HOST RESPONSE AGAINST SALMON LOUSE - slrc · 2015-07-06 · Norwegian University of Life Sciences (NMBU) Department of Basic Sciences & Aquatic Medicine HOST RESPONSE AGAINST SALMON

Norwegian University of Life Sciences (NMBU)Department of Basic Sciences & Aquatic Medicine

HOST RESPONSE AGAINST SALMON LOUSE

what have we learned so far?

Stanko Skugor1, Helle Holm1, Anne Kari Osmo2, Aleksei Krasnov3,

Øystein Evensen1, Simon Wadsworth2

[email protected]

1. SLRC-Sea Lice Research Centre, Faculty of Veterinary Medicine and

Biosciences, Norwegian University of Life Sciences (NMBU), Norway

2. SLRC-Sea Lice Research Centre, Ewos Innovation, Norway

3. Nofima AS, Norway

NMBU

overview host protective responses

role of different organs�

hormonal regulation�

comparative studies:

resistant vs susceptible�

breeding for higher resistance�

protection by diet�

testosteroneestradiol cortisol

NMBU

Atlantic salmon can be more resistant

resistance

Host smell less attractive

Host less nutritious for lice

Appropriate immune response

1

2

3

PHYTOCHEMICALS

IRON DEPRIVATION

KILLING MECHANISMS

good news:

Main themes to focus on in the future�

interplay of

various

mechanisms

no single

mechanism of

protection

NMBU

Type 2

immunity

fairly well

understood

not so well

understood

Type 1

immunity

Immune responses to parasites

contain the extracellular parasites,

not kill

rapid repair of damaged tissues

kill microbial pathogens

various killing mechanisms

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NMBU

role of different organs during infection�

� proteases

� Accute phase

� Immunoglobulins

SPLEEN

dayspostinfection

profiles of immunoglobulin genes

days3 5 101 15

Scetchy knowledge

Organs involved in different functions:

immune

metabolic

detoxifying...

Genomics tools:

large number of gene expression changes

why? how?

detailed description of

processes during infection

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Parasites are sensed early

Biphasic protease profile at molting �immunomodulation

NMBU

Gen

e ex

pres

sion

days

� MHCI

3 5 101 15

• suppression and diversion

• early activation of immune responses

• immunoglobulin/antibody production

increases

SKIN

copepod chalimus


NMBU

Gen

e ex

pres

sion

days

� MHCI

� T-cells

3 5 101 15

SKIN

copepod chalimus





increases

NMBU

Gen

e ex

pres

sion

days

� MHCI

� T-cells� Ig

3 5 101 15

SKIN

Receptors D1 D3mannose receptor, C type 2 8.88 17.18C-type lectin domain family 4 member E 3.04 19.39mannose-specific lectin precursor -1.11 2.22CD209 antigen-like protein D 2.10 1.92mannose receptor C type 1 precursor 2.73 1.37rhamnose-binding lectin WCL1 4.21 1.34

Gene expression profiling of A. salmon skin in vici nity to lice

Salmon recognition receptors – promote Type 2 immunity

copepod chalimus





increases

NMBU

conclusionsrole of different organs during infection�

killing mechanisms

Type 1 Type 2

Inflammatory

Regulatory

modified from Diaz and Allen (2007) Eur. J. Immunol 37: 3319–3326

(Th17)

RESISTANCE

Typical A. salmon immune response to lice: not protective

(Th2)(Th1)

(Treg)

Active immunosuppression by salmon louse

OK in wild populations

Needed in farmed settings

Two-dimensional map of immune responses

Type 2-modified

Atlantic salmon’sresponse to lice

Pink salmon and coho’s response to lice

Lice/fish

0

5

10

15

20

25

30

35

40

45

mature (n=28) nonmature (n=22)

lice/CF

liceField study:

Host-pathogen relationship

Sexually mature salmon � less lice

Do sex hormones provide protection

against lice?

