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TRANSCRIPT
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Lecture 7: Immune Response of
Aquatic Organisms
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Preliminary Concepts Disease problems have grown proportionally with
the intensive or expansive culture of aquaculturespecies
Why?1) Increased stocking densities (lower profit margins)2) Infected carriers (largely broodstock)
3) Infected facilities (GMPs being followed?)
4) Poor nutrition (we are way behind)
5) Substandard water quality (traditional) Biggest problem: greater susceptibility via weakening of
resistance under intensive culture conditions
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The Immune Response For fish, response to a foreign agent is rather similar to
that of mammals; shrimp, very rudimentary
Response can be highly specific (a specific antibody for a
specific antigen) is known as the immune response. The immune system scans the body to identify any
substance (natural/synthetic or living/inert) that itconsiders foreign
Differentiates between self and non-self Works with several types of white blood cells, located
throughout the body, that work together in a highlyintegrated way
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Definitions
resistance: any type of barrier within the hostthat allows it to resist the pathogen
innate or natural immunity: attributed to
inherited ability to produce antibodies withoutstimulation by antigens
acquired immunity: host is stimulated bycontact with antigens
passive immunity: acquired through the useof antibodies from other animals (vaccination)
we will add another term today, tolerance
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ImmuneResponseSystem
Made up of two cellular systems: 1) cell-mediated immunity (Tcells) and 2) humoral antibody system (B cells)
Both work by identifying antigens (foreign proteins or
glycoproteins)
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Immune Response Sequence: 1
Begins when macrophageencounters this non-selfentity (e.g., virus):macrophage literallyeats the substance,
digests it and displayspieces of the invader onits surface. Thesepieces are antigens.
Meanwhile, other viral
particles are at work,infecting nearby hostcells.
Source: Cancer Research Institute
(2002)
www.cancerresearch.org/immhow.html
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Immune Response Sequence: 2
Antigenic fragments alerta specific type of Tlymphocyte (helperT) to begin
choreographed attackof intruder
Helper recognizes antigenparticles and binds tothe macrophage viaan antigen receptor
Helper T cells are uniqueto a specific antigen
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Immune Response Sequence: 3
This binding stimulatesproduction ofchemical substancessuch as interleukin-1
(IL-1), tumor necrosisfactor (TNF) bymacrophage
Helper T cells generatesinterleukin-2 andgamma interferon(IFN-y)
All substances facilitateintercellular
communication
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Astonishing Synchronization
TNF steps up production of IL-1, it alsocauses fever in homeotherms
TNF and IL-1 are cytokines (cellular)
IL-1 also causes fever but additionally formsimmune cell clusters and stimulates thehelper T cell to release IL-2
IL-2 causes T cells to release gamma
interferon which, in-turn, activatesmacrophages
IL-2 also instructs other helper T cells andkiller T cells to multiply
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Immune Response Sequence: 4
As mentioned IL-2 instructshelper Ts andkiller Tstomultiply
Proliferating helper Ts release
substances that cause Bcells (another type oflymphocyte) to multiply andproduce antibodies
Meanwhile, many invader cells
have been consumed bymacrophages, but otherdaughter viral particleshave escaped and areinfecting other cells
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Immune Response Sequence: 5
Killer T cells start shooting holesin the surface of infected hostcells
Antibodies released by B cells bindin a lock-and-key fashion toantigens on the surface ofinvaders that have escapedmacrophages (Ag-Abcomplex).
Makes it easier for macrophagesand special killer lymphocytes
to destroy unwelcomedentities.
