Development of nanocarriers for oral vaccination in fish
By: Janhavi Kailas Vanjari
Under the guidance of:
Dr. Jyutika M. Rajwade
Nano bioscience group
Agharkar Research Institute
Submitted to
Vellore Institute of Technology (VIT Bhopal)
Feb 2023
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Supervisor Certificate
This is to certify that the work presented in the Thesis titled “Development of
nanocarriers for oral vaccination in fish” is the bonafide work of “Janhavi Kailas
Vanjari Registration Number 19BOE10062 VIT Bhopal University” is a record of
original research carried out by her under my supervision and guidance in partial
fulfillment of the requirements of the Bachelor of Technology in Bioengineering.
Dr. Jyutika Rajwade
Agharkar Research Institute
Supervisor
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Declaration of Originality
I, Janhavi Kailas Vanjari, bearing the Roll Number 19BOE10062 hereby declare that this thesis entitled
“Development of nanocarriers for oral vaccination in fish” represents my original work carried out as a
undergraduate student at VIT Bhopal University. To the best of my knowledge, it contains no material
previously published or written by another person, nor any material presented for the award of any other
degree of VIT Bhopal University or any other institution. Any contribution made to this thesis by others,
with whom I have worked at VIT Bhopal University and Agharkar Research Institute, is explicitly
acknowledged in the thesis. Works of other authors cited in this dissertation have been duly
acknowledged under the section ''References''.
I am fully aware that in case of any non-compliance detected in the future, the VIT Bhopal University
may withdraw the degree awarded to me on the basis of the present thesis.
April 09, 2023
Janhavi Kailas Vanjari
Agharkar Research Institute
VIT Bhopal University
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ACKNOWLEDGEMENT
This thesis has evolved from support of great number of people and all of them deserve special mention
in this thesis. I am very much please do take this opportunity to extend my regards to them. It is indeed
difficult to put down on paper my heartfelt gratitude towards those people who have aided me in the
completion of my project. I wish to express my deep sense of appreciation to Dr. Jyutika Rajwade,
Agharkar Research Institute Pune. For valuable guidance and support during the course of study and for
extending the laboratory facility during this tenure. I am grateful to Dr. Siddhartha Maiti and Dr. Neetu
Kalra VIT Bhopal University for never ending moral support. I will never forget the valuable support
given by them, which I never expected from anyone other than my parents. I am also thankful to my labmates at Agharkar Research Institute, and classmates for their kind co-operation and help during
research work.
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Contents
Description
Page No.
Supervisor certificate……………………………... 02
Declaration of originality ………………………….. 03
Acknowledgment…………………………………… 04
Contents…………………………………………… 05
List of Tables ……………………………………… 07
List of Figures………………………………………. 08
Abbreviations………………………………………. 09
Abstract……………………………………………. 10
Introduction…………………………………….. ….10
Chapter 1
Vaccines
1.1 History of vaccines………………………………. ..11
1.2 Need for Vaccination humans……………................12
1.3 Need for vaccination in animals………………………12
1.4 Need for vaccination in aquaculture…………………..13
1.5 Routes of vaccine administration………………………14
Chapter 2
Immune system
2.1 Types of immunity………………………….15
2.2 Immune system in fishes………………………….16
2.3 Types of immunity in fish………………………….17
2.4 Immunoglobin…………………………18
2.5 GALT………………………….20
2.6 GIALT………………………….21
Chapter 3
Fish stressors………………………….22
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Chapter 4
Vaccine in fish
4.1 Routes of Vaccine administration in fish………………………….23
4.2 Types of vaccine in fish………………………….27
Chapter 5
Commercially important fish………………………….29
Chapter 6
Commercialized vaccines………………………….34
Aim of Thesis………………………..34
Instruments………………………35
Methodology………………………….40
Results and Discussions………………………….41
Conclusion………………………….45
References………………………….45
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List of Table
Table 1
Commercially Important fish
Table 2
Protein estimation by biuret method
7|Page
Table of Images
Image 1
Different routes of vaccine administration
Image 2
Route of vaccine administration in fishImmersion
Image 3
Routes of vaccine administration in fishImmersion
Image 4
Routes of vaccine administration in fishInjection
Image 5
Routes of vaccine administration in fish- Oral
delivery
Image 6
Instrument - Centrifudge
Image 7
Instrument - Nanodrop
Image 8
Instrument - Gel Electrophoresis
Image 9
Instrument - Lyphoilizer
Image 10
Instrument - Sonicator
Image 11
Instrument – Zeta sizer
Image 12
Instrument – Gel Imager
Image 13
Protein estimation graph by biuret method
Image 14 A, B, C, D
Physiochemical
characterization
nanoparticles A-
Nanoparticle
of
HAP
Tracking
Analysis; B- Zeta Potential; C- FTIR Spectrum;
D- SEM
Image 15
SEM image of HAP after protein adsorption
assay
Image 16
Adsorption Efficiency of Bovine serum albumin
and Hydroxyapatite Nanoparticle in PBS,
Citrate, and carbonate buffer.
Image 17
At different temperature (25, 30, 35, 40); B: At
different Temperature
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Abbreviation
HPV- Human papillomavirus vaccines
IG- Immunoglobulins
IgA- Immunoglobulin A
IgM- Immunoglobulin M
IgD- Immunoglobulin D
HepA- Hepatitis A
HepB- Hepatitis B
MMR- Measles, Mumps, Rubella
IP- Intraperitoneal
IM- Intramuscular
NTA- Nanoparticle Tracking Analysis
SEM- Scanning Electronic Microscope
FTIR- Fourier Transform Infrared
NP- Nanoparticles
HAP NP- Hydroxyapatite nanoparticles
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Development of nanocarriers for oral vaccination in fish
Abstract
In order to maintain a sustainable aquaculture, both economically and environmentally,
disease prevention and management are essential. This study provides a summary of
the advancements made in fish vaccination and the development of oral vaccines using
nanotechnology, with a focus on the latter as a useful tool for the successful
advancement of aquatic animal bioproduction.
Key Words: Vaccination, Vaccinology, Bioproduction, Oral Vaccine, Nanotechnology
Introduction
Vaccination is a protective process that can shield us from infections and disease. It
plays a crucial role in saving a large population from the disease outbreaks. In response
to vaccination, the host elicits an immune response (akin to a natural infection) in form
of antibodies. Our immune system and vaccines collaborate to protect us from a variety
of diseases and infections. The majority of vaccines include a virus or bacteria in
weakened, inactivated (killed), or minuscule amounts that cannot spread disease1
known as an antigen. When antigen is introduced in body, the body identifies it as
foreign particle and starts to make antibodies against them and neutralize them. Vaccine
antigens activate immune cells, Immune cells are developed from bone marrow and
stem cells. It helps to fight against the diseases. T cells and B cells are two important
components of immune system. T cells which is also called as T lymphocytes or
Thymocyte have important role in immune system which are Directly killing infected
host cells, activating other immune cells and producing cytokines and regulating
immune response. B cells are responsible for making a protein called as antibody, B
cells can also recruit other cells to help destroy an infected cell. The immune system
has a special ability known as immunological memory that allows it to "remember"
information about a stimulus and mount a powerful defense when the stimulus is reexposed.2
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1. History of Vaccines
The term "vaccination" originated with Edward Jenner's invention of the smallpox vaccine in
1796. Smallpox was the first vaccine that was ever created [Jenner E. An inquiry into the causes
and effects of the variolae vaccinae, a disease discovered in some of the western counties of
England particularly Gloucestershire, and known by the name of cow pox. London: Sampson
Low; 1798]. In 1977 Salk & Salk first time introduced the term vaccination Later, In order to
express the interdisciplinary aspect of disease prevention based on microorganisms stimulating
the immune system to prevent infectious diseases in individuals and populations.3
The accomplishments of numerous scientists who worked in academic institutions andresearch
labs are documented in the history of fish vaccination. The scientific literature presents and
documents how they are made. Most early vaccine experiments inaquaculture were centred on
killed vaccines. Duff, who looked into oral immunisationof cutthroat trout Oncorhynchus clarkii,
used a dead Aeromonas salmonicida vaccine as the first instance of a fish vaccine being used. A
dead Yersinia ruckeri vaccine administered by immersion against enteric redmouth disease was the
first commerciallyapproved vaccine for fish.4
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2.
