Fish Immunology:

The Atlantic cod, Gadus morhua L.

Bergljót Magnadóttir, MSc, PhDzoologist – fish immunologist

Head of the Department of Virology and Molecular

Biology

Institute for Experimental Pathology,

University of Iceland,

Reykjavík, Iceland

Invited talks at 3 universities in china in May 2009

This talk:

1. The Institute – Keldur

1. The immune system of fish

1. Cod – Gadus morhua L.

1. The immune system of cod

1. The Institute Keldur http://www.keldur.hi.is

• Veterinary Institute established in 1948

• The Institute belongs to The University of Iceland, under the Ministry of Education with close links to The Icelandic Food and Veterinary Authority

• Staff: 50 – 60

• 4 departments•Administration

•Bacteriology, parasitology, pathology

•Fish Diseases

•Virology and molecular biology

The Institute - Keldur

The Institute - Keldur

The role of the Institute

• Basic research in veterinary biology and medicine

• Health control (screening for infections) and diagnostic services for all animals

• To produce some vaccines and antiserum for the farming industry

• To provide laboratory animals for research in Iceland.

Examples of routine screenings for diseases

• Scrapies in sheep and cattle

• Campylobacter and salmonella in chickens

• Avian influenca (H5N1) in wild migrating birds

• Renebacterium salmoninarumin salmon and trout

• Viral and other infections in imported animals (pets)‏

Example of diagnostic/post mortem

services

2 polar bears landed on the north coast of Iceland in

June 2008 and post mortem was carried out from Keldur

Examples of research projects

• The MaediVisna virus of sheep:

evolution of the APOBEC viral

protection proteins in mammalia

• Summer eczema in Icelandic

horses abroad

• The parasitic load of wild

ptarmigan (Lagopus muta)‏

• The pathogenicity of atypical

furunculosis in fish

• The immune system of cod

This talk:

• The Institute – Keldur

• The immune system of fish

• Cod – Gadus morhua L.

• The immune system of cod

2. The Immune system of fish

• The immune system fights infections and

controls biological equilibrium (homeostasis)‏

• Studies of the immune system and immune

response of fish at different stages of

development are important

– contribution to comparative and developmental

immunology and

– for the fish farming industries, for vaccine

production and good husbandry practices

Aquired immune system(specific)‏

450 m.y.

Innate immune system (non-specific)‏

VERTEBRATA

INVERTEBRATA

Mammalia

Gnathostomata

ElasmobranchiiTeleosteii

Dipnoi

ReptiliaAvesAmphibia

Branchiopoda

PolyzoaEchinoderma

Agnatha

The evolution of the immune system

• Appeared first in vertebrates with

jaws (fish) (450 m.y.)‏

• T- & B-lymphocytes, TCR and

Immunoglobulins (antibodies) are

key factors

• Germline diversity, RAG

(recombination activation gene)‏

• Memory

• Delayed response but long lasting

Acquired immune system(adaptive/specific)‏

Innate immune system(non-specific)‏

• Evolutionary ancient defence

system, the only defence of

invertebrates

• Phagocytes, natural killer

cells and pattern recognition

proteins and receptors are key

factors

• Germline encoded and

genetically passed on

• No memory

• Immediate response

Fish v. mammalia

• The immune system of fish is less sophisticated

• Specific antibody response takes weeks (10 – 15) compared to days/hours

• Explains fish immunologists’ interest in the innate immune system:

• Fish antibody response gives a valuable “slow motion” view of innate response and the communication between the innate and acquired systems

kidney

spleen

GALT

thymus

IgM + “IgD”

thymus

spleen

lymphnodes

germinal centers bone

marrowGALT IgM

IgG’s

IgA’s

IgE

IgD

Components of the immune systems

Humoral components

Lectins

Complement system

Anti-proteases

Lysozyme

Antibacterial peptides

Cytokines and chemokines

Cellular components

Immunoglobulins (IgM in fish)‏

Phagocytes

Natural killer cells

Innate system Aquired system

Antigen presenting cells

B-lymphocytes

T-lymphocytes

This talk:

• The Institute – Keldur

• The immune system of fish

• Cod – Gadus morhua L.

