stress. mortality pattern general adaptation syndrom استرس مجموعه ای از...

stressstress

Mortality patternMortality pattern

General adaptation syndromGeneral adaptation syndrom

استرس مجموعه ای از واکنشهای فیزیولوژیک که بوسیله آن •حیوان تالش می کند تا سوخت و ساز طبیعی خود را در برابر

تغییرات فیزیکی یا شیمیایی ایجاد شده حفظ کند.بوسیله یک متغیر محیطی تحمیل شده و باعث امتدادو •

استمرار واکنشهای سازگاری ماهی در ماورائ حدود طبیعی و اغتشاش در فعالیتهای فیزیولوژیک ماهی شده و شانس بقا

کاهش می یابد.

What about stress??

• Stressors (handling, sustained exercise such as escape from predator pursuit) cause release of adrenaline (epinephrine) - for mediating escape, etc.

• Adrenaline causes diffusivity of gill epithelium to increase (become “leaky” of water & ions)

• This accentuates the normal osmoregulatory challenge for FW or marine fishes

پاسخ اولیه:فعالیت سیستم عصبی-ترشحی و •رهایی کاتکول امینها و هورمون

کورتیکواستروئیدیپاسخ ثانویه: پاسخ فیزیولوژیک به این •

هورمونها)افزایش خروجی کاردیاک، مصرف o2)ذخیره انرژی ،

پاسخ سوم: اثر بر عدم رشد، آسیب به اندامها •مانند تولید مثل و سیستم ایمنی

Permanent disabilityPermanent disability

Reversible effectReversible effect

Limit of compensationLimit of compensation

Limit of normal adjustmentLimit of normal adjustment

تغییرات :مراحل

خطر • زنگمقاومت•تحلیل•

Endocrine Systems of FishesEndocrine Systems of Fishes

HypothalamusHypothalamus

• Corticotropin releasing hormon (CRH): Corticotropin releasing hormon (CRH): adrenocorticotropin hormon (ACTH)

• Thyrotropin releasing hormon (TRH): Thyrotropin releasing hormon (TRH): thyroid stimulating hormon (TSH)

• Growth hormon releasing hormon Growth hormon releasing hormon (GHRH): (GHRH): growth hormon (GH)

• Gonadotropin releasing hormon (GnRH): Gonadotropin releasing hormon (GnRH): Gonadotropin hormon (Gn)

Pituitary Gland - Master GlandPituitary Gland - Master Gland

Linked with hypothalamus of brainLinked with hypothalamus of brain Produces hormones that affect other Produces hormones that affect other

endocrine tissues - indirect influenceendocrine tissues - indirect influence Produces hormones that affect non-Produces hormones that affect non-

endocrine tissues directlyendocrine tissues directly

Pituitary GlandPituitary Gland

Indirect influenceIndirect influence ACTH - adrenocorticotrophic ACTH - adrenocorticotrophic

hormonehormone stimulates interrenal tissue production stimulates interrenal tissue production

of cortisolof cortisol

TH - thyrotrophic hormoneTH - thyrotrophic hormone stimulate thyroid production of stimulate thyroid production of

thyroxin (growth, metamorphosis-i.e. thyroxin (growth, metamorphosis-i.e. flounder)flounder)

GTH- gonadotrophic hormoneGTH- gonadotrophic hormone stimulates gonads to produce stimulates gonads to produce

androgens/estrogensandrogens/estrogens

Pituitary Gland

• Effects non-endocrine tissues directly– pigmentation - melanophore stimulating

hormone (MSH)• affects long-term control of color

– osmoregulation - prolactin, vasotocin• controls fresh/saltwater systems

– growth – somatotrophic hormone• stimulates > length, cell multiplication

Thyroid GlandThyroid Gland

• isolated follicles distributed in connective tissue along isolated follicles distributed in connective tissue along ventral aortaventral aorta

• controls metabolic ratecontrols metabolic rate• affects metamorphosis, maturationaffects metamorphosis, maturation• facilitates switch between fresh & salt waterfacilitates switch between fresh & salt water

Gonads

• gamete and sex hormone production• controls gametes maturation• cause formation of secondary sex

characteristics: color, shape, behavior• in fish, several sex hormones also serve as

pheromones - e.g. goldfish males respond to hormones released with ovulation

Other endocrine tissues in fishesOther endocrine tissues in fishes

chromaffin chromaffin tissues-located near kidneys & hearttissues-located near kidneys & heart produce adrenaline/noradrenaline – “fight or flight”produce adrenaline/noradrenaline – “fight or flight” increases blood flow through gills, ventilation rateincreases blood flow through gills, ventilation rate

interrenal (inside kidney) tissuesinterrenal (inside kidney) tissues produce cortisol, cortisone - stress response produce cortisol, cortisone - stress response

hormones (reduce inflamation)hormones (reduce inflamation)

BRAINBRAIN

hypothalamushypothalamus

Anterior pitutaryAnterior pitutary Systemic systemSystemic system

Internal tissueInternal tissue Suprarenal tissueSuprarenal tissue

CorticosteroidsCorticosteroidscortisolcortisol

CathecholaminsCathecholaminsAdrenalin, noradrenalinAdrenalin, noradrenalin

Blood glucose, fatty acids, lactateBlood glucose, fatty acids, lactate

ImmunosuppressionImmunosuppressionLiver glycogenLiver glycogenLymphocytesLymphocytes

Atrophy of lymphaticsAtrophy of lymphatics

Heart rateHeart rateDisterbed osmoregulationDisterbed osmoregulation

Liver glycogenLiver glycogen

Cortisol depletionCortisol depletionDegeneration of internal secretory tissueDegeneration of internal secretory tissue

LesionsLesionsmortalitymortality

Osmoregulatory Systems in Osmoregulatory Systems in FishesFishes

Maintaining homeostasis with respect to solute concentrations and

water content

Introduction

• Homeostasis = maintaining steady state equilibrium in the internal environment of an organisms

• Much is done involuntarily by action of hormones, enzymes and osmoregulatory processes. Although occasionally fish do just “pick up and move” if environmental conditions are unfavorable.