Need for Vaccination (humans)
Humans need vaccination to protect themselves from deadly diseases and to acquire
immunity against these diseases. Being vaccinated also reduces the risk of spreading
infectious diseases. As the need for smallpox vaccination grew in the nineteenth
century, the first mass vaccination campaigns were held over limited intervals to
effectively and quickly protect the large number of susceptible people. Even though
routine immunisation services have been strengthened in many countries because to the
Expanded Program on Immunization policies and GAVI (Global Alliance for Vaccines
and Immunization) assistance, the difficulty of quickly and effectively protecting
populations through mass vaccination persists even two centuries later. The ability to
quickly boost population immunity (herd immunity) in the face of an ongoing or
impending outbreak can limit the morbidity and mortality that could result, especially
when there has been no routine immunisation or when populations have been relocated
and regular immunisation services have been disrupted. This is perhaps the most widely
accepted justification for mass vaccination. The ability to quickly expand vaccination
coverage with a new vaccine when it is introduced into routine immunization programs
and to achieve the herd immunity levels necessary to satisfy global targets for
eradication and mortality reduction is a second significant use of mass vaccination. In
order to achieve both national and international objectives in the control of vaccinepreventable disease, mass vaccination and routine immunisation remain essential
partnerships in the twenty-first century.5
3. Need For Vaccination (Animals)
Several diseases spread via animal-human contact. Wild, domesticated, and farmanimals are reservoirs/intermediate hosts of many viruses during their life cycle.Animal
welfare and public health have both benefited greatly from the use of veterinaryvaccines.
They also lessen animal suffering, enable efficient food animal production tofeed the
world's expanding population, and significantly reduce the need for antibioticsto treat
both food and companion animals. During vaccination the animals are exposed to
disease-causing organisms at a young age, training their immune systems to recognize
the infectious pathogen against which they have received vaccinations. Immunization
contributes to the economic and social stability of farmers and the communities they
serve. A vaccine is a practical and affordable way to prevent animaldiseases; in general,
they are safe, effective, and have minimal serious adverse effects.They are beneficial
for long-term protection since illnesses can be avoided and financialburden of diseases
can be significantly reduced. The value of veterinary vaccinations goes beyond just
the immunization of pets, herds of livestock, companion animals etc.because many of
them also protect people from anthropozoonoses, i.e., diseases that affect both humans
and animals.6
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4. Need For Vaccination in aquaculture
The farming of aquatic species, such as fish, mollusks, crustaceans, and aquatic plants,
is known as aquaculture. In order to increase productivity, farming usually involves
some type of intervention in the rearing process, such as frequent stocking, feeding,
predator protection, etc. Farming also denotes that the stock being cultivated is owned
by a person or a business. For statistical purposes, aquatic organisms that are harvested
by a person or corporate entity that has owned them throughout their entire rearing
period are considered to be part of aquaculture, whereas aquatic organisms that are
exploitable by the general public as a common property resource, with or without the
necessary permits, are considered to be part of fisheries. fishes are good source of food
and meat. Fishes and aquaculture can contribute in reducing global food shortage.
Fishes are important for human because, humans can feed on them. And also, they
support economies and create diversity of aquatic system. We must take care of them
because they are a crucial component of the natural system. They also contribute to
natural diversity. Fish is an excellent food source for many people. It is a good source
of high-quality, low-fat protein and contains a variety of essential vitamins and
minerals, such as omega-3 fatty acids, vitamins D and B2 (riboflavin), iron, zinc, iodine,
magnesium, potassium, calcium and phosphorus. Eating fish can help lower blood
pressure and reduce the risk of heart attack and stroke. Not only is fish a great source
of nutrition, but it can also be an enjoyable and tasty way to get the nutrients your body
needs. Fish illnesses continue to be a significant financial problem in commercial
aquaculture around the world despite numerous efforts to cutting-edge treatment.
Although antibiotics or chemotherapy may be used to treat diseases, there are some
obvious downsides, such as safety concerns and problems with drug resistance. The use
of vaccines in the worldwide aquaculture industry helps to maintain environmental,
social, and economic sustainability by effectively avoiding a wide range of bacterial
and viral infections.
Considering that they are a popular and extensively consumed source of protein. As a
result, we need to take steps to make sure they are free of any dangerous infections
causing mortality in fish. Fish vaccination will increase production and have a longlasting preventive impact. We will receive healthier fish and fish food as a result.
Additionally, the need of antibiotics will decrease as a result of vaccination. It will boost
the cost-benefit ratio and protect the farmer's investment.
Fishes have an under-developed immune system and diseases can easily be transmitted
through water.
We need to understand the immune system and immune response of fishes thoroughly
to develop vaccines.
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5. Routes of Administration of Vaccines
Image 1
5.1 Intramuscular
Immunizations delivered intramuscularly are injected at a 90° angle into the
muscle layer of the body. The large number of blood capillaries found in muscle
results in an enhanced blood supply, which makes it possible for the vaccine to
spread easily. Muscles also have dendritic immune cells, which deliver antigens
and start a sustained immune response. Other than moderate and transient
redness and soreness at the injection site, intramuscular vaccinations often have
fewer side effects.
Example: HPV, HepA, HepB
5.2 Intradermal
A 5–15° angle is used for injecting intradermal vaccinations into the dermis and
epidermis of the skin. Antigen-presenting cells (APCs), which are assumed to
play a crucial role in generating an effective and protective immune response to
particular vaccinations, are abundant in the epidermal and dermal layers of the
skin. Dendritic cells, B cells, and macrophages are the three primary APCs.
APCs deliver particular antigens to immune system cells in order to trigger an
immunological-mediated response that results in the production of memory
cells and antibodies.
In order to assure the safety and effectiveness of the vaccine, intradermal
vaccination is a special technique of delivery.
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Example: Bacille Calmette-Guérin and rabies vaccines.
5.3 Subcutaneous
The fatty layer beneath the skin's surface is where subcutaneous vaccines are
injected at a 45° angle. Because subcutaneous tissue has a lower blood supply
than muscle, vaccines intended for subcutaneous injection are absorbed more
slowly and steadily.
Example: MMR vaccine
5.4 Oral Delivery
Oral vaccines can come in either tablet or liquid formulation. It activates
immune cells in the mucosal membranes lining the gastrointestinal tract whichis
beneficial in protecting against diseases that affect the gut For Example Polio
Vaccine
6. Types of Immunity
Immunity is classified under two categories that are Active and Passive
Immunity. Active Immunity is then further sub-categorized in Natural
Immunity and Vaccine based Immunity.
6.1 Active
The process of exposing the body to an antigen to create an adaptive immune
response is known as active immunity; the response takes days or weeks to
develop but may be persistent, even lifetime. Active immunity is typically
categorized as either acquired or natural.
We can acquire two types of active immunity
6.1.1
Natural immunity is obtained when we are exposed to a disease and our
immune system starts to producing Antibodies against that disease that is
known as natural immunity. Natural immunity stays for a longer period of
time as compared to vaccine based immunity.
6.1.2
Vaccine based immunity is the type of immunity acquired from vaccines.
Vaccine based immunity tends to be renewed after a certain period of time.
6.2 Passive
Direct Immunity we acquire when we are given direct antibodies instead of our immune
system producing it. Passive immunity is the process of supplying IgG antibodies to
fight infection; it offers instant but transient protection, lasting no more than a few
weeks to three or four months. Typically, passive immunity is categorized as either
acquired or natural. The placental transfer of maternal tetanus antibody, primarily IgG,
gives the newborn baby natural passive immunity for a few weeks or months until such
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antibody is lost and destroyed. The procedure of getting serum from immune people,
pooling it, concentrating the immunoglobulin fraction, and then injecting it to protect a
susceptible person is known as acquired passive immunity.
7. Immune System of Fishes
Skin is regarded as a key immunological organ in fish because it acts as the first line of
protection against microbes and other stresses. Fish skin is made up of the dermis and
a skin mucosa layer, which secretes mucus and is covered in calcified scales.7 These
offer protection from environmental pathogens that can cause bodily harm and disease,
which is further enhanced by a mucus layer that contains bactericides and fungicides.
The mucous membrane regenerates continuously. It aids in clearing away waste and
prevents parasites from clinging to the fish. Skin of fish is also responsible for exchange
of nutrients, gametes, gases, odorants and hormones. The skin mucosa has a distinctive
shape and a strong metabolic activity. Different mucosal immunity systems have been
developed in fish skin. First, mucus coats it to keep infections from adhering to the
skin's surface. Second, it contains a wide range of antibacterial substances, such as
proteins, enzymes, lectins, C-reactive proteins, immunoglobulins, complementproteins,
and lysozyme and proteolytic enzymes. Third, certain immunocompetent cells, such as
epithelial, mucus, club, and goblet cells, are present in the dermis and epidermis and
are responsible for the skin-associated lymphoid tissue (SALT).8
Still, pathogens are able to enter the fish's body through digestive system or physical.
Diseases can occasionally persist even though the digestive system has active enzymes
and a pH level that is particularly unfavorable to pathogens. Anaerobic fermentation
and active enzymes can damage the gut wall and weaken it to the point where pathogens
can enter if stress causes the gut to tighten up.
