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The immune defence of fish: Studies of Atlantic cod (Gadus morhua L.)

Bergljót Magnadóttir

Tilraunastöð H.Í. Í meinafræði að Keldum

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This talk:

•The evolutionary status and immune system of fish (teleosts/bony fish)

•The immune system and cod: An overview of studies at Keldur

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The innate immune system (non-specific)

450 m.y.

VERTEBRATAINVERTEBRATA

Mammalia

Gnathostomata

Elasmobranchii

Teleosteii

Dipnoi

Reptilia

Aves

Amphibia

Agnatha

Polyfera

Mollusca

Nematoda

Echinoderma

ArthropodaAmphioxus

1000 m.y.

150 m.y.

1 m.y.

The adaptive immune system

(aquired/specific)

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The innate immune system (non-specific)

450 m.y.

VERTEBRATAINVERTEBRATA

Mammalia

Gnathostomata

Elasmobranchii

Teleosteii

Dipnoi

Reptilia

Aves

Amphibia

Agnatha

Polyfera

Mollusca

Nematoda

Echinoderma

ArthropodaAmphioxus

1000 m.y.

150 m.y.

1 m.y.

The adaptive immune system (aquired/specific)

5450 m.y.

VERTEBRATAINVERTEBRATA

Mammalia

Gnathostomata

Elasmobranchii

Teleosteii

Dipnoi

Reptilia

Aves

Amphibia

Agnatha

Polyfera

Mollusca

Nematoda

Echinoderma

ArthropodaAmphioxus

1000 m.y.

150 m.y.

1 m.y.

The adaptive immune system (aquired/specific)

Dunkleosteus

The innate immune system (non-specific)

•Improved systemic defence

and homeostatic regulation

•Improved defence against

changable antigens (viral

infections) and complex

antigens (parasitic infections)

•RAG* enzymes

•IgSF**

•T- & B- cell markers

•MHC I & II

•Thymus

*Recombination activating gene

**Immunoglobuin super family

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Fish v. mammals

• The immune system of fish is less sophisticated:• Limitations of the genetic machinery (restricted antibody repertoire)

• Slower metabolic rate (exothermic), fewer & slower leukocytes

• Organization of clonal selection is limited (no lymphnodes or germinal centres), no Ig class switch

• Some differences in the innate and adaptive parameters of fish compared to mammals may to some extent compensate for thesedeficiencies

• Specific antibody response can take weeks (10 – 15) and secondary response is similar to to the primary response

• Fish rely on the innate system in the first line of defence

kidney

spleen

GALT

thymus

IgM (IgD, IgT. . .)

thymus

spleen

lymphnodes

germinal centers bone

marrowGALT

IgM

IgG’s

IgA’s

IgE

IgD

RAG enzymes, IgSF,

B- & T- lymphocytes,

MHC, thymus

E.g. C3 & CRP isotypes,

flexible tetrameric IgM,

phagocytic lymphocytes

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The immune system of fish

• Studies of fish have proved important in comparative and developmental immunology

• The immune response of fish gives a valuable “slow motion” view of the innate response, the activation of the adaptive system and of the communication between the innate and adaptive systems

• Requirements of the fish farming industry are important and the main source of funding

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There is fish – and fish

• Evolutionary status– The different orders of modern teleosts go back 100 – 200 m.a. and

have adapted to varied environments and habitats. Each new species in aquaculture or immunological research demands a fresh start!

• Genetic make-up– The activity of immune parameters can show great individual

variations within the same species

• Age, size, sex

• Maternal effects

• Environmental effects– Temperature, season, water quality, pollution . . .

• Stress – Mating, migrations, captures . . .

– Confinement, density, handling, transport . . . .