Topics

• Osmoregulation• Endocrine system• Thermal regulation

Four osmoregulatory strategies in fishes

1. Isosmotic (nearly isoionic, osmoconformers)2. Isosmotic with regulation of specific ions3. Hyperosmotic (fresh H20 fish)

4. Hyposmotic (salt H2O fish)

– ionic conc. approx 1/3 of seawaterionic conc. approx 1/3 of seawater– drink copiously to gain waterdrink copiously to gain water– Chloride cells eliminate NaChloride cells eliminate Na++ and Cl and Cl-- – kidneys eliminate Mgkidneys eliminate Mg++++ and SO and SO44

==

advantages and disadvantages?advantages and disadvantages?

Osmotic regulation by marine teleosts...Osmotic regulation by marine teleosts...

Saltwater teleosts:

drinkdrink

active tran.active tran.passive diff.passive diff.

NaNa++, Cl, Cl--

MgMg++++, SO, SO44==

HH22OO

NaNa++, Cl, Cl--

NaNa++, Cl, Cl--

chloride cellschloride cells

MgMg++++, Ca, Ca++++ , SO , SO44==

kidneyskidneys

HH22OO

NaNa++, Cl, Cl--

gutgut

Chloride Cell fig 6.2:

pavementpavementcellcell PCPCPCPC

activeactive

passivepassive

chloride cellchloride cell

accessory accessory cellcell

sea watersea water

internalinternalmitochondriamitochondria

tubular systemtubular system

NaNa++

KK++ NaNa+ + KK+ + ATPaseATPase

NaNa++, , ClCl--

NaNa++

ClCl--

ClCl--

ClCl--ClCl--

gutgut

carriercarrier

NaNa++

NaNa++

NaNa++

pumppump

++

– Ionic conc. Approx 1/3 of seawater– Don’t drink– Chloride cells fewer, work in reverse – Kidneys eliminate excess water; ion loss– Ammonia & bicarbonate ion exchange mechanisms

advantages and disadvantages?

Osmotic regulation by FW teleostsOsmotic regulation by FW teleosts

Freshwater teleosts:Freshwater teleosts: activeactive

passivepassive

HH22OO

NaNa++, Cl, Cl--

NaNa++, Cl, Cl--

don’tdon’tdrinkdrink

waterwater

kidneyskidneysIon exchangeIon exchange

pumps; beta chloride cellspumps; beta chloride cells

saltssalts

gutgut

Chloride Cell fig 6.2:

epitheliumepitheliumcellcell ECECECEC

activeactive

passivepassive

chloride cellchloride cell

epitheliumepitheliumcellcell

Fresh waterFresh water

internalinternal

NaNa++ ClCl--

pump

pump

pump

pump

NHNH44++ or H or H++ HCOHCO33

--

• NH4 H +NH3 glutamine•D-Aminase

• HCO3 H2CO3 H2O+ CO2

•Carbonic anhydrase enzyme

Ion Exchange MechanismsIon Exchange Mechanisms

gill membranegill membrane

freshwaterfreshwater interiorinterior

activeactive

pumppump

activeactive

pumppump

NaNa++

ClCl--

NHNH44++ or H or H++

HCOHCO33--

ATPATP

ATPATP

Euryhaline

• Diadromous fishes (spend part of life in salt water, part in freshwater – catadromous (migrate seaward) or anadromous (migrate up river)

• hormone-mediated changes associated with metamorphosis - convert from FW adaptations to SW or vice versa, depending on direction of migration

How to reduce stress in stressed fishes?• Minimize the osmotic challenge by placing fish

in conditions that are isosmotic– add salt to freshwater, e.g. in transporting fish or

when exposing them to some other short-term challenge

– dilute saltwater for same situation with marine species

Thermoregulation in Fishes

Temperature effects on fish

• Temperature exhibits the greatest influence on fish’s lives!

• Affects metabolism• Affects digestion• Signals reproductive maturation and behavior

Fish are conformers (well, sort of...)

• Body temperature is that of the environment• Each species has particular range of temperatures

that they can tolerate and that are optimal• Big difference between what you can tolerate and

what you thrive in...

Behavioral Thermoregulation in Fishes

• Although fish are ectotherms, they can alter their body temperature by moving to habitats with optimal temperature

Hot Fishes

• Some fish can maintain body temperature greater than ambient - tunas, billfishes, relatives (nearly endothermic)

• Use retia (similar to rete mirable) in swimming muscles to conserve heat, exchange O2, etc.

• Red muscle is medial rather than distal• Billfishes have warm brains - heat organ from

muscles around eye

Practical application

• You’re management decisions and actions must account for fish responses to temperature gradients and limitations

Immune SystemImmune System

Introduction

• Obviously, the immune system is important in homeostatic processes.

• Immune systems of fish have two components: non-specific and specific.

• As we will see, both are involved in protecting fish from visible as well as invisible disease causing agents.

Non-specific immunity

• Skin & Scales—specific solid layers of protection from pathological and chemical stressors.