Environment has an impact on a fish's immune system's effectiveness. Because colder
water slows the system down, sick fish often experience "fever symptoms" and migrate
to warmer environments. The infection may or may not be affected by colder water;
either way, if it does not slow down the bacteria and the immune system, death is
unavoidable. The antiviral chemical interferon and C-reactive protein, which instantly
fight germs and viruses, give fish some broad immunity. The fish's body organizes its
defenses as soon as a pathogen is identified. To start, the entry point is sealed up to
address any osmoregulatory issues and prevent the foreign body from progressing.
Damaged cells at the entry point produce histamines and other chemicals that promote
inflammation and force the blood cells to constrict. A physical barrier is simultaneously
constructed by fibrinogen, a blood protein, and clotting factors. The same area is drawn
to white blood cells, which then pick up the foreign objects and transport them to the
spleen and kidney for processing. Still bacteria can invade these defenses. They may do
so by secreting poisons that assault and kill white blood cells or by creating a dissolving
agent that dissolves the fibrin and makes it easier for an infection to spread. To combat
each unique antigen, the kidney and spleen produce antibodies (invading disease).
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8. Types of Immunity in Fish
An antigen is either present in or created by a fish vaccination. The fish's innate and/or
adaptive immune systems are then stimulated to respond to a specific pathogen as a
result of this component. Over 10,000 scientific publications on fish vaccines have been
published in the last 20 years alone, reflecting the growth of research into fish
immunology and vaccines over the 20th century. Fishes acquire two types of immunity;
Non-specific which is also called as Innate immunity and the specific immunity called
as Adaptive Immunity.
8.1 Innate Immunity
The first line of protection the fish's immune system has against the various diseases
that endanger their balance is innate or non-specific immunity. The three basic parts of
this defense system are mucosal immunity, humoral components, and cellular
components. Since it is in charge of fixing the complement, opsonization, and activating
the cytotoxic response, among other things, these immunoglobulins are essential for the
innate immune system's defense against bacteria and viruses. Monocytes (e.g.,
macrophages) and granulocytes (e.g., neutrophils) as well eosinophil granulocytes that
resemble the mammalian mast cells are the kind of cells that are involved in innate
immunity.
8.2 Mucosal Immunity
The fish immune system is heavily dependent on mucosal immunity, which serves as a
barrier and keeps pathogens from entering the body. The mucus that covers the surface
of these creatures' bodies, as well as their gills and other tissues, offers mechanical and
physical protection in addition to containing numerous immunological components
such antimicrobial peptides, complement factors, and immunoglobulins.
8.3 Adaptive Immunity
Early in infancy, the acquired immune system begins to form, but it relies on gene
recombination to produce specificity antibodies to match the pathogens encountered.
There is growing evidence that fish and mammals have a common mechanism.
Antigen-presenting cells (APCs), which absorb and degrade foreign antigens invading
the tissue, provide antigens that T cells respond to. Any cell that can take up and present
antigen, such as monocytes (macrophages and dendritic cells) or B cells, is an APC. In
T cell dependent activation, T helper (Th) cells that express the CD4 complex (CD4+
cells; CD=cluster of differentiation) regulate the generation of antibodies by B cells.
The pathogen's surface is marked for eradication by the antibodies created following B
cell activation. When an infected cell displays a "non-self" antigen, cytotoxic T cells
(Tc) that express the CD8 complex (CD8+ cells) can also directly respond by secreting
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poisonous substances that cause cell lysis. Through the T cell receptor (TCR)
complexes, the T cells communicate with the antigen that has been delivered. T cells
express CD3, a component of the complex that transmits signals from the TCR and is
essential for T cell activation and proliferation.
9. Immunoglobins (Ig’s)
Ig’s are highly specialized proteins that can identify a wide range of antigens from
bacteria, viruses, and other disease-causing organisms. They then enlist the help of
other cells and molecules to kill these pathogens. Ig’s are made up of two heavy (H)
and two light (L) chains, with the exception of some camelid and nurse shark antibodies
that don't have L chains.
The constant portion of the heavy chain, which in Teleosts contains C, C, and C/C,
encoding IgM, IgD, and IgT/IgZ, respectively, specifies the effector function of a
particular antibody. There are four different L chain types found in bony fish, of which
Igκ and Igσ are present in the majority of teleost species, Igλ has been lost in the majority
of teleost lineages during species divergence (cod, catfish, and rainbow trout are notable
exceptions), and Ig-2 was recently discovered in the coelacanth.
This particular reaction in fish begins with the T cells' recognition of the major
histocompatibility complex (MHC) molecules. Immunoglobulins belonging to the
superfamily of MHC molecules function as receptors. MHC genes are not related and
can be found on different chromosomes in the fish immune system, in contrast to higher
mammals where they are combined into a single chromosome. Teleosts have the two
types of MHC receptors that have been discussed in relation to upper vertebrates. All
nucleated cells have MHC I molecules, which are connected to endogenous antigens
and are recognized by CD8 or cytotoxic T lymphocytes. The MHC II molecules, which
are only present in antigen-presenting cells, are associated to external antigens
(dendritic cells, macrophages, and B lymphocytes). The immune system can have a sort
of "memory", which makes a subsequent exposure to the same antigen result in a
stronger and longer-lasting reaction. Two phases are necessary for B lymphocyte
activation (figure 3). They need to be able to identify external antigens through type II
MHC, but they also need a CD4 T lymphocyte or helper to present those antigens. After
becoming activated, B lymphocytes change into plasma cells that can secrete several
immunoglobulin subtypes.9
Teleost Produce 3 Types of Immunoglobulins that are IgM, IgD and IgT/IgZ.
The most prevalent immunoglobulin in fish immune systems is IgM, which, Teleost
have a tetramer comprising two heavy chains and two light chains that make up their
IgM. (2H:2L).
9.1 IgM
IgM is mostly present in all vertebrates except in African coelacanth because they
carries IgW H chain loci.
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Most teleosts have tetrameric IgM as their predominant serum Ig type. Since teleost
IgM lacks the J chain, covalent (disulfide) linkages are mostly used to connect its
monomers together. The affinities of trout IgM to antigens have been found to increase
with increased disulfide polymerization, and these trout IgM have also been found to
have longer half-lives. Depending on factors such water temperature and quality, fish
species, size, stress, stimulation, and immunisation, teleost serum IgM concentration
might vary (0.6–16 mg/ml). Trout's overall blood IgM concentration and its parasitespecific IgM binding ability both increased dramatically during parasitic infection, and
the proliferation of IgM+ B cells in the head kidney also increased. These findings
illustrated the regulatory roles of IgM in teleost systemic immunity. IgM has also been
demonstrated to exist as a tetramer in the various rainbow trout mucus types at various
concentrations, including the gut mucus (0.075mg/ml), skin mucus (0.0046 mg/ml), gill
mucus (0.02mg/ml), pharyngeal mucus (0.072mg/ml), and nose mucus (0.28mg/ml)
Nevertheless, IgM remains the most prevalent Ig of the three in all teleost mucus, and
it has been demonstrated that parasite-specific IgM binding in pharyngeal mucus
increases following parasitic infection. Although less frequently than IgT, it has been
discovered that IgM can coat bacteria in several forms of mucus. Additionally, teleosts'
IgM expression shows some universal characteristics. For instance, Ig-producing cells
often occur in the following tissues in the following order: head kidney, spleen, and
ultimately MALT. Cytoplasmic IgM is also typically expressed earlier than surface
IgM. Ontogenetic IgM expression patterns in zebrafish are as follows: surface Ig
transcript at 7 days post-fertilization (dpf), IgH chain transcript in the pancreas at 10
days post-fertilization (dpf), sIg transcript at 13 days post-fertilization (dpf), IgH chain
transcript in the head kidney at 19 days post-fertilization (dpf), and detectable humoral
Ig at (28 dpf).
9.2 IgD
The number of C δ domains varies greatly among various fish species, in contrast to the
limited (i.e., often two or three) C δ domains in mammals. The increased number of C
δ domains in teleosts may offer a wider range of structural choices to synthesize more
flexible H chain products than eutherian chains, which have two domains joined by a
hinge. Additionally, it was discovered that in δ bony fish was produced exclusively by
splicing C1 between rearranged VDJ and Cδ1, resulting in a chimeric H chain sequence.
Notably, practically every teleost Ig transcript has been shown to include Cµ1. In
channel catfish, two distinct IgH genes are used to produce membrane-bound and
secreted IgD. It's interesting to note that an IgH transcript without a V-region encodes
the secreted delta form. The second gene has a germline-recombined VDJ, but the
signal sequence is immediately spliced into Cδ1 in the mRNA, suggesting that the
secretory version might not have antigen recognition capabilities.
