48. 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

49. Micro algal Recombinant vaccines
Antigens expressed in the chloroplast or anchored to the surface of plasma
membrane
Safe and inexpensive mechanism 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.

50. 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)

51. 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

53. 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

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

55. 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

56.  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

57. CONCLUSIONS & FUTURE DIRECTIONS

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

59. 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.

60. THANK YOU