• Mucus secretion—traps microorganisms; preventing entry into body cavity or circulation

• Macrophages (phagcytes) and cytotoxic cells—part of the inflamatory response which destroy pathogens within the body before they can do harm.

Immune Immune Response Response

SystemSystem

• Made up of two cellular systems: 1) cell-mediated immunity (T cells) and 2) humoral antibody system (B cells)

• Both work by identifying antigens (foreign proteins or glycoproteins)

Specific Immune ResponseSpecific Immune Response

• More of an active response where an “invader” is detected and destroyed.

• Primary organs: kidney, thymus, spleen, intestine.

• Antigens—invading compounds which provoke an immune response.

Source: Cancer Research Institute (2002) www.cancerresearch.org/immhow.htmlSource: Cancer Research Institute (2002) www.cancerresearch.org/immhow.html

Specific immune response: What if something does get in??

• White blood cells called B lymphocyte cells (B cells) and T lymphocyte cells (T cells)—bind to foreign cells and begin replication and attachement to (sort of markers for things to come...).

• Occasionally, invader actually goes trough a macrophage first...then B cell responds

• Once B cells replicate, antibodies are produced which bind specifically to pathogens and tag them for destruction (eating) by macrophages!

“Looks like meat’s back on the menu

boys!!!”

Lecture 7: Lecture 7: Immune Immune Response of Aquatic Response of Aquatic

OrganismsOrganisms

Preliminary ConceptsPreliminary Concepts Disease problems have grown proportionally Disease problems have grown proportionally

with the intensive or expansive culture of with the intensive or expansive culture of aquaculture speciesaquaculture species

Why?Why?1(1( Increased stocking densities )lower profit margins(Increased stocking densities )lower profit margins(2(2( Infected carriers )largely broodstock(Infected carriers )largely broodstock(3(3( Infected facilities )GMPs being followed?(Infected facilities )GMPs being followed?(4(4( Poor nutrition )we are way behind(Poor nutrition )we are way behind(

5(5( Substandard water quality )traditional(Substandard water quality )traditional( Biggest problemBiggest problem: : greater susceptibility via greater susceptibility via

weakening of resistance under intensive culture weakening of resistance under intensive culture conditionsconditions

The Immune The Immune ResponseResponse

For fish, response to a foreign agent is rather similar to For fish, response to a foreign agent is rather similar to that of mammals; shrimp, very rudimentarythat of mammals; shrimp, very rudimentary

Response can be highly specific )a specific antibody for Response can be highly specific )a specific antibody for a specific antigen( is known as the a specific antigen( is known as the immune immune responseresponse..

The immune system “scans” the body to identify any The immune system “scans” the body to identify any substance )natural/synthetic or living/inert( that it substance )natural/synthetic or living/inert( that it considers foreignconsiders foreign

Differentiates between “self” and “non-self”Differentiates between “self” and “non-self” Works with several types of white blood cells, located Works with several types of white blood cells, located

throughout the body, that work together in a highly throughout the body, that work together in a highly integrated wayintegrated way

DefinitionsDefinitions resistanceresistance: any type of barrier within the : any type of barrier within the

host that allows it to resist the pathogenhost that allows it to resist the pathogen innate or natural immunityinnate or natural immunity: attributed : attributed

to inherited ability to produce antibodies to inherited ability to produce antibodies without stimulation by antigenswithout stimulation by antigens

acquired immunityacquired immunity: host is stimulated by : host is stimulated by contact with antigens contact with antigens

passive immunitypassive immunity: acquired through the : acquired through the use of antibodies from other animals use of antibodies from other animals )vaccination()vaccination(

we will add another term today, we will add another term today, tolerancetolerance

Immune Immune ResponsRespons

e e SystemSystem

Made up of two cellular systems: Made up of two cellular systems: 1) cell-mediated immunity 1) cell-mediated immunity (T cells)(T cells) and and 2) humoral antibody system (B cells)2) humoral antibody system (B cells)

Both work by identifying antigens (foreign proteins or Both work by identifying antigens (foreign proteins or glycoproteins)glycoproteins)

Immune Response Immune Response Sequence: Sequence: 11

Begins when Begins when macrophagemacrophage encounters this non-encounters this non-self entity )e.g., self entity )e.g., virus(: macrophage virus(: macrophage literally “eats” the literally “eats” the substance, digests it substance, digests it and displays pieces of and displays pieces of the invader on its the invader on its surface. These pieces surface. These pieces are are antigensantigens..

Meanwhile, other viral Meanwhile, other viral particles are at work, particles are at work, infecting nearby infecting nearby host host cellscells..

Source: Cancer Research Institute (2002) www.cancerresearch.org/immhow.html


Antigenic fragments Antigenic fragments alert a specific type alert a specific type of T lymphocyte of T lymphocyte )“helper” T)“helper” T( to begin ( to begin choreographed choreographed attack of intruderattack of intruder

Helper recognizes Helper recognizes antigen particles antigen particles and binds to the and binds to the macrophage via an macrophage via an antigen receptorantigen receptor

Helper T cells are unique Helper T cells are unique to a specific antigento a specific antigen


This binding stimulates This binding stimulates production of production of chemical chemical substances such as substances such as interleukin-1interleukin-1 )IL-1(, )IL-1(, tumor necrosis tumor necrosis factorfactor )TNF( by )TNF( by macrophagemacrophage

Helper T cells generates Helper T cells generates interleukin-2interleukin-2 and and gamma interferongamma interferon )IFN-y()IFN-y(

All substances facilitate All substances facilitate intercellular intercellular communicationcommunication

