Upload
others
View
9
Download
0
Embed Size (px)
Citation preview
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 !