• The type of pathogen

Various factors influence the immune system and immune response of fish

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The diminishing stocks of wild cod

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1

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3

4

1968 2005

North Atlanticm

illi

on

to

ns

0

100

200

300

400

1981 2008

Icelandic waters

tho

us

an

d t

on

s

Growing interest in cod aquaculture in the

past 15 – 20 years and with it - a growing

interest in the immune system of cod

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The requirements of the cod farming industry

• We need to know more about the immune system of cod at different developmental stages and under different environmental conditions– To secure the optimum activity of the

natural immune defence of cod in aquaculture

– To improve survival of cod larvae culture (the main bottleneck)

– To develop and improve prophylactic measures (vaccination, immunostimulation, probiotics etc.) for larvae and fully grown cod

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The fish and facilities

• Cod at different developmental stages has come from the Marine Institute’s Experimental Station at Staður, Grindavík (+ some wild fish samples)

• Facilities for experiments at Staður are limited to non-infective experiments

• Facilities at Sandgerði for challenge experiments are being improved

• Facilities for keeping fish at Keldur are very limited

– This and the distance from Staður and Sandgerði has restricted the studies we have been able to do

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The immune system of cod: 5 aspects

1. Physical components

2. Cellular components

3. Humoral components

4. Ontogeny

5. The acute phase response

http:/www.hi.is/gadus

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1. Physcical components: The cod mucus

Humoral defence components in cod mucus:

– Relatively rich in antibodies (IgM* 1.5 mg/g mucus)

– Strong enzyme activity (esp. at low temp.)

– Bactericidal effects

– Lectins/pentraxins and lysozyme

Pentraxins Gelatinase activity

lysozyme

* Mucosal IgM

is identical to

serum IgM with

respect to

redox forms

and

glycosylation

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2. Cellular componentsGranuloma formation

• Granuloma formation is very characteristic of cod’s defence against bacterial infection

•Macrophages and epithelioid

cells start to organize around

bacterial colonies

•They form several layers around

the bacteria with a thin

congregation of fibroblast cells

on the periphery

•Granuloma formations are seen

within 3 weeks of experimental

infection

Photographs: Bjarnheiður Guðmundsdóttir

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2. Cellular components*Phagocytic activity (The Amplex Red Assay)

• Phagocytic activity is relatively high in adult cod and shows fast response to stimulation

• Phagocytic activity appears early in ontogeny (< 4 dph)

• Stress and acute phase response supresses phagocytic activity

• Other work on cod leukocytes has mainly been in developing cultural and analytical methods and testing anti-cod antibodies in cell sorting experiments (FACS)

*Sigríður Guðmundsdóttir

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3. Humoral components

• We have measured the following humoral parameters in cod under different conditions and produced mono-specific antibodies against some(*):– IgM* and specific & natural antibody activity

– Haemolytic activity, ApoLP-AI* and C3*

– Lysozyme activity

– Anti-trypsin activity

– Iron binding capacity

– Pentraxins (CRP-PI & CRP-PII)*

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3. Humoral componentsIgM + specific and natural antibody response of cod

1 2 3 4 5 6 7

• Tetrameric IgM is the main immunoglobulin, it has several redox forms

• IgD-like cell receptor has also been described

• Cod has a high serum concentration of IgM (5-20 mg/ml)

• High natural antibody activity (anti-TNP-BSA)

• Shows poor specific antibody response

• Vaccination gives limited protection if any

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I. Cod vaccinated against Aeromonas salmonicida ssp. achromogenes (Asa)* and challenged with Asa 13 weeks later

II. Comparison of the activity and affinity of specific and natural antibodies of cod

*Causes atypical furunculosis in cod

An example of the specific and natural antibody response of cod in a vaccination

trial at Keldur (Sandgerði)

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I. Cod vaccinated against Asa and challenged with Asa

challengevaccination termination*

0 13 w 19 w

Specific antibody response Natural antibody response

Anti-Asa antibodies Anti-TNP-BSA antibodies

* survivors

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II. The activity and affinity of specific and natural antibodies (tested on the same 5 cod sera)

Asa AsaTNP-BSA TNP-BSA

Activity* Affinity**

The affinity – i.e. strength of the antigen-antibody binding

of specific antibodies is weaker than of the natural

antibodies

**thiocyanate elution*ELISA

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4. The ontogenic development of cod

• Samples collected from fertilisation

until 57 days post hatching

• SDS-PAGE and Western blotting of

larvae lysates

• Zymograms of larvae lysates

• Immuno-, in situ- and enzyme-

histology

• Planed: qrtPCR gene expression

analysis of various innate

parameters

The appearance of innate parameters

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Pentraxins

cathepsins

4. The ontogenic development of cod

Complement C3 Phagocytes

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Pentraxins

cathepsins

4. The ontogenic development of cod

Complement C3 Phagocytes

•No maternal transfer of IgM or C3

•Cod is not fully immunologically competent

until 80 – 90 days after hatching

•Need to look for protective methods other than

vaccination for cod larvae & fry during the first

3 months (the main bottleneck in cod aquaculture)

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• What stimulant/probiotic to use?