Astonishing SynchronizationAstonishing Synchronization

TNF steps up production of IL-1, it also TNF steps up production of IL-1, it also causes fever in homeothermscauses fever in homeotherms

TNF and IL-1 are cytokines )cellular(TNF and IL-1 are cytokines )cellular( IL-1 also causes fever but additionally IL-1 also causes fever but additionally

forms immune cell clusters and forms immune cell clusters and stimulates the helper T cell to release IL-2stimulates the helper T cell to release IL-2

IL-2 causes T cells to release gamma IL-2 causes T cells to release gamma interferon which, in-turn, activates interferon which, in-turn, activates macrophagesmacrophages

IL-2 also instructs other helper T cells and IL-2 also instructs other helper T cells and “killer” T cells“killer” T cells to multiply to multiply


As mentioned IL-2 instructs As mentioned IL-2 instructs helper T’s and helper T’s and “killer T’s”“killer T’s” to multiplyto multiply

Proliferating helper T’s release Proliferating helper T’s release substances that cause substances that cause B B cellscells )another type of )another type of lymphocyte( to multiply lymphocyte( to multiply and produce and produce antibodiesantibodies

Meanwhile, many invader cells Meanwhile, many invader cells have been consumed by have been consumed by macrophages, but other macrophages, but other “daughter” viral particles “daughter” viral particles have escaped and are have escaped and are infecting other cellsinfecting other cells


Killer T cells start shooting Killer T cells start shooting “holes” in the surface of “holes” in the surface of infected host cellsinfected host cells

Antibodies released by B cells Antibodies released by B cells bind in a lock-and-key bind in a lock-and-key fashion to antigens on the fashion to antigens on the surface of invaders that surface of invaders that have escaped have escaped macrophages )macrophages )Ag-Ab Ag-Ab complexcomplex(.(.

Makes it easier for macrophages Makes it easier for macrophages and special killer and special killer lymphocytes to destroy lymphocytes to destroy unwelcomed entities.unwelcomed entities.

Binding of antibodies with Binding of antibodies with antigens signals release of antigens signals release of a blood component, a blood component, complementcomplement, to puncture , to puncture virus membrane )death(virus membrane )death(


Finally, as the infection is Finally, as the infection is brought under control, brought under control, yet another type of T yet another type of T cell, the cell, the suppressor T suppressor T cellcell, tells B cells, helper , tells B cells, helper T’s and killer T’s to turn T’s and killer T’s to turn offoff

Most immune cells die, but a Most immune cells die, but a few remain in the body, few remain in the body, called called memory cellsmemory cells

They will be able to respond They will be able to respond more quickly the next more quickly the next time the body is invaded time the body is invaded by the same foreign by the same foreign substancesubstance

Immune Response in FishImmune Response in Fish Cultured finfish and shellfish account for Cultured finfish and shellfish account for

approximately 25% of world aquatic animal approximately 25% of world aquatic animal productionproduction

With intensification comes a deterioration in With intensification comes a deterioration in culture environment, leading to increased culture environment, leading to increased incidence of diseaseincidence of disease

Poor water quality affects the fish immune system Poor water quality affects the fish immune system in a negative wayin a negative way

The status of being The status of being immuneimmune is “an inherited ability is “an inherited ability to resist infection” )Shoemaker et al., 2000(to resist infection” )Shoemaker et al., 2000(

I.e., recognition of “non-self” or a foreign agent, I.e., recognition of “non-self” or a foreign agent, with subsequent response and memory in with subsequent response and memory in vertebratesvertebrates

Immune Response in FishImmune Response in Fish Fish are the most primitive vertebrates, but had to Fish are the most primitive vertebrates, but had to

develop an immune system for protectiondevelop an immune system for protection the only exception was cold water species: due to low the only exception was cold water species: due to low

bacterial generation time at lower temperaturesbacterial generation time at lower temperatures those living under schooling conditions and in warm those living under schooling conditions and in warm

environments needed a highly developed responseenvironments needed a highly developed response all fish pathogens contain all fish pathogens contain antigensantigens: viral particles, : viral particles,

bacteria, fungi, toxins and animal parasitesbacteria, fungi, toxins and animal parasites

Immune Response in FishImmune Response in Fish

Immune response in fish includesImmune response in fish includes: : – expansion of cells for the immune responseexpansion of cells for the immune response– expression of the cells and molecules )e.g., expression of the cells and molecules )e.g.,

antibody(antibody(– the coordination of the response by regulatory the coordination of the response by regulatory

substancessubstances Study of fish immunity and disease resistance Study of fish immunity and disease resistance

is relatively young compared to mammalsis relatively young compared to mammals Early work was largely comparative, now Early work was largely comparative, now

focuses on understanding how immune focuses on understanding how immune system responds to foreign agents or how system responds to foreign agents or how innate resistance can be selected for by innate resistance can be selected for by breeding programsbreeding programs

Response of Fish Following Response of Fish Following an Encounter with a an Encounter with a

PathogenPathogenFish Contacts Pathogen

Innate ImmunityFailure

(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)

Immune Tissues and Immune Tissues and OrgansOrgans

Most important immunocompetent organs: Most important immunocompetent organs: thymus, kidney )head, trunk(, spleen and liverthymus, kidney )head, trunk(, spleen and liver

Immune tissues in these organs not well Immune tissues in these organs not well defined )Manning, 1994(defined )Manning, 1994(

ThymusThymus: develops T-lymphocytes )helpers, : develops T-lymphocytes )helpers, killers; similar to other vert’s(, indirect killers; similar to other vert’s(, indirect evidence evidence