IgD's immunoprotective function in teleosts is still not well understood. Although
several studies have speculated about IgD's function in teleost gills, it is known that this
antibody plays no part in specific immunity in rainbow trout's gills and PM during
parasite infection. A subset of commensal microbiota can also be coated by teleost sIgD
in mucosal tissues, including the gut, gills, BM, and PM. However, mucosal bacteria
19 | P a g e
are coated by sIgD far less frequently than sIgT. These findings imply that teleost sIgD
may possibly play a role in maintaining mucosal homeostasis.
9.3 IgT
Similar to IgA in mammals and IgX in frogs, IgT functions as a mucosal-associated Ig
in bony fish. IgT subclasses have also been observed in other teleost fish, including
stickleback (Gasterosteus aculeatus) and carp, in addition to salmonid species
(Cyprinus carpio). Various immune reactions have been seen, and these responses may
change based on the species and the pathogen delivery system/study vaccine used.10
Other ig isotypes have had comparable outcomes. The IgH V domain is known to
rearrange in teleost B cells either to Dτ-Jτ-Cτ/ζ to encode a / chain or to Dμ/δ-Jμ/δ-CμCδ encode μ and δ chains. Similar to IgD, different teleost species had variable numbers
of / domains.
10. GALT- Gut Associated Lymphoid Tissue
The greatest portion of the digestive tract, the gut, is directly connected to the outside
environment and may serve as a major entry point for pathogens in both mammals and
teleost fish. GALT functions as a local immune response environment for pathogen
defense in teleost fish and is a critical part of the mucosal immune system. Despite
differences in gut structure amongst teleost species, there are generally three primary
parts to the gut. The first segment's enterocytes serve as cells that absorb dietary protein.
The uptake of macromolecules and enterocytes is mediated by the second segment.
The GALT varies remarkably amongst vertebrate groups. For instance, unlike
mammals, chickens have caecal tonsils, and even among mammals, the GALT of
chickens show a significant anatomical variability. In general, both dispersed and
organized lymphoid tissue comprises the GALT of higher vertebrates. Fish, on the other
hand, do not have a well-organized GALT, and as a result, they do not have peyer's
patches (PP) or mesenteric lymph nodes (MLNs). Although lymphoid accumulations
were found in the lamina propria of the amphibian urodele Pleurodeles waltlii, the
presence of PP or MLN in amphibians has not yet been proven. In fish, lymphoid cells
can be found along the alimentary canal in a dispersed fashion. Nevertheless, the LP
and IEL compartments are recognized. Recent research has accumulated updated
information on the teleost fish GALT, including a description of all immune cell types
found there and further information about several cartilaginous and bony fish. In
general, teleost gut LP contains a wide range of immune cells, including macrophages,
granulocytes, lymphocytes, and plasma cells, whereas the IEL compartment is
primarily made up of T cells and a small number of B cells. The halibut (Hippoglossus
hippoglossus) is an exception, where the epithelium of the second segment of the
intestine contains a wide variety of leukocytes.
Similar to how the mammalian GI tract is divided into separate segments, the fish GI
tract also exhibits immunological variations. The majority of our understanding in that
field focuses on how particles are absorbed differently in the anterior gut (also known
as the foregut or first segment) and the posterior gut (hindgut or second segment). In
20 | P a g e
cod (Gadus morhua L.), there are obvious immunological variations between the
rectum and the second segment of the gut. However, the geographic breakdown of the
populations of gut immune cells in teleosts is far from comprehensive. The distribution
of Igs classes and B cell subsets in various regions of the GI tract is not well understood.
It is important to note that the pH levels along the fish's GI system alter significantly.
11. Gill Associated Lymphoid Tissue (GIALT)
Teleost fish has four pairs of gill arches made up of several gill filaments providing an
incredibly effective approach to expand the surface area where oxygen can be taken in
from the water. The gills also have other roles besides breathing, such as
osmoregulation, pH balance regulation, ammonia excretion, hormone regulation, and
detoxification. Notably, due to their constant exposure to water, teleosts' gills are
constantly tested by infections and environmental pollutants/toxins, both of which drive
the teleost GIALT to mount an immune response. Additionally, multiple studies have
shown that numerous innate and adaptive immune molecules or cells engaged in
immune-related pathways, such as Igs and antibody-secreting cells, are present in
teleost gills.11
Various fish species' GIALTs have been found to contain small and big lymphocytes,
neutrophils, eosinophilic granulocytes, and cells that secrete antibodies.
Lipopolysaccharide (LPS) and phytohemagglutinin (PHA) were used to induce
mitogenic responses in gill cell suspensions from the dab (Limanda limanda), which
revealed a lack of B-cells and a predominance of T-cells. A very thin epithelium that is
supported by pillar cells creates secondary lamellae. Erythrocytes can now pass via a
capillary gap created by this. Since, lymphoid cells are uncommon in this region. It has
been proposed that interlamellar veins and mammalian lymphatic capillaries are
physiologically, if not embryologically, identical due to their obvious physical
similarities. It's interesting to note that Amphyoxus, a basal chordate, is also known to
have gill-associated lymphoid tissue.
Salmonids have an interbranchial lymphoid tissue (ILT) in addition to the lymphoid
tissue present within the gill lamellae. This lymphoid tissue is organized similarly to
the thymus: an epithelial covering covers it, and trabecular walls run through it.
Therefore, at least in salmonids, GIALT is composed of organised lymphoid regions
between gill arches as well as distributed leukocytes within the lamellar epithelium.
It has been demonstrated that the region around the gill cover produces more mucus
than any other spot on the body. Additionally, the related microbial population on fish
gills is less varied than that on the skin in the case of gibel carp (Carassius auratus
gibelio) and bluntnose black bream (Megalobrama amblycephala).
21 | P a g e
12. Fish Stressors
Various stresses are applied to fish during the production process. Even if we are able to prevent
some of them, particularly environmental ones like abrupt temperature changes or poor water
quality, and maintain the proper culture densities, other ones, particularly those linked to
management methods, are more challenging to stop. Withinfarms, everyday procedures like tank
cleaning, fish classification, vaccination, and transportation are vital, but they can cause animals a
lot of stress and impair their abilityto produce. The immunological response of fish during stressful
times can be enhanced by immunostimulant drugs. They are products built on compounds with
active botanicalorigins, such as immunostimulant pronutrients, which can be added to feed to
enhancethe immune system's innate and adaptive responses and lessen the negative consequences
of stress. Because the immune response and disease resistance decline, stress, especially chronic
stress, is known to have a deleterious impact on a variety of productive species, including
aquaculture species.
Fish stressors can be categorized into three main categories: environmental, social, and
reproductive, and physical or management.
Sudden fluctuations in temperature and oxygen levels, variations in water quality, and
dense populations of cultures are all examples of environmental influences. In cages or
ponds, social elements allude to the construction of a dominance order. For aquaculture
species, the reproductive season has also shown to be a very stressful time, hence
production systems should definitely take this into consideration. Fish immune systems
can be impacted by physical or handling variables, including animal transportation—
both inside and between facilities—as well as activities like tank cleaning,
immunization, and animal classification.
The three stages of the stress state are the initial stage, the endurance stage, and the
exhaustion stage. The hypothalamus-hypophysis axis is activated when an animal is
exposed to a stressful substance because distinct signals are conveyed from the sensitive
organs to the central nervous system. These aquatic creatures' kidneys contain
chromaffin cells, which secrete catecholamines, and inter renal cells, which secrete
cortisol. When this neural axis is activated, both of these substances are released. The
immune system's response to stress is mediated by catecholamines and cortisol. By
raising heart rate, blood flow, energy availability (glucose levels), and boosting the
immunological response, these chemicals are secreted to get the animal ready for a
potential challenge. If the stressor doesn't go away, animals move into the resistance
phase, where alterations in metabolism and enzymatic secretion allow them to keep the
alert state active. If the stressful stimuli persist for a long time and the animal cannot
maintain this stage, the immune response declines, which is known as the exhaustion
stage. The animal can no longer produce large amounts of the chemicals needed for
defense systems at this point (lysozyme, complement system, IgM, leukocytes, etc.).
22 | P a g e
13. Routes of Vaccine Administration in fish
There are three different routes to administer vaccine in fishesA. Immersion
A quick and effective way to immunize fish against infection is with this kind of vaccine. Live
suspensions of attenuated bacteria, vector vaccines, or live bacterial vaccines are the vaccines
used for immersion type vaccination. The immersion category of
commercially available
vaccinations includes live and formalin-inactivated bacterialvaccines. Fish are briefly submerged
in a diluted vaccine solution before being put intothe culture unit, e.g., in pond or net pen.12
Both the dip and bath vaccination methods can be used for immersion immunization.