• How should it be administered?– Bathing, in the feed, injection

• When and how often?– Repeated treatment can be counter

effective

• How do we measure the effects?– The effects on immune parameters,

survival, growth, disease resistance

4. The ontogenic development of cod

Immunostimulation and probiotics

We have tried bathing

cod larvae in LPS from

Asa, in various

polysaccharides from

sea weed, algae, licens

and shellfish,

we have tried bathing in

non-pathogenic bacteria

isolated from cod

(probionts) and

we have tried to

introduce stimulants in

the feed of cod fry.

Variable results and

limited successSee also recent papers by Helen Lauzon, Matís

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5. The acute phase response of cod2 recent experiments at Keldur:

1. Cod infected with Asa

2. Cod injected with turpentine oil

• Samples collected for 6 – 10 days, the effects on cortisol and immune parameters analysed

– Serological analysis of humoral components

– Analysis of RB activity

– Quantitative real time PCR analysis

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5. The acute phase response of cod Serum cortisol

Asa infected cod

Turpentine injected cod

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5. The acute phase response of codSerology and RA activity

Parameter Infected Turpentine

early (1d) late 6d) early (3d) late (7d)

Nat.ab’s

IgM

Anti-tryps.

CRP-PI

CRP-PII

RB activity nd nd

Suppressive effects on humoral immune parameters and RB activity

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Gene expression analysis, turpentine injected fish

Primers Kidney Spleen

early (1h) late (>3d) early (1h) late (>3d)

CRP-PI

CRP-PII

IL-1β

Transferrin

C3

ApoLP-AI

Cathelicidin

5. The acute phase response of cod

Preliminary results but it seems that:

•Cod shows a relatively slow humoral and

cellular response to infection and acute phase

induction

•The increase in serum cortisol may be a key

modulator and

•Pentraxins are not typical acute phase

proteins in cod

Stimulatin effects on the gene expressin esp. in kidney

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Some points about the immune system of cod

• In aquaculture cod (unlike salmon) is known as a stessful fish and a poor responder to vaccination

• Other points about cod:– Vaccination gives variable protection (often limited),

depends on the pathogen and on individual cod– Cod is not particularly susceptible to infectious diseases– There is no deficiency in the number, structure,

organization, diversity or expression of Ig H- or L-chain compared to other teleosts

– Deficient MHC Class II is a prime suspect as well as possible deficiency in cytokine activity

• Is cod a primitive fish?

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Gnathostomata

Chondrichthyes

Actinopterygii

Teleostei

coelacanths

sturgeon

sharks

codlantern fish salmonids

carp eel

Cartilaginous fishes

The evolutionary status of cod

Cod may have taken a

different line in immune

defence from other teleost

species

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Physically strong and

immunologically

active mucus

High, relatively constant

level of humoral

innate parameters

High level of natural

antibodies with relatively

strong affinity

Active phagocytes/macrophages

and epithelial cells quickly form

granuloma around the pathogen

Great heterogeneity

of some PRP like

the pentraxins

The immune defence of cod

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What are we working on now?

• SALCOD: The effects of variable salinity during larval stages on immune parameters of cod (The Marine Institute)

• The gene expression and IH analysis of pentraxins and IL-1β in cod larvae (qrtPCR)

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Co-Workers

The “fish” group at Keldur

Agnar Steinarsson,

Marine Institute

Sigríður Guðmundsdóttir

Sigurður Helgason

Bjarnheiður Guðmundsdóttir

Sigríður Steinunn Auðunsdóttir

Eggert Gunnarsson o.fl. á Keldum

Agnar Steinarsson o.fl. á Stað

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Tank you – takk!