KidneyKidney: important in both immunity and : important in both immunity and hematopoiesis, site of blood cell differentiationhematopoiesis, site of blood cell differentiation– Early immune response handled by entire kidneyEarly immune response handled by entire kidney– With maturity, anterior used for immune response; With maturity, anterior used for immune response;

posterior for blood filtration, urinary activitiesposterior for blood filtration, urinary activities


KidneyKidney )cont.(: )cont.(: – blood flows slowly through kidney and antigens blood flows slowly through kidney and antigens

are “trapped” or exposed to reticular cells, are “trapped” or exposed to reticular cells, macrophages, lymphocytesmacrophages, lymphocytes

– Anterior is where “memory” occurs )Secombs Anterior is where “memory” occurs )Secombs et al., 1982(et al., 1982(

SpleenSpleen: secondary to kidney, involved in : secondary to kidney, involved in immune reactivity and blood cell immune reactivity and blood cell formation, contains lymphocytes and formation, contains lymphocytes and macrophagesmacrophages

LiverLiver: could be involved in production of : could be involved in production of components of the complement cascade, components of the complement cascade, important in resistance; not real clearimportant in resistance; not real clear


Mucus and skinMucus and skin: natural barriers, has : natural barriers, has molecules with immune actions:molecules with immune actions:– LysozymeLysozyme– ComplementComplement– Natural antibodies )Ab( and immunoglobulins Natural antibodies )Ab( and immunoglobulins

)Ig()Ig(– Specific antibodies tentatively reported in mucus Specific antibodies tentatively reported in mucus

of of Ictalurus punctatusIctalurus punctatus )Lobb, 1987(; )Lobb, 1987(; Oncorhyncus mykissOncorhyncus mykiss )St. Louis-Cormier et al., )St. Louis-Cormier et al., 1984(1984(

– Zilberg and Klesius, 1997( showed mucus Zilberg and Klesius, 1997( showed mucus immunoglobulin elevated in immunoglobulin elevated in II. . punctatuspunctatus after after exposure to bacteriaexposure to bacteria

Natural Immunity and Natural Immunity and Disease ResistanceDisease Resistance

1(1( Non-specific immune cellsNon-specific immune cells• Monocytes and tissue macrophagesMonocytes and tissue macrophages: most : most

important cells in immune response, produce important cells in immune response, produce cytokinescytokines )Clem et al., 1985(, primary cells involved in )Clem et al., 1985(, primary cells involved in phagocytosis and first killing of pathogens upon first phagocytosis and first killing of pathogens upon first recognition and subsequent infection )Shoemaker et recognition and subsequent infection )Shoemaker et al.,1997(al.,1997(

• NeutrophilsNeutrophils: primary cells in early stages of : primary cells in early stages of inflammation )Manning, 1994(, neutrophils produce inflammation )Manning, 1994(, neutrophils produce cytokines to recruit immune cells to damaged or cytokines to recruit immune cells to damaged or infected area; neutrophils are phagocytic in infected area; neutrophils are phagocytic in I. PunctatusI. Punctatus, , kill bacteria by extracellular mechanismskill bacteria by extracellular mechanisms

• Natural killer cellsNatural killer cells: use receptor binding to target : use receptor binding to target cells and lyse them; important in parasitic and viral cells and lyse them; important in parasitic and viral immunityimmunity


2(2( PhagocytosisPhagocytosis: most primitive of : most primitive of defense mechanisms, occurs in stagesdefense mechanisms, occurs in stages

Movement by Movement by chemotaxischemotaxis )directional( or )directional( or chemokinesischemokinesis )non-d( of phagocytes in )non-d( of phagocytes in response to foreign objectresponse to foreign object

Attachment via Attachment via lectinslectins Engulfment of the foreign agent )simple Engulfment of the foreign agent )simple

movement into the phagocyte(movement into the phagocyte( Killing and digestionKilling and digestion

• Oxygen-independent mechanisms: low pH, Oxygen-independent mechanisms: low pH, lysozyme, lactoferrin, proteolytic/hydrolytic lysozyme, lactoferrin, proteolytic/hydrolytic enzymesenzymes

• Oxygen dependent mechanismsOxygen dependent mechanisms


3(3( Nonspecific Humoral MoleculesNonspecific Humoral Molecules: : MoleculeMolecule CompositionComposition Mode of ActionMode of Action

LectinsLectins Specific sugar-Specific sugar-binding proteinsbinding proteins

Recognition, Recognition, precipitation, precipitation, agglutinationagglutination

Lytic enzymesLytic enzymes Catalytic proteins Catalytic proteins lysozyme, etc.lysozyme, etc.

Hemolytic and Hemolytic and antibacterial antibacterial activityactivity

Transferrin/Transferrin/lactoferrinlactoferrin

GlycoproteinGlycoprotein Iron bindingIron binding

CeruloplasminCeruloplasmin Acute-phase Acute-phase proteinprotein

Copper bindingCopper binding

C-reactive proteinC-reactive protein Acute-phase Acute-phase proteinprotein

Activation of Activation of complementcomplement

InterferonInterferon proteinprotein Resistance to Resistance to viral infectionviral infection


Lytic enzymes are antibacterial molecules that Lytic enzymes are antibacterial molecules that cleave the cleave the 1,4 linkages n-acetyl muramic and n- 1,4 linkages n-acetyl muramic and n-acetyl glucosamine in bacterial cell wallsacetyl glucosamine in bacterial cell walls

Lysozyme )another enzyme( works on Gram-positive Lysozyme )another enzyme( works on Gram-positive bacteria, complement on Gram-negativebacteria, complement on Gram-negative

