fish health: immune system and vaccine development · • atypical furunkulosis - can be difficult...
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Fish health: Immune system and vaccine development
Vaxxinova Norway and IMR
Sonal Patel
Workshop on Atlantic halibut aquaculture, 11.-12. September
Co-funded by the Seventh
Framework Programme
of the European Union
Thanks to colleagues at IMR and Vaxxinova
Audun
Rolf HariAina
Ann Cathrine
Ina
Ingrid SisselJoachimElin
Stig
Kristian
Eivind
Colleagues at Austevoll research station
Prevention strategies
Knowledge about the immune system and disease/pathogen:
•When?•How? •For which pathogens?
Egg Adult
Parasites
• Myxosporidia –High mortality in 2015, no detection since 2016
• Ichthyobodo spp. (”Costia”),Trichodina sp
• Combined with- bacterial gill health problems
Formalin treatment
Diseases
Fiskehelserapportene, VI
Disease 2012 2013 2014 2015 2016 2017
Notifiable
VER 1 1 0 0 0 0
Non-notifiable
IPNV 2
Moritella viscosa X - - - - -
ARV X? X
• Vibriosis, IPN and atypical furunculosis: three most important diseases
• Atypical furunkulosis - Can be difficult to completely get control over in landbased facilities
Furunculosis 1 - - X? 9 7
Bacterial diseases
• Atypical Aeromonas salmonicida – type II
• Vibrio logei
• Vibrio (Allivibrio) wodanis
• Vibrio splendidus
• Vibrio tapetis
Viral diseases
• Viral haemorrhagic septicaemia virus (VHSV)
• Infectious pancreatic necrosis virus (IPNV)
• Nervous necrosis virus (NNV)
• Aquareovirus (ARV)1
0 d
ph
45
dp
h
66
dp
h
94
dp
h
12
2 d
ph
15
9 d
ph
24
dp
h
Nodavirus
Aquareovirus
Aquareovirus (AHRV)
• Norway:
• First aquareovirus from a marine coldwater fish spp.
• Clinical signs: Lethargy, darkening
• Necrosis in the liver and pancreas
• 90–100 days after fertilization
• 80-90% mortality
• Canada (1998): 100 mg – 1 g, ~56% mortality, +bacterial infections
• Scotland (2003): Weaned, 1000 dd, >95% mortality
Cusack et al, 2001, Ferguson 2003, Blindheim et al, 2015
Nervous necrosis virus (NNV)
• Betanodavirus, Nodaviridae family
• Viral encephalopathy and retinopathy (VER)
• Central nervous system (CNS) and retina
• Abnormal swimming pattern and loss of appetite
• Larvae and juvenile stages affected by VER
Grotmol et al. 1999: Bath challenged halibut yolk-sac larvae
Size: 25-30 nmGenom: RNA1 3100nt
RNA2 1400nt
The cells in the immune system
Pluripotent stem cell
Myeloid stem cell Lymphoid progenitor
Y
Red blood cells
Megakaryocyte
Dendritic cell
Mast cell
Eosinophil
Neutrophil
Basophil
Natural
killer cell
Macrophage Plasma cell
Dendritic
cell
T helper
cell
Platelets
T cytotoxic
cell
B
lymphocyte
T lymphocyte
• Developement of lymphoid organs
• Adaptive immunity:
• Detection of specific B- and T-cell markers – RNA
• Detection of IgM - protein
egg adult
Sequential sampling over 6 months
Development of immune system
Days post hatching
Halibut larvae during metamorphosis
0
10
20
30
40
50
60
70
80
90
100
59 66 73 80 87 94 102 108 115
Dis
trib
uti
on
of
ind
ivid
ua
ls(%
)
Sæle et al, 2004
Hematopoietic precursor
CD8+ CD4+
CD8+ TC-cell CD4+ TH-cell
RAG expression
ON
Kidney
Blood
Thymus
Blood
Peripheral tissues
33 dph
42 dph
66-73 dph
87 dph
Halibuttimeline:
T-cell development
CD3 TCRβ TCRαCD4/CD8
ETP
DN1
DN2 + +
DN3 + +
DN4 + +
DP + + + +
Immunocompetence ?
