slide on marine fish disease

7/29/2019 slide on marine fish disease

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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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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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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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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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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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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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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 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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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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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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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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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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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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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

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