Fish are immersed in a solution with a high concentration of vaccine for typically 30
seconds while receiving a dip vaccination. In contrast, during bath vaccination, fish are
exposed to a lower vaccine concentration for a longer period of time, typically one to
several hours. The dip immersion method of vaccination is preferred because it can
quickly immunize a larger number of fish. Immersion vaccination is frequently used
and advised, especially for smaller fishes, for fry weighing between 0.5 and 5 g since it
is efficient, quick, practical, less stressful, and affordable. It also requires less handling
stress and provides protection for a long time. For antigen uptake in immersion
vaccination, a number of tools have been developed, including multiple puncture
instruments, hyperosmotic dips, and ultrasound-mediated uptake devices.
The immersion method provides short term immunity. Immunity takes between three
and twelve months to develop. For the culture of some fish species, this is not ideal.
Consequently, a booster dose is needed. Due to a number of issues, including a longer
time period, higher expense, stress, and difficulty using various immune stimulating
drugs and adjuvants, this procedure cannot be used with larger fish.
Advantages: It can be administered very easily and it is economically.
Disadvantages: The protection effect does not last long, and there are chances to get
vaccinated for second time.
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Image 2
[Source:https://www.vetcare.gr/ARTPRES/Fish_Vaccination_Strategies.htm]
24 | P a g e
B. Injection
When injectable vaccinations are administered, just a little amount of antigen
can be directly injected into the fishes by intraperitoneal (IP) or intramuscular
(IM) delivery techniques. When compared to the immersion procedure, the
duration of protection is longer in this strategy. In addition, injections allow for
the concentration and distribution of substances such as bacterial antigens,
bacterial cells, adjuvants, and transporters that are not achievable with other
vaccine delivery methods. Since intraperitoneal injection is the most effective
and prolific method of immunizing fish, it has been used to administer the
majority of contemporary vaccines. Adjuvants, particularly oil adjuvants, are
utilized in IP injection because they provide better protection than the
immersion approach. The vaccination is administered intraperitoneally to
sedated fish. Fish are commercially vaccinated using injection guns that can be
manually or automatically operated. As a result, each operator may inject 1000–
2000 fish in a single hour. The size of the fish determines how much is injected.
The IM delivery approach, which involves manually injecting fish with a needle
or, alternatively, using a device like compressed air, is the recommended way
for DNA immunisation of fish. Fish growers favor intramuscular vaccination
over other methods. One drawback of this approach is that the stress brought on
by the vaccine results in mortality. Longer-lasting protection is provided via
intramuscular immunization. Typically, 0.1 or 0.2 ml are injected per fish,
providing protection throughout the production cycle. The brief reduction in
feeding, adhesion formation, unintentional gut puncture, laboratory-intensive
nature, and sores that may develop at the injection site, which might serve as an
entrance point for secondary infections are additional drawbacks of
immunization by injection. Moreover, this procedure is impractical for fish
weighing less than 5 g. The biggest disadvantage of injectable vaccines is that
it is not financially feasible to administer them multiple times during the fish
production cycle. Additionally, due of their immature immune systems, they
cannot be given to fish during the first phases of development.13
Advantages: Low volume is sufficient; it provides long term protection unlike
immersion.
Disadvantages: It is a labor intense technique only skilled person can do it and
minimum size is required.
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Image 4
[Source: https://tvmnews.mt/en/news/maltese-company-produces-vaccine-whichcan-lead-to-reducing-the-amount-of-fish-which-die-globally/]
C. Oral Delivery
One technique for immunizing fish is the oral vaccination approach, in which
the vaccine is initially added to the meal before being given to the fish. The oral
immunization method, is economical, particularly when it comes to larger fish.
The efficacy of an oral vaccine is lower than those of immersion and injection.
Adding the oral vaccine to the feed or putting it on top of the meal are two ways
to administer it. The vaccine can be bio-encapsulated, combined with the feed,
or sprayed on top of it. Special consideration must be given to the antigens that
will be included in the diet. Vaccines must be top-dressed on the feed to avoid
antigen leaching from the pellet. The antigen delivery in fish feed has some
advantages, including low stress, cost effectiveness, simplicity, and safe
administration at all stages to fish of various sizes. Different microencapsulation techniques are analyzed and tested for sensitive antigens. The
vaccination suspension is incubated with rotifers, Artemia nauplii, or copepods
before being fed to the fry. They are living, non-selective filter feeders that will
collect the antigen in their gastrointestinal system before changing into live
microcapsules. In order to improve immunity against several chronic endemic
diseases, oral vaccines can also be given as a booster shot after primary
immunization. In these diseases, humoral immune responses, as opposed to
26 | P a g e
cellular and innate immunological responses, are primarily responsible for
immunity.14
Advantages: They are easily administered and gives minimal stress on fishes.
Based on the above advantages and disadvantages of different routes, we have
taken oral delivery as the route of administration in this project.
Oral vaccination is the most important type of aquaculture vaccination. Oral
vaccinations can be given by bio-encapsulating them in live feeds like rotifers,
daphnia, and artemia or by enriching prepared feed. Oral vaccines can also be
given by encapsulating them in a wide variety of polymers. Oral vaccinations
are created in formulated feeds either by spray coating the antigen over the feed
or by including it into the feed during production for co-processing. Antigen
administration via oral ways has the benefits of being stress-free and easy to
provide to numerous fish at once.15
Image 5
[Source: https://link.springer.com/article/10.1007/s10499-022-01004-4.]
14. Types of Vaccines
14.1 Attenuated (Live) Vaccines
Attenuated vaccines are developed when disease causing pathogen are weakened
genetically or chemically to acquire humoral and cellular immunity. These vaccines are
fairly strong because they simulate infection by the local pathogens and trigger
powerful immune reactions. AQUAVAC-ESC for catfish, Renogen employing
Renibacterium salmoninarum antigen, and Koi Herpesvirus 3 (KHV 3) for carp in Israel
are a few examples of commercialised attenuated vaccines. However, it is claimed that
some live attenuated vaccinations may cause pathogen features to change or that the
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organisms present in these vaccines may revert to their original state. Consequently, it
is crucial to guarantee the safety of a live attenuated vaccination.16
14.2 Inactivated Vaccines
The bacterium that causes the disease is used in killed form to create inactivated
vaccines. Then, as a result of our immune system, antibodies are produced against the
pathogen. Consequently, the immune system will start producing antibodies against the
infection the next time it enters the body because the preceding pathogen's memory is
retained. Inactivated vaccines are less dangerous and more affordable. When compared
to other vaccination forms, inactivated vaccines may generate lower or shorter-lived
immunity because they do not survive in the environment or in the vaccinated fish. Due
to the inadequate activation of cellular immunity within the fish species, weak
immunogenicity of inactivated vaccines may require the use of adjuvants or numerous
booster vaccinations to generate protective immunity. Phagocytic antigen presentation
cells (APCs) launch the process of clearing out activated immune cells and initiating a
humoral immune response once they have been supplied. Immunosuppressive
passenger antigens, immune-enhancing adjuvant-caused toxicities, decreased
immunogenicity due to protein denaturation, and systemic responses to specific
adjuvants are all drawbacks of inactivated vaccines.17
As mentioned above, in order to immunize trout, Oncorhynchus, Duff et al. (1942)
undertook research and created the first inactivated vaccine, containing Aeromonas
salmonicida. The first commercially available inactivated fish vaccine was for Yersinia
ruckeri, which causes enteric red mouth disease. This was followed by the creation of
immersion vaccines for salmon and trout vibriosis (caused by Vibrio spp.). The same
method was used to develop the first salmonid vaccines, which were given via
immersion, to inactivate the germs that caused infections in Atlantic salmon (Salmo
salar).
14.3 Recombinant Vaccines
Recombinant vaccination is made to produce plenty of a certain substance, usually a
pure pathogen part that successfully elicits an immune response. As opposed to
inactivated vaccines, which use the entire pathogen, subunit vaccine has protein, toxin
or carbohydrate to promote a healthy immune system. The usage of recombinant
vaccines has significantly increased as a result of recent developments in genetic
engineering and expression systems. Using genetic engineering Escherichia coli can be
tailored to have the genes for producing antigens, in enormous amounts.18
14.4 Subunit Vaccines
Subunit vaccines cannot replicate in the host, there is no risk of pathogenicity to the
host or non-target species. Subunit vaccines benefit from employing only antigenic
components for vaccination. It is possible to make subunit vaccines in a highly defined
condition, and they can be freeze-dried to facilitate non-refrigerated transit and storage.