Binding of antibodies with antigenssignals release of a bloodcomponent, complement, topuncture virus membrane
(death)
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Immune Response Sequence: 6
Finally, as the infection isbrought under control, yetanother type of T cell, thesuppressor T cell, tells B
cells, helper Ts and killerTs to turn off
Most immune cells die, but a fewremain in the body, calledmemory cells
They will be able to respondmore quickly the next timethe body is invaded by thesame foreign substance
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Immune Response in Fish
Cultured finfish and shellfish account forapproximately 25% of world aquatic animalproduction
With intensification comes a deterioration in cultureenvironment, leading to increased incidence ofdisease
Poor water quality affects the fish immune system ina negative way
The status of being immuneis an inherited ability toresist infection (Shoemaker et al., 2000)
I.e., recognition of non-self or a foreign agent, withsubsequent response and memory in vertebrates
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Immune Response in Fish
Fish are the most primitive vertebrates, but had todevelop an immune system for protection
the only exception was cold water species: due tolow bacterial generation time at lower temperatures
those living under schooling conditions and in warmenvironments needed a highly developed response
all fish pathogens contain antigens: viral particles,bacteria, fungi, toxins and animal parasites
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Immune Response in Fish
Immune response in fish includes: expansion of cells for the immune response
expression of the cells and molecules (e.g.,
antibody) the coordination of the response by regulatory
substances
Study of fish immunity and disease resistance isrelatively young compared to mammals
Early work was largely comparative, now focuseson understanding how immune system respondsto foreign agents or how innate resistance can beselected for by breeding programs
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Response of Fish Following anEncounter with a Pathogen
Fish Contacts Pathogen
Innate Immunity
Failure (Disease
and Death) Initiation and Instruction of the
Specific Immune Response
Success (No Disease
or Infection)
Humoral Response
(Extracellular Pathogens
and Toxins)
Cell-Mediated Immune
Response (Intracellular
Pathogens and Viruses)
Acquired Immunity,
Immunologic Memory,
and Protection (Survival)
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Immune Tissues and Organs
Most important immunocompetent organs: thymus,kidney (head, trunk), spleen and liver
Immune tissues in these organs not well defined
(Manning, 1994) Thymus: develops T-lymphocytes (helpers, killers;
similar to other verts), indirect evidence
Kidney: important in both immunity and
hematopoiesis, site of blood cell differentiation Early immune response handled by entire kidney
With maturity, anterior used for immune response; posteriorfor blood filtration, urinary activities
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Immune Tissues and Organs Kidney (cont.):
blood flows slowly through kidney and antigensare trapped or exposed to reticular cells,macrophages, lymphocytes
Anterior is where memory occurs (Secombs etal., 1982)
Spleen: secondary to kidney, involved inimmune reactivity and blood cell formation,
contains lymphocytes and macrophages Liver: could be involved in production of
components of the complement cascade,important in resistance; not real clear
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Immune Tissues and Organs
Mucus and skin: natural barriers, hasmolecules with immune actions: Lysozyme
Complement
Natural antibodies (Ab) and immunoglobulins (Ig)
Specific antibodies tentatively reported in mucusofIctalurus punctatus(Lobb, 1987); Oncorhyncus
mykiss(St. Louis-Cormier et al., 1984) Zilberg and Klesius, 1997) showed mucus
immunoglobulin elevated in I. punctatusafterexposure to bacteria
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Natural Immunity andDisease Resistance
1) Non-specific immune cells
Monocytes and tissue macrophages: most importantcells in immune response, produce cytokines (Clem et al.,
1985), primary cells involved in phagocytosis and firstkilling of pathogens upon first recognition and subsequentinfection (Shoemaker et al.,1997)
Neutrophils: primary cells in early stages ofinflammation (Manning, 1994), neutrophils produce
cytokines to recruit immune cells to damaged or infectedarea; neutrophils are phagocytic in I. Punctatus, killbacteria by extracellular mechanisms
Natural killer cells: use receptor binding to target cellsand lyse them; important in parasitic and viral immunity
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Natural Immunity andDisease Resistance
2) Phagocytosis: most primitive of defensemechanisms, occurs in stages Movement by chemotaxis (directional) or
chemokinesis (non-d) of phagocytes in responseto foreign object
Attachment via lectins
Engulfment of the foreign agent (simple
movement into the phagocyte) Killing and digestion
Oxygen-independent mechanisms: low pH, lysozyme,lactoferrin, proteolytic/hydrolytic enzymes
Oxygen dependent mechanisms
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Natural Immunity andDisease Resistance
3) Nonspecific Humoral Molecules:Molecule Composition Mode of Action
Lectins Specific sugar-
binding proteins
Recognition,
precipitation,agglutination
Lytic enzymes Catalytic proteinslysozyme, etc.