Acute-phase proteins are serum proteins: Acute-phase proteins are serum proteins: ceruloplasmin responsible for binding of copper, ceruloplasmin responsible for binding of copper, usually generated as the result of stressusually generated as the result of stress

Nutrition also influences levels of C-reactive proteinNutrition also influences levels of C-reactive protein


4)4) ComplementComplement:: consists of 20 or more chemically consists of 20 or more chemically different serum proteins + glycoproteins having different serum proteins + glycoproteins having enzyme functionenzyme function

originally named “complement” because it was originally named “complement” because it was considered a biological substance considered a biological substance complementingcomplementing the the action of antibodyaction of antibody

Instead, antibodies actually activate a series of Instead, antibodies actually activate a series of reactions in serum known as the “reactions in serum known as the “complement complement cascadecascade.”.”

interacts with either a specific antibody, or acts non-interacts with either a specific antibody, or acts non-specifically on surface molecules of bacteria, viruses specifically on surface molecules of bacteria, viruses and parasites; both pathways exist in fish )Sakai, and parasites; both pathways exist in fish )Sakai, 1992(1992(

ActionAction: clears antigenic molecules, immune : clears antigenic molecules, immune complexes, participates in inflammation and complexes, participates in inflammation and phagocytosisphagocytosis

Humoral Immunity in FishHumoral Immunity in Fish DefinedDefined:: the antibody response to foreign the antibody response to foreign

antigensantigens Fish posses B-cells )surface immunoglobulin-Fish posses B-cells )surface immunoglobulin-

positive cells(, similar to mammals in structurepositive cells(, similar to mammals in structure Surface IgM of B-cells serves as receptor for Surface IgM of B-cells serves as receptor for

antigen recognition and is of same specificity as antigen recognition and is of same specificity as the antibody molecule that will be produced the antibody molecule that will be produced )Janeway and Travers, 1994()Janeway and Travers, 1994(

Unlike crustaceans, fish possess immunologic Unlike crustaceans, fish possess immunologic memory )Arkoosh and Kaattari, 1991(memory )Arkoosh and Kaattari, 1991(

Their primary and memory response both use the Their primary and memory response both use the same IgM molecule, with eight antigen binding same IgM molecule, with eight antigen binding sites, a potent activator of complementsites, a potent activator of complement

Cell-Mediated Immunity Cell-Mediated Immunity in Fishin Fish

Used to eliminate intracellular pathogens )e.g., Used to eliminate intracellular pathogens )e.g., bacteria, virus, parasites(bacteria, virus, parasites(

Relies on contact of the foreign invader with the Relies on contact of the foreign invader with the subsequent presentation of an antigen having subsequent presentation of an antigen having the same the same major histocompatability complexmajor histocompatability complex )MHC I or II( to T-helper cells )REM?( )MHC I or II( to T-helper cells )REM?(

Once T-helper cells are stimulated, the produce Once T-helper cells are stimulated, the produce cytokines that result in stimulation of cytokines that result in stimulation of effectoreffector cellscells )cytotoxic lymphocytes( or macrophages )cytotoxic lymphocytes( or macrophages

Cytokines stimulate aforementioned cells and Cytokines stimulate aforementioned cells and also recruit new cells to the area, activate themalso recruit new cells to the area, activate them

Work quite well against bacteria, important Work quite well against bacteria, important against against Edwardsiella ictaluri Edwardsiella ictaluri )Shoemaker, et al., )Shoemaker, et al., 1999(1999(

Factors Influencing Disease Factors Influencing Disease Resistance and Immune Response of Resistance and Immune Response of

FishFish11

GeneralGeneral SpecificSpecific

GeneticsGenetics Individuals may exhibit differences in innate Individuals may exhibit differences in innate resistance and acquired immunityresistance and acquired immunity

EnvironmeEnvironmentnt

Temperature, season, photoperiodTemperature, season, photoperiod

StressStress Water quality, pollution, density, handling and Water quality, pollution, density, handling and transport, breeding cyclestransport, breeding cycles

NutritionNutrition Feed quality and quantity, nutrient Feed quality and quantity, nutrient availability, use of immunostimulants, availability, use of immunostimulants, antinutritional factors in feedsantinutritional factors in feeds

FishFish Age, species or strains, individualsAge, species or strains, individuals

PathogenPathogen Exposure levels, type )parasite, bacterial, Exposure levels, type )parasite, bacterial, viral(, virulenceviral(, 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.

Factors Affecting Immune Factors Affecting Immune Response: Response: temperaturetemperature

Resting fish body temperature is near Resting fish body temperature is near ambientambient

pathogen generation time is temperature pathogen generation time is temperature dependentdependent

fishes living in cold temperatures have fishes living in cold temperatures have little need for an immune responselittle need for an immune response

coldwater fishes do not produce coldwater fishes do not produce immunoglobulinsimmunoglobulins

immune response slower at cold immune response slower at cold temperatures )up to 28 days!(temperatures )up to 28 days!(

Factors Affecting Immune Factors Affecting Immune Response: Response: ageage

Immune competency develops relatively Immune competency develops relatively slowly in animalsslowly in animals

mammals obtain antibodies through mammals obtain antibodies through mother’s milk for up to six weeksmother’s milk for up to six weeks

not the case with fishnot the case with fish rainbow trout are found to be immune rainbow trout are found to be immune

competent at an early age )0.3g(competent at an early age )0.3g( significancesignificance: immunization of very : immunization of very

young fish is practicalyoung fish is practical

Passive Immunity: Passive Immunity: vaccinationvaccination

Most immunizing substances developed Most immunizing substances developed for fish have been for fish have been bacterinsbacterins

these are killed, whole-cell suspensions of these are killed, whole-cell suspensions of pathogenic bacteriapathogenic bacteria

some practical viral vaccines exist )e.g., some practical viral vaccines exist )e.g., for CCV, see subsequent notes on for CCV, see subsequent notes on viruses(viruses(

probably will take place through injection probably will take place through injection of avirulent viral strainsof avirulent viral strains

immunization against animal parasites immunization against animal parasites might also eventually be possiblemight also eventually be possible