28 | P a g e
Subunit vaccines can target immune responses against certain microbial determinants
and allow the insertion of unnatural components.19
Subunit vaccines offer many positive attributes, but frequently, they are less effective
at eliciting a strong immune response than dead or live whole cell preparations. This is
because there aren't many antigens replicated or exposed to in order to represent a full
cell vaccination, and because there aren't many components that are represented and
capable of triggering an immune system. Since the streamlined (synthetic, recombinant,
and/or highly purified) antigenic components of the vaccine typically lack
immunogenicity, some subunit vaccines rely on efficient adjuvants to elicit the
appropriate immunity, and may even require multiple booster immunizations to ensure
long-term protective immunity.20
14.5 DNA Vaccines
There is no need for the laborious and difficult steps necessary to obtain a pure antigen
from an expression system since DNA vaccination leverages the transcriptional
machinery of the vaccine recipient as an expression system. As a result, DNA vaccines
are being considered as a novel approach in vaccine development for fish, animals, and
humans. Injecting the target DNA (often a plasmid) into the body results in the
production of a target antigen that resembles the native form and can elicit an immune
response. Thus, a sequence encoding the virus is included in the structures used as DNA
vaccines. Recent advances in DNA synthesis technology have led to the development
of plasmids that can express many antigens at once. The DNA vaccine can increase
both humoral and cellular immunity. In addition, it was proposed that the vaccinated
DNA randomly incorporates into host chromosomes, possibly having negative effects.
However, recent research findings suggest that the DNA vaccines can be used in fish.
A few DNA vaccines are commercialized, which include the salmon IHNV vaccine
from Aqua Health Ltd. which is accepted in the USA and Canada.21 In a DNA vaccine
the gene of interest is flanked by promoter and terminator regions that enhance
expression within eukaryotic cells, and plasmid is multiplied within bacterial cells22.
The cellular and humoral immune systems can be highly activated by DNA vaccine.
15. Commercially Important Fishes
Fish Name Biological Name Nutrients Content Picture
Cod
Gadus morhua
Cod livers are
processed to make
cod liver oil, an
important source
of vitamin A,
vitamin
D,
vitamin E and
omega-3
fatty
acids (EPA and
DHA).
29 | P a g e
Grass Carp Ctenopharyngod Vitamin-E,
on idella
Vitamin-B12,
thiamin,
and
riboflavin, Iodine,
selenium,
phosphorus,
calcium,
zinc,
potassium,
and
magnesium.
Silver
Hypophthalmich vitamin-E,
Carp
thys molitrix
vitamin-B12,
thiamin,
and
riboflavin, Iodine,
selenium,
phosphorus,
calcium,
zinc,
potassium,
and
magnesium.
Cyprinus carpio It has essential
Common
Carp
fatty
acids,
protein, minerals
and
fat-soluble
vitamins
like
vitamin A, E and
D. Carp fish is
moderately high;
100 g holds 127
calories and 17.8
g/100 g (32% of
RDI) of protein.
Nile tilapia Oreochromis
Tilapia is packed
niloticus
with vitamins and
minerals
like
choline, niacin,
vitamin
B12,
vitamin
D,
selenium,
and
phosphorus. It is
also a good source
of omega-3 fatty
acids
Shrimp is rich in
Whiteleg
Penaeus
Pritein, selenium,
Shrimp
vannamei
choline,
and
vitamin B12. It
also contains good
amounts of niacin,
zinc, vitamin E,
and vitamin B6. It
30 | P a g e
ia
also
good
source of iodine
It is rich in
vitamins B3, B5,
B6, B12, vitamin
D, vitamin E and
selenium.
Atlantic
salmon
Salmo salar
Rohu
Labeo rohita
Omega 3 fatty
acids and vitamins
A, B, and C
Yellowfin
tuna
Thunnus
albacares
Japanese
anchovy
Engraulis
japonicus
selenium, zinc,
manganese and
vitamin C, which
help to boost
immunity.
Omega-3
fatty
acids.
Niacin.
Vitamin
B12.
Calcium.
Longtail
tuna
Thunnus tonggol Manganese,
selenium, vitamin
C and zinc
Giant tiger Penaeus
prawn
monodon
Vitamin
D
,
Calcium, Iron and
Potassium
Atlantic
horse
mackerel
Trachurus
trachurus
vitamins A, B3,
B6, B9 and B12,
and vitamin D
Albacore
Thunnus
alalunga
selenium, Vitamin
B3
(niacin),
Vitamin B12,
Vitamin
B6,
protein,
phosphorus,
Vitamin D and
potassium.
31 | P a g e
American Placopecten
sea scallop magellanicus
Omega-3
fatty
acids,
Vitamin
B12,
Calcium,
Iron,
Magnesium,
Phosphorous,
Potassium, Zinc,
Copper, Selenium
Manganese,
Vitamin
B12,
Vitamin
B6,
Vitamin
K,
Vitamin
A,
Vitamin
C,
Vitamin
E,
Selenium, Zinc,
Magnesium and
Calcium.
vitamin
D,
calcium, vitamin
B12, and protein
Bombay
Duck
Harpadon
nehereus
Madeiran
sardinella
Sardinella
maderensis
Bonga shad
Ethmalosa
fimbriata
Vitamin
A,
Vitamin
E,
Vitamin
K,
Thiamin,
Riboflavin,
Niacin,
Vitamin
B6,
Folate, Vitamin
B12, Pantothenic
Acid,
Choline,
Calcium,
Goldstripe
sardinella
Sardinella
gibbosa
Calcium, vitamin
B12, vitamin D,
vitamin
E,
magnesium,
potassium, and zinc
Bigeye tuna
Thunnus obesus
Pacific cod
Gadus
macrocephalus
B-Complex
vitamins,
Vitamins A and D
as well as iron,
selenium
and
phosphorus.
Vitamin
B12.,
Niacin.
Phosphorus.,
Selenium.
32 | P a g e
Black carp
Mylopharyngod
on piceus
Omega-3
fatty
acids,
Choline,
Iodine
vitamin A, E and
D.
Indian
sardine
Sardinella
longiceps
selenium,
and
vitamin B-12
Chanos chanos
calcium
(Ca),
magnesium (Mg),
sodium (Na) and
potassium
(K).
iron, zinc, copper
(Cu)
and
manganese (Mn),
and the main
vitamins present
include A, B1 and
B12.
vitamin
A,
vitamin
C,
vitamin D
Milkfish
oil
Big head Hypophthalmich
carp
thys nobilis
Catla
Catla catla
Zinc, potassium,
iodine, vitamins,
selenium,
and
Vitamin A.
Crucian
carp
Carassius
carassius
Calcium,
Potassium,
Vitamin
Vitamin
Sodium.
Atlantic
herring
Clupea
harengus
C,
A,
Vitamin
B12,
Vitamin B9.
Source of table: Wikipedia
33 | P a g e
16. Commercialized Vaccines
There were only two commercialized vaccines in 1980s but now there are 26
commercialized vaccines worldwide for fishes. The first commercially available
vaccination was released in 1976.23 The Yersinia ruckeri vaccine, which was developed
to treat enteric red mouth disease, was the first commercially available vaccination for
aquaculture.24
There are numerous fish species for which vaccines are available, including tilapia
(Oreochromis niloticus/mossambicus), amberjack (Seriola dumerili), yellowtail
(Seriola quinqueradiata), and catfish (Ictalurus punctatus), as well as Vietnamese
catfish (Pangasionodon hypophthalmus), sea bass (Dicentrarchus labrax), and sea
bream. Although live attenuated vaccines are authorised for use in catfish in the USA,
the majority are whole cell vaccines that have been formalin-killed.
When using commercial vaccines on fish, there are a number of important factors to
take into account, including the type of fish, immune system status, life cycle and
production, when diseases occur, farming technology (handling, mechanization, etc.),
environment (such as temperature, salinity), stress factors, nutrition, and financial
advantages. The Responsible Use of Medicines in Agriculture Alliance offers
recommendations for the administration of fish vaccines. Most commercial vaccines
are delivered via intraperitoneal injection and contain adjuvants.25
17. Aim Of Thesis
Oral Vaccination is the safest method and vaccine can be delivered to the animals via
feed. Thus oral vaccination would be a painless procedure to the fish and easy for
farmers as well. To achieve slow release of antigen over a longer period, safe
carriers/adjuvants are recommended. The proposed work the aim is to prepare benign
Hydroxyapatite nanoparticles, coating them with antigen and to be used for oral
immunization of fish.
18. Hydroxyapatite Nanoparticle
Hydroxyapatite nanoparticles (HAp Nps) have been successfully used in numerous
biomedical applications during the past ten years. The synthesis and characterization of
HAp Nps using several studies, including TEM, FTIR, and XRD, are the main topics
of the current study. According to the TEM findings, the particles were rod-shaped and
ranged in size from 20 to 100 nm. The O-H group, amine group, calcium and phosphate
group, as well as other groups, were all visible in the FTIR spectrum.26
19. Instruments
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19.1 Centrifuge
Principle: The centrifuge operates on the sedimentation principle, which states that
under the influence of gravitational force (g-force), things separate based on their
densities. It is possible to separate things using isopycnic, ultrafiltration, density
gradient, phase separation, and pelleting techniques.