7 39

hormonal regulation

Laboratory challenge study:

Host-pathogen relationship

• Fish fed testosteron (T), estrogen (E)

& control diet

• Skin and plasma analysed

0

5

10

15

20

25

30

35

40

estrogen group testosteronegroup

control

Number of lice/fish

E T C

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NMBU

Estradiol:

promotes

Type 1 immunity

NMBU

-1

-0,5

0

0,5

1

1,5

E1 T1 E2 T2 E3 T3

MHC class I protein

Tapasin - Ident 25

Beta-2-microglobulin

MHC class I antigen

MHC class II

H-2 class II histocompatibility antigen gamma

chain

Mhc-UAA

Mhc-UAA

Antigen presentation

IFN inducible genes –effector mechanisms

-1

-0,8

-0,6

-0,4

-0,2

0

0,2

0,4

0,6

0,8

1

1,2

E1 T1 E2 T2

Tyrosine-protein kinase

Kruppel-like factor 2a

Interferon regulatory factor 1

Very large inducible GTPase 1-3

Novel protein containing a TLD domain - Ident

50Retinoic acid-inducible gene-I

Novel nacht domain containing protein - Ident

72Novel zinc finger protein - Ident 47

Retinoic acid-inducible gene-I

Interferon gamma inducible protein 30

Interferon-induced GTP-binding protein MxA

Interferon gamma inducible protein 30

Bloodthirsty - Ident 56

52 kDa Ro protein-4

T-cell activation

-2

-1,5

-1

-0,5

0

0,5

1

E1 T1 E2 T2

Hemoglobin subunit beta-2

Hemoglobin alpha adult-1

Hemoglobin subunit beta

Novel alpha globin





Hemoglobin subunit alpha

Novel alpha globin 1








hemoglobins


Reduced

flow of

red blood

cells in skin?

NMBU

Testosterone

promotes

Type 2 effector

mechanisms

TISSUE REMODELLING


NMBU

Type 1IMMUNITY Type 2

IMMUNITY

Highly pro-inflammatory response

Regulatory /anti-inflammatory responses

ESTRADIOL

TESTOSTERONE

Type 2-modified Control

50% less lice

30% less lice

Immunosuppression � SUSCEPTIBILITY


?

NMBU

comparative studies�

Differences in resistance between salmonid species

Atlantic

Chum

Pink

Coho

Sockeye

highly susceptible

fully resistant

Behaviour

Nutritional habits

Skin properties

Lice preferences

Immune responses

Life history adaptations

Differences might be due to:

fastest growth

least fertile

shortestocean-phase

more energy to invest

in immunity

NMBU


Coho salmon vs Atlantic salmon

3 70 141

days

lice

num

ber

EWOS infestation model Chile, 2015

� Atlantic

� Coho

Field study (sea cages)

all fish infected with Caligus rogercreseyi Very similar to previous findings

with L. salmonis

NMBU


scale

dermis

epidermis

Engulfed louse (Caligus)

Massive cell proliferation

neutrophils

14 days after infectionCoho salmon

Infiltration of

immune cells

(neutrophils)

EWOS infestation model Chile, 2015

NMBU

Atlantic salmon, PAS, 100X

Chalimus

21 days

Moderate cell proliferation

Moderate influx of inflammatory cells


Atlantic salmon, H&E, 100X

Weak cell proliferation

No influx of inflammatory cells

Atlantic salmon

Potential for selective breeding

Majority of individuals but- variation exists!

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NMBU

Atlantic

Coho


Braden et al, 2015

Can this be modulated by

breeding or diet

mucus

cell #

mucus

cell #

mucus

cell #

?

Academic unit


Feeding on Pacific salmon

similar to starvation

susceptibility

Atlantic > sockey > coho

• lice do not like sockey

• sockey has weak inflammation

L. salmonis microarray study on different hostsBraden et al, 2015

Sockeye

NMBU

Atlantic, chum & pink salmon

Sutherland et al, 2014

down-regulation of iron

genes in head kidney of

pink salmon

resistant pink salmon:

best iron deprivation strategy


NMBU


experiment:

Lice number � anti-correlated to immunity

Type 1 (anti-viral defence pathways)