Duration of Passive Duration of Passive ImmunityImmunity

Typical response is of short durationTypical response is of short duration very dependent upon environmental very dependent upon environmental

temperaturetemperature primary response to injection is usually primary response to injection is usually

only a few weeksonly a few weeks secondary injections nine weeks after secondary injections nine weeks after

primary have resulted in maintenance of primary have resulted in maintenance of protective antibody protective antibody titerstiters, as in higher , as in higher animalsanimals

Part 2:Part 2: Immune Response Immune Response in Shrimpin Shrimp

As mentioned, fish and shrimp differ As mentioned, fish and shrimp differ significantly in their ability and degree to significantly in their ability and degree to which they carry out this responsewhich they carry out this response

the capacity to recognize, expand the specific the capacity to recognize, expand the specific recognition, express specific recognition, and recognition, express specific recognition, and coordinate defense is much lower in shrimpcoordinate defense is much lower in shrimp

mistakemistake: often drug manufacturers and : often drug manufacturers and scientists assume that fish and shrimp have scientists assume that fish and shrimp have the same the same immune competencyimmune competency

thus, inappropriate decisions have been thus, inappropriate decisions have been made on how defense mechanisms might be made on how defense mechanisms might be enhanced in shrimpenhanced in shrimp

Immunoreactive Immunoreactive Molecules of the ShrimpMolecules of the Shrimp Shrimp blood is known as Shrimp blood is known as hemolymphhemolymph it contains both oxygen-carrying molecules it contains both oxygen-carrying molecules

))hemocyaninhemocyanin( and immunoreactive ( and immunoreactive molecules known as molecules known as lectinslectins

lectins are glycoproteins )sugar + protein( lectins are glycoproteins )sugar + protein( that bind with the sugar portion of other that bind with the sugar portion of other molecules, particularly foreign onesmolecules, particularly foreign ones

these lectins have broad specificity, meaning these lectins have broad specificity, meaning they will bind with a broad range of other they will bind with a broad range of other molecules, not just sugarsmolecules, not just sugars

for example, they can bind with the sugar for example, they can bind with the sugar moeity of lipopolysaccharides, or moeity of lipopolysaccharides, or beta-beta-glucansglucans

Immunoreactive Immunoreactive Molecules in ShrimpMolecules in Shrimp

Gram negative bacteria )e.g., Gram negative bacteria )e.g., Vibrio sp.Vibrio sp.) ) and yeasts which contain beta-glucans can be recognized by lectins

they also happen to recognize viruses and other infectious agents with surface glycoproteins

after recognizing the foreign agent, the lectin will agglutinize it, rendering it ineffective

the specificity for binding by a lectin cannot be increased as with antibodies


The only way the immune response in shrimp can be The only way the immune response in shrimp can be enhanced is by putting more lectins in the enhanced is by putting more lectins in the bloodstreambloodstream

after the infection is over, the cells that produce after the infection is over, the cells that produce lectins completely lack the ability to remember the lectins completely lack the ability to remember the infectious agentinfectious agent

so, immune response in shrimp is so, immune response in shrimp is notnot an acquired one an acquired one another characteristic of lectins is that once bound to another characteristic of lectins is that once bound to

a sugar on the foreign agent, the complex is easily a sugar on the foreign agent, the complex is easily phagocitizedphagocitized

the phagocytic cell is known as the phagocytic cell is known as hemocytehemocyte

Shrimp Hemocyte Shrimp Hemocyte ResponseResponse

As mentioned, the primary defense cells in As mentioned, the primary defense cells in shrimp are called shrimp are called hemocyteshemocytes

certain hemocytes have the ability to certain hemocytes have the ability to phagocytize foreign cells, others to encapsulate phagocytize foreign cells, others to encapsulate and render agents ineffectiveand render agents ineffective

the defense mechanisms of shrimp are thus the defense mechanisms of shrimp are thus more primitive and singular in their ability to more primitive and singular in their ability to control infectioncontrol infection

this means that stress is more likely to this means that stress is more likely to negatively impact shrimp defenses against negatively impact shrimp defenses against infectioninfection

no backup systems available when primary no backup systems available when primary system fails!!system fails!!


blocking attachment by use of drugs blocking attachment by use of drugs or diets containing beta-glucans might or diets containing beta-glucans might prevent the binding of foreign agentsprevent the binding of foreign agents

along with lectins, shrimp have along with lectins, shrimp have lysozymelysozyme, an anti-bacterial enzyme, an anti-bacterial enzyme

lipolytic enzymes against viruseslipolytic enzymes against viruses

A Brief History of Shrimp A Brief History of Shrimp ImmunologyImmunology

Bacteria and fungi are dealt with by Bacteria and fungi are dealt with by appropriate measures )e.g., similar for appropriate measures )e.g., similar for most aquaculture animals(most aquaculture animals(

Most work Most work hashas dealt with bacterial dealt with bacterial pathogenspathogens

Relatively few parasites: cuticular Relatively few parasites: cuticular excretions and molting get rid of themexcretions and molting get rid of them

Most problems lie with prevention Most problems lie with prevention and/or treatment of virusesand/or treatment of viruses

Shrimp ImmunologyShrimp Immunology As mentioned, shrimp have both a As mentioned, shrimp have both a

cellular and humoral response to viruses:cellular and humoral response to viruses:– Certain proteins respond to Certain proteins respond to -glucan -glucan

)component of bacterial cell wall()component of bacterial cell wall(– Hemocytes attack bacteria, release Hemocytes attack bacteria, release

compounds causing browning reaction in the compounds causing browning reaction in the HPHP

But… no antibodies generated!But… no antibodies generated! No No defensedefense against viruses has to date against viruses has to date

been described in any detailbeen described in any detail ConclusionConclusion: there must be some defense : there must be some defense

that has been overlooked!that has been overlooked!