Image 6
19.2 Nanodrop
Principle: The sample retention mechanism used by the NanoDrop
microvolume technology depends on the surface tension characteristics of the
sample being examined to create a liquid column. For optimal column
formation, the sample must make contact with both the top and lower optical
measuring surfaces.
Image 7
19.3 Gel Electrophoresis
35 | P a g e
Principle: On application of electric charge, each molecule having different size
and charge will move through the gel at different speeds. The porous gel used
in this technique acts as a molecular sieve that separates bigger molecules from
the smaller ones. Smaller molecules move faster across the gel while the bulkier
ones are left behind. The mobility of the particles is also controlled by their
individual electric charge. Two oppositely charged electrodes that are part of
the system pull molecules of towards them on the basis of their charge.
Image 8
19.4 Lyophoilizer
Principle: Lyophilization or freeze drying is a process in which water isremoved
from a product after it is frozen and placed under a vacuum, allowing the ice to
change directly from solid to vapor without passing through a liquid phase. The
process consists of three separate, unique, and interdependent processes;
freezing, primary drying (sublimation), and secondary drying.27
36 | P a g e
Image 9
19.5 Sonication
In order to stir up particles in liquids, sonicators are high-frequency (20 kHz)
devices. Many operations, including mixing, cleaning, degassing, cell
disruption, and sample preparation, are made easier by the use of these devices.
Image 10
19.6 Zeta Potential
Zeta potential determination is an important method of characterising
nanocrystals that may be used to calculate the surface charge and comprehend
the physical stability of nanosuspensions.28 To study interactions between
colloid and electrolyte, one uses the zeta potential. The essential idea is that
counter-ions, which have an oppositely charged surface, are attracted, whereas
similarly charged particles are repelled. The Zetasizer equipment is used to
measure the zeta potential.
37 | P a g e
Image 11
19.7 NTA
The Nanoparticle Tracking Analysis (NTA) method uses Brownian motion and
light scattering properties to determine the distribution of nanoparticle sizes in
materials suspended in liquid. A sample chamber that is lit by a laser beam with
a unique form is loaded with particles suspended in liquid. The Stokes Einstein
equation is used by the Nanoparticle Tracking Analysis (NTA) program to
compute the hydrodynamic diameters of many particles that are analysed
simultaneously and individually (particle-by-particle).
19.8 Gel Imager
The idea behind an automatic gel imaging system is that when ultraviolet light
(with a wavelength of 254nm–302nm) is focused on a gel that has been dyed
with ethidium bromide, the dye intercalates with the groove in the DNA,
becomes excited, and generates fluorescent light.
38 | P a g e
Image 12
19.9 DLS
The method of dynamic light scattering, sometimes referred to as photon
correlation spectroscopy or quasi-elastic light scattering, principally detects the
Brownian motion of macromolecules in solution brought on by bombardment
from solvent molecules and links this motion to the size (or D) of particles29
When laser light strikes macromolecules in a dynamic light-scattering
apparatus, the incident light scatters in all directions, and a detector measures
the strength of the scattering. Given that the macromolecules are constantly
moving in solution, the monochromatic incident light will experience a
phenomenon known as Doppler widening.30One of two phases will emerge from
the scattered light: mutually destructive phases that cancel each other out, or
mutually constructive phases that result in a measurable signal.
19.10 SEM
The SEM is a system that forms a picture using electrons rather than light,
resulting in a significantly magnified image. An electron gun at the top of the
microscope produces an electron beam. The microscope is maintained in a
vacuum and the electron beam travels through it in a vertical path. The beam is
focused downward towards the sample as it passes via electromagnetic fields
and lenses. Electrons and X-rays are ejected from the sample after the beam
strikes it.
39 | P a g e
Methodology
A. Synthesis of HAP Nanoparticles
Dissolve 2M of calcium chloride in DMSO for 30 min. add orthophosphoric
acid dropwise to the solution. Maintain Ca:P atomic ratio at 1.67. add stabilizing
agent here, we have used acetyl acetone. Again, stir it for 1 hour. By using liquid
ammonia adjust the pH to 10. Continue stirring until the complete gelation. Add
0.1 wt% arginine (R) + 0.3wt % glucose (G) + 0.05 wt % polyethylene glycol
(PEG). Stir for 2 hours, to get the final product wash it by ethanol.31
B. Physiochemical characterization of Nanoparticle
Characterization of HAP-NP were carried out by Nanoparticle Tracking Device
(NTA), Dynamic Light Scattering (DLS), Scanning Electron Microscope
(SEM) and Fourier Transform Infrared (FTIR). By Zeta potential analyzer, the
zeta potential of HAP NPs was analyzed. By combining DI water with the NP
suspension, the samples were made. Fourier transform infrared spectroscopy
(FTIR) was used to determine the functional groups that were present in the
produced compounds over the 4000-450 cm-1 range.
C. Protein estimation by Biuret
A colorimetric method designed specifically for proteins and peptides is the biuret
method. Copper salts in an alkaline solution combine with molecules that have two or
more peptide bonds [–CO-NH-] to generate a purple complex. The amount of peptide
bonds reacting and therefore the quantity of protein molecules in the reaction system
are reflected in the absorbance that results. As a result, the biuret reaction with proteins
is measured by spectrometer at 540nm.
D. Nanoparticle protein (BSA) adsorption assay
Adsorption of albumin on HAP NPs were studied. By mixing (1mg) of HAP
NPs with (100μL) of (2.25g/dL) albumin solution and made up the solution to
1000μL with distilled water. And then was sonicated for 30 min.
E. Characterization of HAP Nanoparticle BSA complex
There is no reliable method for determining the surface charge of microscopic particles
in liquid. The standard procedure is to locate a particle's electric potential anywhere in
the diffuse layer, far from the particle surface. The sliding or shear plane is the term used
to describe this area in relation to particle movement in liquid. Zeta potential, a crucial
characteristic for colloids or nanoparticles in suspension, is the potential measured at this
plane. Its worth is highly correlated with particle surface shape and suspension stability.
As a result, surface adsorption research and studies of product stability make extensive
use of it.32 By zeta potential analyzer, HAP-NP and BSA protein complex was analyzed.
F. Nanoparticle BSA release study assay
Studied the BSA Conjugate at different temperature (25, 30, 35 and 40 °C)
these conjugates were kept for 30 min Also, at different pH (7, 8, 9) studied
the protein concentration using BCA assay.
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Results And Discussion
A. HAP NPs synthesis
HAP NPs were synthesized according to the protocol. 1mg of hydroxyapatite
nanoparticles were synthesized.
B. Protein estimation by biuret method
For Protein concentration (200,400,600,800,1000 µg/mL) the absorbance was
measure at 540nm. And protein estimation graph was plotted.
concentration
(µg/mL)
Absorbance
(540 nm)
200
0.016
400
0.038
600
0.062
800
0.09
1000
0.116
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Image 13; Protein estimation graph, Absorbance(540nm) Vs Protein
concentration(μg/mL).
C. Zeta Potential and Characteristic of Nanoparticles
Physiochemical characterization of nanoparticles was found out by NTA, FITR, DLS
and SEM. The Average diameter of the HAP Nanoparticles was measured using
Nanoparticles Tracking Analysis was measured between 50-70nm (Image14 A), Using
DLS zeta potential was measure and it came out to be -1.56 (Image14 B). when
characteristics of HAP Nanoparticles were measured by FITR spectrum (Image14 C),
it showed that HAP double peaks near 600cm-1 are due to the bending nodes of P-O
bonds in phosphate group with contribution from the OH group of apatite group at about
3600-3200cm-1 (). Analyzing with SEM, the Nanoparticle out to be round and size was
approx. 200nm (Image14 D)
Refer to Image
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Image 14: Physiochemical characterization of HAP nanoparticles ANanoparticle Tracking Analysis; B- Zeta Potential; C- FTIR Spectrum; D- SEM
D. SEM of HAP Nanoparticles after Protein Adsorption Assay
Bovine serum albumin was chosen as the standard protein for the experiment. SEM
was performed of HAP and BSA conjugate. And from the image we can conclude that
BSA has not affected the morphology of HAP. The Attached image15 gives us the
result for SEM and protein adsorption assay.
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Image 15; SEM image of HAP after protein adsorption assay
E. Protein Adsorption Efficiency
The attached graph shows BSA- HAP adsorption efficiency at different concentration
(5,10,15,20,25). Phosphate buffered saline, Citrate and Carbonate buffers were used to
resuspend the nanoparticles. The BSA adsorption efficiency was greatest in citrate
buffer at all concentrations tested.