• The immune system of cod

Vertebrates

Agnatha Gnathostomata

Chondrichthyes

Actinopterygii

TeleosteiDipnoiTetrapoda

hagfish

lungfish

coelacanths

sturgeon

sharks

codlantern fish salmonids

carp eel

cartilaginous fishes

The evolutionary status of cod

3. Atlantic cod, Gadus morhua L.

Iceland

Beijing

Iceland and the Cod

A mainstay of Iceland’s economy

Diminishing wild stocks

0

1

2

3

4

1968 2005

0

100

200

300

400

1981 2008

North AtlanticIcelandic waters

mil

lion

to

ns

thou

san

d t

on

s

Increasing interest in cod aquaculture

Cod aquaculture in Iceland

• Experimental cod farming

started around 1990

• At present 11 on-growing

commercial farms of both

wild and hatchery

produced cod

• Not yet commercially

viable

Cod aquaculture – common problems

• Losses during initial rearing i.e.

egg and larval stages (> 70%)‏

– Natural causes

– Technical problems (feeding)‏

– Infections

• Losses during on-growing stages

– High densitiy

– Handling stress

– Diseases

We have to know more about

the immune defence system of cod

This talk:

• The Institute – Keldur

• The immune system of fish

• Cod – Gadus morhua L.

• The immune system of cod

4. The immune system of cod(Research started at Keldur in 1992)‏

http:/www.hi.is/gadus

The immune system of cod: 4 aspects

• Physical components

– Cod mucus

• Cellular components

– Cod phagocytes and

granuloma formation

• Humoral components

– The specific and natural

antibody response of cod

• Ontogeny

• Development and immuno-

stimulation

IgM

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

Pentraxin

Gelatinase activity

And anti-bacterial peptides!

lysozyme

Phagocytic activity(The Amplex Red Assay)‏

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

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

• Stress and acute phase induction inhibits phagocytic activity

Cellular components

• Granuloma formation is 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

Humoral components

The specific and natural antibody response of cod

• Specific antibodies e.g. against a pathogen, vaccine or induced antigen

• Non specific antibodies present without an apparent antigenic stimulation

• In fish natural antibodies have anti-hapten activity e.g. against TNP-BSA

• Cod is known as a poor or non responder in terms of specific antibody production

• Cod has a high anti-TNP activity and a high concentration of IgM in its serum

•Two examples from our laboratory

a) Cod immunised with protein in adjuvant

Immunisation

Blood sampling0 18

months

No specific antibody response but stimulation of natural antibody

response (anti-TNP-LPH) probably by the adjuvant

b) Cod vaccinated against Asa and challenged with Asa

challengevaccination termination

0 13 w 19 w

Specific antibody response Natural (non-specific) response

Anti-Asa antibodies Anti-TNP-BSA antibodies

Specific antibodies of cod

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

The ontogenic development of

the immune system 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

• And this spring:

qrtPCR analysis of various

immune parameters

Pentraxins

cathepsins

No maternal IgM in eggs

Innate parameters before and soon after hatching

Thymus and lymphocytes 40 – 50 d.p.h.

Cod is fully immunologically competent

70 – 80 days after hatching depending on temperature

and feeding regime

Immunostimulation and probiotics

Live micro organisms

which when administered

in adequate amounts are

beneficial for the host

Probionts

Polysaccharides of

bacterial, plant or

crustacian origin that will

stimulate the innate

immune system through

pattern recognition

proteins or receptors

Immunostimulants

Immunostimulation and probiotics

• What stimulant/probiotic to use?

• How should it be administrated?

– Bathing, in the feed

• 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

Immunostimulation and disease resistance

Cod larvae bathed in LPS

from Asa and challenged

with Asa 145 days later

% accumulative death

Cod larvae bathed in LPS

from Asa or probiotic

bacteria and challenged

about 100 days later

How does cod manage?

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

Cod relies on the quantity rather than

the quality of its immune response

What are we working on now?

• The acute phase response (APR)

of cod

• The gene expression of immune

parammeters in cod larvae

(qrtPCR)‏

• The effects of variable salinity

during larval stages on immune

parameters of cod

Co-WorkersThe fish research group at Keldur

Sigríður Guðmundsdóttir – fish immunology & Rene bacterium

Sigurður Ingvarsson – head of the Institute

Árni Kristmundsson – parasitology & Rene bacterium

BM – fish immunology, head of Virology- & Molecular Biology

Sigurður Helgason – parasitology & pathology, head of the Dept.

Fish Diseases

Bjarnheiður Guðmundsdóttir – fish bacteriology, vaccines

Johanna & Bryndis – PhD students

Ivar, Sigríður & Harpa – MSc students

Edda & Þórunn - technicians Agnar Steinarsson,

Marine Institute

Thank you! 谢谢 ! Takk !