Shrimp ImmunologyShrimp Immunology There is also little histological response to There is also little histological response to

viruses: blood cells don’t go to locationviruses: blood cells don’t go to location Viral infections are persistent, remain Viral infections are persistent, remain

evident for life of shrimpevident for life of shrimp Despite having no set specific response to Despite having no set specific response to

specific viral pathogens, shrimp appear to specific viral pathogens, shrimp appear to have a have a high tolerance to themhave a have a high tolerance to them

Case in point: historical information on Case in point: historical information on viral epizootics in Southeast Asiaviral epizootics in Southeast Asia

What’s Going On?What’s Going On?

Our current management practice is to Our current management practice is to look for look for SPFSPF, , high-healthhigh-health animals for animals for stocking pondsstocking ponds

Most PL’s derived from new sources, not Most PL’s derived from new sources, not from survivorsfrom survivors

The history of each batch is important The history of each batch is important to know!to know!

ImplicationImplication: perhaps SPF animals are : perhaps SPF animals are not appropriate!not appropriate!

““Normal” ShrimpNormal” Shrimp If you sample a normal shrimp pond in SE Asia, If you sample a normal shrimp pond in SE Asia,

88% of shrimp are infected with a virus88% of shrimp are infected with a virus 53% have been infected with two to three viruses53% have been infected with two to three viruses Survival now )after multiple years in population( Survival now )after multiple years in population(

has returned to a more or less normal levelhas returned to a more or less normal level Does this indicate Does this indicate resistanceresistance or or tolerancetolerance?? ResistanceResistance = no sign of pathogen in individual; = no sign of pathogen in individual;

however, virus can be detected in tissueshowever, virus can be detected in tissues ConclusionConclusion: something different from resistance: something different from resistance

Theory of Viral Theory of Viral AccomodationAccomodation

Shrimp viral response is an active processShrimp viral response is an active process Involves binding of viron to receptor site Involves binding of viron to receptor site

that triggers some kind of “memory”that triggers some kind of “memory” Binding is not related to infection receptorBinding is not related to infection receptor Memory causes reduced Memory causes reduced apoptosisapoptosis Subsequent binding turns off ability of Subsequent binding turns off ability of

virus to induce death in hostvirus to induce death in host Death is prevented, but not infectionDeath is prevented, but not infection Viral replication can take place, but no Viral replication can take place, but no

deathdeathApoptosis: the process of cell death which occurs naturally as part of the normal development, maintenance and renewal of tissues within an organism. Occurs when a virus infects a cell.

Dr. Tim Fleigel

Viral Infection is a Phased Viral Infection is a Phased ProcessProcess

InitialInitial: brief and evolutionary with acute : brief and evolutionary with acute mortality via apoptosis, leads to mortality via apoptosis, leads to intermediate phaseintermediate phase

IntermediateIntermediate: virus and host live : virus and host live together, but without mortality; better together, but without mortality; better host survivors replicate so population is host survivors replicate so population is positively selected for against viruspositively selected for against virus

FinalFinal: hard to find virus, mutual : hard to find virus, mutual existence governed by genetic factorsexistence governed by genetic factors

AccomodationAccomodation

Higher virulence is naturally Higher virulence is naturally selected againstselected against

No resistance to infection = No resistance to infection = reduced or low virulencereduced or low virulence

PointPoint: no pressure on virus to : no pressure on virus to become virulentbecome virulent

PointPoint: may increase competition : may increase competition for new viruses to enter host!for new viruses to enter host!

What to Do???What to Do???

Use survivors as a source of broodstockUse survivors as a source of broodstock Expose progeny to virus or Expose progeny to virus or tolerenetolerene to to

develop tolerance )avirulent virus(develop tolerance )avirulent virus( When? Possibly at Zoea 3 or earlierWhen? Possibly at Zoea 3 or earlier How? Tolerene developed specifically How? Tolerene developed specifically

for each virusfor each virus ImplicationsImplications: for larval rearing, it means : for larval rearing, it means

introduction of a introduction of a tolerenetolerene in proper in proper formform

Virology Summary: Virology Summary: Shrimp vs. FishShrimp vs. Fish

No clear response to No clear response to virusesviruses

Survivors remain Survivors remain infectedinfected

Pathogen persistsPathogen persists Survivors infectious Survivors infectious

to othersto others Tolerance is a normal Tolerance is a normal

situationsituation No antibodiesNo antibodies Multiple active Multiple active

infections are normalinfections are normal

Specific response to Specific response to virusesviruses

Survivors often don’t Survivors often don’t remain infectedremain infected

Pathogen removed Pathogen removed from bodyfrom body

May or may not be May or may not be infectious to othersinfectious to others

Tolerance not normalTolerance not normal Antibodies presentAntibodies present Usually only one Usually only one

virus at a timevirus at a time

SHRIMP FISH

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