BSA-HAP Adsorption Efficiency
100
90
% Adsorption efficiency
80
70
60
50
Ads. Eff. In PBS
40
Ads. Eff. In citrate
30
Ads. Eff. In carbonate
20
10
10
15
20
25
HAP conc. (mg/ml)
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Image 16; Adsorption Efficiency of Bovine serum albumin and Hydroxyapatite
Nanoparticle in PBS, Citrate, and carbonate buffer.
F. Study of stability of conjugate at different temperature and pH
After investigating the conjugate, we can conclude that the conjugate is stable at the
given temperature (25, 30, 35, 40 °c) and pH (7, 8, 9) (Image 17 A and B)
Fig 17 A; At different temperature (25, 30, 35, 40); B: At different Temperature
Conclusion
Here, the concept of vaccination was reviewed. Information on the different types of
vaccines, modes of vaccine delivery was presented. The immune response post
vaccination was accounted. Need for vaccination in fish was emphasized with specific
development in the field. To generate oral vaccines for use in aquaculture,
hydroxyapatite nanoparticles were proposed.
As part of experimental work, hydroxyapatite nanoparticles were synthesized and
characterized. As a model antigen, BSA was loaded on the HAP. This concept can be
extended to the purified antigens. The field of fish vaccination has made incredible
strides recently. The majority of the products are first-generation vaccines, but scientific
research and invaluable practical experience will provide a strong foundation for the
creation of better products that will support the economic, social, and environmental
sustainability of global aquaculture.
References
1
Plotkin, S. L., & Plotkin, S. A. (2012). A short history of vaccination. Vaccines, 4.
Ratajczak, W., Niedźwiedzka-Rystwej, P., Tokarz-Deptuła, B., & Deptuła, W. (2018).
Immunological memory cells. Central-European journal of immunology, 43(2), 194–203.
2
45 | P a g e
3
Salk J, Salk D. Control of influenza and poliomyelitis with killed virus vaccines. Science
1977;195:834e47.
4
Gudding R., Goodrich T. The History of fish vaccination. In: Gudding R., Lillehaug A.,
Evensen Ø., editors. Fish Vaccination. 1st ed. John. Wiley & Sons, Inc.; New. York, NY, USA:
2014. pp. 1–11.
5
Heymann, D. L., & Aylward, R. B. (2006). Mass vaccination: when and why. Mass
Vaccination: Global Aspects—Progress and Obstacles, 1-16.
6
Lombard, M., Pastoret, P. P., & Moulin, A. M. (2007). A brief history of vaccines and
vaccination. Revue Scientifique et Technique-Office International des Epizooties, 26(1), 29-48.
7
Xu Z, Parra D, Gómeza D, Salinas I, Zhang YL, Jørgensen L, et al. Teleost skin, an ancient
mucosal surface that elicits gut-like immune responses. Proc Natl Acad Sci U S A (2013)
110:13097–102. doi: 10.1073/pnas.1304319110
8
. Parra D, Reyes-Lopez FE, Tort L. Mucosal Immunity and B Cells in Teleosts: Effect of
Vaccination and Stress. Front Immunol (2015) 6:354
9
Yu, Y., Wang, Q., Huang, Z., Ding, L., & Xu, Z. (2020). Immunoglobulins, Mucosal
Immunity and Vaccination in Teleost Fish. Frontiers in Immunology, 11.
. Danilova N, Bussmann J, Jekosch K, Steiner LA. The immunoglobulin heavy-chain locus in
zebrafish: identification and expression of a previously unknown isotype, immunoglobulin Z. Nat
Immunol (2005) 6:295–302. doi: 10.1038/ni1166
10
11
Yu, Y., Wang, Q., Huang, Z., Ding, L., & Xu, Z. (2020). Immunoglobulins, mucosal
immunity and vaccination in teleost fish. Frontiers in immunology, 11, 567941.
12
Adams, A. (2019). Progress, challenges and opportunities in fish vaccine development.
Fish & shellfish immunology, 90, 210-214.
13
Ma, J., Bruce, T. J., Jones, E. M., & Cain, K. D. (2019). A review of fish vaccine
development strategies: Conventional methods and modern biotechnological
approaches. Microorganisms, 7(11), 569.
14
Duff DCB. The oral immunization of trout against Bacterium salmonicida. J Immunol
1942;44:87e94
15
Mutoloki S, Munang’andu HM, Evensen Ø (2015) Oral vaccination of fish - antigen
preparations,
uptake,
and
immune
induction.
Front
Immunol
6:519.
https://doi.org/10.3389/fimmu.2015.00519).
16
Marsden, M. J., Vaughan, L. M., Fitzpatrick, R. M., Foster, T. J., & Secombes, C. J. (1998).
Potency testing of a live, genetically attenuated vaccine for salmonids. Vaccine, 16(11-12),
1087-1094.
17
Karlsen, M., Tingbø, T., Solbakk, I. T., Evensen, Ø., Furevik, A., & Aas-Eng, A. (2012).
Efficacy and safety of an inactivated vaccine against Salmonid alphavirus (family
Togaviridae). Vaccine, 30(38), 5688-5694.
18
Sun, Y., Liu, C. S., & Sun, L. (2010). Identification of an Edwardsiella tarda surface antigen
and analysis of its immunoprotective potential as a purified recombinant subunit vaccine and a
surface-anchored subunit vaccine expressed by a fish commensal strain. Vaccine, 28(40), 66036608.
19
[Hansson M., Nygren P.A.K., Stahl S. Design and production of recombinant subunit
vaccines. Biotechnol. Appl. Biochem. 2000;32:95–107. doi: 10.1042/BA20000034.].
20
. [Holten-Andersen. L., Doherty T.M., Korsholm K.S., Andersen P. Combination of the
cationic surfactant dimethyl dioctadecyl ammonium bromide and synthetic mycobacterial cord
factor as an efficient adjuvant for tuberculosis subunit vaccines. Infect. Immun. 2004;72:1617.
doi: 10.1128/IAI.72.3.1608-1617.2004.]
21
. [Hølvold, L. B., Myhr, A. I., & Dalmo, R. A. (2014). Strategies and hurdles using DNA
vaccines to fish. Veterinary research, 45, 1-11.]
46 | P a g e
22
Kurath G. Biotechnology and DNA vaccines for aquatic animals. Rev. Sci. Tech. Off. Int.
Des. Epizoot. 2008;27:175–196.
23
Ma, J., Bruce, T. J., Jones, E. M., & Cain, K. D. (2019). A Review of Fish Vaccine
Development Strategies: Conventional Methods and Modern Biotechnological Approaches.
Microorganisms, 7(11), 569.
24
Bullock GL, Anderson DP. Immunization against Yersinia ruckeri, cause of enteric red
mouth. In: de Kinkelin P, editor. Symposium on fish vaccination. Paris: OIE; 1984. p. 151e66.
25
Ma, J., Bruce, T. J., Jones, E. M., & Cain, K. D. (2019). A Review of Fish Vaccine
Development Strategies: Conventional Methods and Modern Biotechnological Approaches.
Microorganisms, 7(11), 569.
26
Jyotsna, & Vijayakumar, P. (2020). Synthesis and characterization of hydroxyapatite
nanoparticles and their cytotoxic effect on a fish vertebra derived cell line. Biocatalysis and
Agricultural Biotechnology, 101612.
27
Ravnik, J., Golobič, I., Sitar, A., Avanzo, M., Irman, Š., Kočevar, K., Cegnar, M.,
Zadraveca, M., Ramšak , M.,Hriberšek, M. (2018). Lyophilization model of mannitol water
solution in a laboratory scale lyophilizer. Journal of Drug Delivery Science and Technology,
45, 28–38.
28
Joseph, E., & Singhvi, G. (2019). Multifunctional nanocrystals for cancer therapy: a potential
nanocarrier. Nanomaterials for Drug Delivery and Therapy, 91–116.
29
Stetefeld, J., McKenna, S. A., & Patel, T. R. (2016). Dynamic light scattering: a practical
guide and applications in biomedical sciences. Biophysical reviews, 8(4), 409–427.
30
[Harding SE, Jumel K. Light scattering. In: Coligan JE, Dunn BM, Ploegh HL, Speicher
DW, Wingfield PT, editors. Current protocols in protein science. New York: John Wiley &
Sons, Inc.; 1998.
31
Deshmukh, K., Ramanan, S. R., & Kowshik, M. (2019). A novel method for genetic
transformation of C. albicans using modified-hydroxyapatite nanoparticles as a plasmid DNA
vehicle. Nanoscale Advances.
32
Xu, R. (2008). Progress in nanoparticles characterization: Sizing and zeta potential
measurement. Particuology, 6(2), 112–115.
47 | P a g e