Hemolytic andantibacterial activity
Transferrin/lactoferrin Glycoprotein Iron binding
Ceruloplasmin Acute-phase protein Copper binding
C-reactive protein Acute-phase protein Activation ofcomplement
Interferon protein Resistance to viral
infection
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Natural Immunity andDisease Resistance
Lytic enzymes are antibacterial molecules that cleavethe 1,4 linkages n-acetyl muramic and n-acetylglucosamine in bacterial cell walls
Lysozyme (another enzyme) works on Gram-positivebacteria, complement on Gram-negative
Acute-phase proteins are serum proteins:ceruloplasmin responsible for binding of copper,
usually generated as the result of stress
Nutrition also influences levels of C-reactive protein
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Natural Immunity andDisease Resistance
4) Complement: consists of 20 or more chemicallydifferent serum proteins + glycoproteins having enzymefunction
originally named complement because it wasconsidered a biological substance complementingtheaction of antibody
Instead, antibodies actually activate a series ofreactions in serum known as the complement
cascade. interacts with either a specific antibody, or acts non-
specifically on surface molecules of bacteria, viruses andparasites; both pathways exist in fish (Sakai, 1992)
Action: clears antigenic molecules, immune complexes,
participates in inflammation and phagocytosis
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Humoral Immunity in Fish
Defined: the antibody response to foreign antigens
Fish posses B-cells (surface immunoglobulin-positivecells), similar to mammals in structure
Surface IgM of B-cells serves as receptor for antigenrecognition and is of same specificity as the antibodymolecule that will be produced (Janeway andTravers, 1994)
Unlike crustaceans, fish possess immunologicmemory (Arkoosh and Kaattari, 1991)
Their primary and memory response both use thesame IgM molecule, with eight antigen binding sites,a potent activator of complement
ll di d i i
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Cell-Mediated Immunity inFish
Used to eliminate intracellular pathogens (e.g.,bacteria, virus, parasites)
Relies on contact of the foreign invader with thesubsequent presentation of an antigen having the
same major histocompatability complex (MHC I or II)to T-helper cells (REM?)
Once T-helper cells are stimulated, the producecytokines that result in stimulation ofeffectorcells(cytotoxic lymphocytes) or macrophages
Cytokines stimulate aforementioned cells and alsorecruit new cells to the area, activate them
Work quite well against bacteria, important againstEdwardsiella ictaluri(Shoemaker, et al., 1999)
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Factors Influencing Disease Resistanceand Immune Response of Fish1
General Specific
Genetics Individuals may exhibit differences in innateresistance and acquired immunity
EnvironmentTemperature, season, photoperiod
Stress Water quality, pollution, density, handling andtransport, breeding cycles
Nutrition Feed quality and quantity, nutrient availability, use ofimmunostimulants, antinutritional factors in feeds
Fish Age, species or strains, individuals
Pathogen Exposure levels, type (parasite, bacterial, viral),virulence
1From Shoemaker et al.,2001. Immunity and disease resistance in fish. In: Nutrition
and Fish Health (Ed.: Lim, C., Webster, C.D.). Food Products Press, NY. Pgs 149-162.
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Factors Affecting ImmuneResponse: temperature
Resting fish body temperature is near ambient
pathogen generation time is temperature
dependent fishes living in cold temperatures have little need
for an immune response
coldwater fishes do not produce
immunoglobulins immune response slower at cold temperatures
(up to 28 days!)
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Factors Affecting ImmuneResponse: age
Immune competency develops relativelyslowly in animals
mammals obtain antibodies through mothers
milk for up to six weeks not the case with fish
rainbow trout are found to be immunecompetent at an early age (0.3g)
significance: immunization of very young fishis practical
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Passive Immunity: vaccination
Most immunizing substances developed forfish have been bacterins
these are killed, whole-cell suspensions of
pathogenic bacteria some practical viral vaccines exist (e.g., for
CCV, see subsequent notes on viruses)
probably will take place through injection ofavirulent viral strains
immunization against animal parasites mightalso eventually be possible
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Duration of Passive Immunity
Typical response is of short duration
very dependent upon environmental
temperature primary response to injection is usually only a
few weeks
secondary injections nine weeks after primaryhave resulted in maintenance of protectiveantibody titers, as in higher animals
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Part 2: Immune Response inShrimp
As mentioned, fish and shrimp differ significantly intheir ability and degree to which they carry out thisresponse
the capacity to recognize, expand the specificrecognition, express specific recognition, andcoordinate defense is much lower in shrimp
mistake: often drug manufacturers and scientistsassume that fish and shrimp have the same immunecompetency
thus, inappropriate decisions have been made onhow defense mechanisms might be enhanced inshrimp
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Immunoreactive Molecules ofthe Shrimp
Shrimp blood is known as hemolymph
it contains both oxygen-carrying molecules(hemocyanin) and immunoreactive molecules
known as lectins lectins are glycoproteins (sugar + protein) that
bind with the sugar portion of other molecules,particularly foreign ones
these lectins have broad specificity, meaning theywill bind with a broad range of other molecules,not just sugars
for example, they can bind with the sugar moeityof lipopolysaccharides, or beta-glucans
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Immunoreactive Molecules inShrimp
Gram negative bacteria (e.g., Vibrio sp.)and yeastswhich contain beta-glucans can be recognized bylectins
they also happen to recognize viruses and otherinfectious agents with surface glycoproteins
after recognizing the foreign agent, the lectin willagglutinize it, rendering it ineffective
the specificity for binding by a lectin cannot beincreased as with antibodies
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Immunoreactive Molecules inShrimp
The only way the immune response in shrimp can beenhanced is by putting more lectins in the bloodstream
after the infection is over, the cells that produce lectinscompletely lack the ability to remember the infectiousagent
so, immune response in shrimp is notan acquired one
another characteristic of lectins is that once bound to asugar on the foreign agent, the complex is easilyphagocitized
the phagocytic cell is known as hemocyte
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Shrimp Hemocyte Response As mentioned, the primary defense cells in shrimp
are called hemocytes
certain hemocytes have the ability to phagocytize
foreign cells, others to encapsulate and renderagents ineffective
the defense mechanisms of shrimp are thus moreprimitive and singular in their ability to controlinfection
this means that stress is more likely to negativelyimpact shrimp defenses against infection
no backup systems available when primary systemfails!!