Type 2 immunity against parasites

22/14

lice countLow resistant fish (LR) High resistant fish (HR)


multivariate analysis 32 genes

gene

expression

of immune

markers

licenumberrelationship

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A

B

Resistant fish:

resist immuno-suppression better

NMBU

23

Skin thickness and mucous cell number in Atlantic salmon


Epidermis

thicknessmucous cell

thinner skin

highly resistant fish (HR)

less mucous cells

Stronger Type 1 immune response is protective

Thicker skin & more mucus cells not linked to protection� �

Conclusion

NMBU

Academic unit

Anti-attachment functional feeds:

developed by Ewos & SLRC

ControlStimulus

copepodidstart point

Low

High

ControlStimulus


ControlStimulus


Low

High


Mode of action:

different plant ingredients1

bioactive phytochemicals2

Mask salmon smell / affect recognition

Modulate lice olfactory genes

Activate protection in host:

right type of immunity

iron / heme metabolism

detox / antioxidant pathways

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Academic unit

1. Sea lice detected host odour and

released hold fast

2. Detection disrupted and no hold fast

released

• In vitro screening of phytochemicals

• frontal filament model in Caligus

Filament extruded in response to A. salmon mucus

Filament not extruded in response to

anti-lice phytochemicals


Academic unit

Ex Vivo tissue model

- masking effect on L. salmonis

Fin from fish

on control

diet

Fin from fish

on anti-

attch. diet

Lice given a choice

(ctrl vs anti-attch.)

Attached cops – CONTROL feed

Attached cops – ANTI-LICE feed

Free swimming cops


0

50

100

150

200

250

A B C

Number of copepoditeson fins from fish fed

control and anti-attch. diet

ATTCH. control diet ATTCH. anti-attachment diet free swimming

65% reduction in attached copepodites

with anti-attch. feed� �

Academic unit

• IRON METABOLISM

• COMPLEMENT

• GRANZYME

• EXTRACELLULAR

METALLOPROTEINASES

• T-CELL

RECEPTORS,

• SIGNALLING

• ANTI-INFLAMMATORY

MARKERS

CONTROL

• MYOFIBERS,

CONTRACTIONHIGH

DOSE

LOW

DOSE

• PRO-INFLAMMATORY

MARKERS

• GLUCOSE METABOLISM

wound healing / Type 2-immunity

Pro-inflammatory mediators / killing mechanisms/ Type 1-immunity

PLSR plot

CONTROL

HIGH DOSE

LOW DOSE30% reduction in lice

number achieved

protection by diet� multivariate analysis

Skin

gene

expression

NMBU

UI HD UI C I LD I HD

52 kDa Ro protein-1 0,97 0,09 -0,15 0,20

Barrier-to-autointegration factor 1,46 -0,57 -0,27 0,66

CD9-1 0,84 -0,09 0,29 0,11

Deoxycytidine kinase 0,59 0,66 0,58 0,80

eukaryotic translation initiation factor 4 gamma, 1 isoform 3-1 0,93 0,49 0,16 0,54

Fish virus induced TRIM-3 0,64 0,02 -0,17 0,21

Fish virus induced TRIM-8 0,72 -0,13 -0,11 0,47

Galectin-9 0,92 -0,15 0,05 0,34

Gig1-1 0,93 -0,31 -0,16 0,34

Gig1-2 1,00 0,02 0,10 0,53

Gig2 2,17 -0,24 0,23 0,51

Gig2-2 1,42 -0,41 -0,03 0,51

Gig2-4 1,57 -0,33 -0,11 0,41

Gig2-7 1,86 -0,38 0,05 0,35

Interferon regulatory factor 3 0,66 -0,09 -0,12 0,11

interferon regulatory factor 7 0,75 -0,37 -0,39 -0,29

Interferon-induced GTP-binding protein Mx 1,44 -0,31 -0,28 0,12

Interferon-induced GTP-binding protein MxB - Ident 29 0,62 0,28 0,27 0,12

Interferon-induced protein 44 0,79 -0,24 -0,02 -0,28

Interferon-induced protein 44-1 1,45 -0,11 -0,03 0,39

Interferon-induced protein with tetratricopeptide repeats 5-2 1,13 -0,22 -0,20 0,25

Low-density lipoprotein receptor-related protein 2 - Ident 31 0,82 0,21 0,18 0,66

Mucin 5AC 2,16 0,05 -0,10 0,55

Mx3 protein 0,91 -0,03 -0,16 0,27

myxovirus resistance 1 1,52 -0,09 0,06 0,69

Nicotinamide phosphoribosyltransferase-1 1,05 -0,20 -0,01 0,21

Novel protein similar to vertebrate hect domain and RLD 3 (HERC3) - Ident 96 1,03 -0,01 -0,03 0,13

Novel protein similar to vertebrate patched domain containing 3 (PTCHD3) 1,38 0,04 -0,20 0,12

PaTched Related family member 1,30 -0,09 -0,30 0,00

Peroxisomal proliferator-activated receptor A-interacting complex 285 kDa-2 0,70 -0,39 -0,13 0,04

Poly (ADP-ribose) polymerase family 14 0,70 -0,03 -0,07 0,18

PRKCA-binding protein 0,74 -0,28 -0,18 -0,56

Probable ATP-dependent RNA helicase DHX58 0,99 -0,61 -0,20 -0,11

Radical S-adenosyl methionine domain-containing protein 2 1,50 -0,44 -0,07 0,64

Radical S-adenosyl methionine domain-containing protein 2; AltName: Full=Virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible; Short=Viperin Radical S-adenosyl methionine domain-containing protein 2, V1,47 -0,34 -0,08 0,66

Radical S-adenosyl methionine domain-containing protein 2; Viperin 1,40 -0,13 0,21 0,64

Receptor transporting protein 3 2,20 -0,14 -0,03 0,52

retinoic acid-inducible gene-I 0,79 -0,08 -0,22 -0,04

RING finger protein 182 0,49 0,19 0,06 0,69

RING finger protein 213 0,69 0,29 0,34 -0,01

Sacsin 1,44 -0,46 -0,15 0,24

Signal transducer and activator of transcription 1 [Oncorhynchus mykiss] 0,68 -0,38 -0,29 -0,05

Signal transducer and activator of transcription 1 0,68 -0,30 -0,18 0,00

similar to hect domain and RLD3 1,32 0,00 -0,27 0,18

sub-family B ATP-binding cassette transporter 2 [Oncorhynchus mykiss] 1,10 0,30 0,05 0,63

thymidylate kinase 0,75 -0,26 -0,09 -0,05

TRIM21-like 1,21 0,24 0,24 0,64

Tripartite motif-containing protein 25 0,47 -0,85 -0,53 0,14

Ubiquitin-like protein-1 1,65 -0,77 -0,30 0,19

Unknown 1,75 -0,26 0,02 0,47

Very large inducible GTPase 1-1 1,45 -0,91 -0,29 0,08



VHSV-induced protein 1,03 -0,15 0,23 0,11

VHSV-induced protein-1 0,96 0,14 -0,07 0,15

VHSV-inducible protein-3 0,99 0,10 -0,06 0,45

VHSV-inducible protein-4 0,83 0,02 0,40 0,53

XIAP-associated factor 1 0,78 -0,10 -0,16 0,21

ZNF 1,25 0,16 0,11 -0,02

**

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BEFORE INFECTIONover 60 IFN- and antiviral genes induced by anti-attachment feed

�

�� Preconditioning fish immunity

by phytochemicals

Academic unit

anti-lice feed � less accesible iron

less accessible iron/heme from host

L. salmonis genome sequenced

L. salmonis cannot make heme!

Iron metabolism

regulation

muscle

• heme oxygenase-1

the most highly induced gene in muscle

Distal kidney

• Ferritin:

potent antioxidant/ iron

sequestration

liver

• Hepcidin:

increased iron storage in liver,

leading to lowered iron plasma

levels

weaker lice


30

NMBU

pro

tect

ion

flo

w anti-attachment feed & improved genetic background

modulated skin immunity

& iron metabolism

masked host

less ”tasty & attractive smelling”

less disturbed salmon

attached lice �

summary�

immunomodulatory

substances released from lice �

Atlantic salmon can be more resistant

Appropriate immune response3 KILLING MECHANISMS

Host less nutritious for lice 2 IRON DEPRIVATION

Host smell less attractive1 PHYTOCHEMICALS

con

clu

sio

n

Academic unit

Thank youfor

your attention

NMBU

liver spleen head kidney

Metabolism of iron and erythropoesis

in head kidney and spleen down-regulation during the whole infection

in liver, initial decrease was followed with the gradual elevation


3

22

33

Days post infection

Host less nutritious for lice 1 IRON DEPRIVATION

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Documents

HOST RESPONSE AGAINST SALMON LOUSE - slrc · 2015-07-06 · Norwegian University of Life Sciences (NMBU) Department of Basic Sciences & Aquatic Medicine HOST RESPONSE AGAINST SALMON