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Immunoreactive Molecules inShrimp
blocking attachment by use of drugs ordiets containing beta-glucans might
prevent the binding of foreign agents along with lectins, shrimp have
lysozyme, an anti-bacterial enzyme
lipolytic enzymes against viruses
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A Brief History of ShrimpImmunology
Bacteria and fungi are dealt with byappropriate measures (e.g., similar for most
aquaculture animals) Most workhas dealt with bacterial pathogens
Relatively few parasites: cuticular excretionsand molting get rid of them
Most problems lie with prevention and/ortreatment of viruses
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Shrimp ImmunologyAs mentioned, shrimp have both a cellular and
humoral response to viruses: Certain proteins respond to -glucan (component of
bacterial cell wall) Hemocytes attack bacteria, release compoundscausing browning reaction in the HP
But no antibodies generated!
No defenseagainst viruses has to date beendescribed in any detail
Conclusion: there must be some defense thathas been overlooked!
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Shrimp Immunology
There is also little histological response toviruses: blood cells dont go to location
Viral infections are persistent, remain evidentfor life of shrimp
Despite having no set specific response tospecific viral pathogens, shrimp appear tohave a have a high tolerance to them
Case in point: historical information on viralepizootics in Southeast Asia
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Whats Going On?
Our current management practice is to lookfor SPF, high-health animals for stockingponds
Most PLs derived from new sources, not fromsurvivors
The history of each batch is important toknow!
Implication: perhaps SPF animals are notappropriate!
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Normal Shrimp
If you sample a normal shrimp pond in SE Asia, 88%of shrimp are infected with a virus
53% have been infected with two to three viruses
Survival now (after multiple years in population) hasreturned to a more or less normal level
Does this indicate resistance or tolerance?
Resistance = no sign of pathogen in individual;however, virus can be detected in tissues
Conclusion: something different from resistance
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Theory of Viral Accomodation
Shrimp viral response is an active process
Involves binding of viron to receptor sitethat triggers some kind of memory
Binding is not related to infection receptor
Memory causes reduced apoptosis
Subsequent binding turns off ability of virus
to induce death in host Death is prevented, but not infection
Viral replication can take place, but no deathApoptosis: the process of cell death which occurs naturally as part of the normal
development, maintenance and renewal of tissues within an organism. Occurs when avirus infects a cell.
Dr. Tim Fleigel
i l f i i h d
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Viral Infection is a PhasedProcess
Initial: brief and evolutionary with acutemortality via apoptosis, leads to intermediatephase
Intermediate: virus and host live together,but without mortality; better host survivorsreplicate so population is positively selectedfor against virus
Final: hard to find virus, mutual existencegoverned by genetic factors
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Accomodation
Higher virulence is naturally selectedagainst
No resistance to infection = reduced orlow virulence
Point: no pressure on virus to becomevirulent
Point: may increase competition fornew viruses to enter host!
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What to Do???
Use survivors as a source of broodstock
Expose progeny to virus or tolerene to
develop tolerance (avirulent virus) When? Possibly at Zoea 3 or earlier
How? Tolerene developed specifically foreach virus
Implications: for larval rearing, it meansintroduction of a tolerene in proper form
Vi l S Sh i
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Virology Summary: Shrimpvs. Fish
No clear response toviruses
Survivors remain
infected Pathogen persists
Survivors infectious toothers
Tolerance is a normalsituation
No antibodies
Multiple activeinfections are normal
Specific response toviruses
Survivors often dont
remain infected Pathogen removed from
body
May or may not beinfectious to others
Tolerance not normal
Antibodies present
Usually only one virusat a time