In situ hybridisation, thymus:
Figure modified from Kindt, 2007
RAG1 42dph
CD4 66dph
CD8α 87dph
Immunity development
LIVE FEED LARVAL STAGE DRY FEED JUVENILEYOLK-SAC LARVAL STAGE
RAG1
CD3ε
CD3ζ
TCRβ
Lck
CD4-2
CD3γδ
TCRα
CD8α
ZAP-70
CD4
CD8β
mR
NA
leve
l rel
ated
to
0 d
ph
(2
-
Ct ) m
RN
A level related
to 0 d
ph
(2-
Ct)
0 3 7 10 14 17 21 24 28 31 35 38 42 45 49 52 59 66 73 80 87 94 102 108 115 122 129 144 1590
100
200
300
400
500
0
1000
2000
3000
4000
5000RAG1
TCRa
TCRß
CD3d
CD3e
CD3
CD4
CD4-2
CD8a
CD8ß
Lck
ZAP-70
dph
RAG1 42dph
CD4 66dph
CD8α 87dph
IgM
66dphIgM
94 dph
IHC
1
Patel et al, 2009;
Øvergård et al 2011
Vaccine trials - Laboratory
Trials:
1. Larvae ~70 dph – tolerance trial
2. Juveniles (25 g) – recombinant recCP by injection
3. Larvae/juveniles (100 dph) – several different formulations – oral
and injection
Unfortunately....
• 99% larvae died – handling stress!!
• Other methods!?
• Trials with bath treatment
• Trials with oral uptake
• ……and analyse uptake of vaccine
Assess protection against nodavirusJuveniles (25 g)
PBS PBS Adjuvant
10 μg recCP
Adjuvant
50 μg recCPAdjuvant
Sampling: 12 weeks post vaccination
Challenge: 15 weeks post vaccination
Sampling: 2, 4, 6, and 8 wpc
Ab production and protection
N = 6
*
*
Time post vaccination
EL
ISA
Ab
so
rban
ce (
OD
49
0)
2 w 5 w 8 w 10 w 12 w0.0
0.4
0.8
1.2
PBS
Adjuvant
recCP(10)
recCP(50)
PBS
Time post challenge
Ct
valu
es (
RN
A2)
2 w 4 w 6 w 8 w
20
25
30
35
40
Adjuvant
Time post challenge
Ct
valu
es (
RN
A2)
2 w 4 w 6 w 8 w
20
25
30
35
40
recCP(10)
Time post challengeC
t valu
es (
RN
A2)
2 w 4 w 6 w 8 w
20
25
30
35
40
recCP(50)
Time post challenge
Ct
valu
es (
RN
A2)
2 w 4 w 6 w 8 w
20
25
30
35
40
Nodavirus RNA2
WP 26 Atlantic halibut
Objective: Determine the effect of delivering recombinant capsid protein during late larval stages on protection to nodavirus (VNN)
Task 26.1 Production of VNN capsid protein
Task 26.2 Monitor and assess immune response and protection
Assessment of vaccine
Protein expression in different systems
E.coli Leishmania tarentolae Tobacco leavesPichia pastoris
Production of antigen
Delivery and challenge
1. purified recCP expressed in E. coli
2. E. coli expressing capsid protein
3. L. tarentolae expressing capsid
protein
4. Pischia expressing capsid protein.
Uptake of protein by Artemia
Day 1 Day 2Pos
ctrl
Notes from lab trials
• Vaccination trials should not be initiated before 100 dph
• A balanced diet during development could be important
• Best practice – hygiene during early phases
• Both E.coli and L.tarantolae are filtered from seawater and taken up by Artemia
Vaccines in practice –Administration and strategy at site
• Which pathogen is causing disease? Choosing the
right isolate
• Farm/farm history….
• When is protection needed (onset and duration of
immunity)?
• Handling of fish – stress
• Effect vs. stress and side effects
• Choose strategy (Bath, immersion/dip, injection)
Depending on the situation at the specific farm
02.10.2018
• Atypical Aeromonas salmonicida
• Vibrio logei
• Vibrio splendidus
• Vibrio tapetis
Relevant bacterial diseases
Vaccine as a solution
02.10.2018
Example from use at a commercial site
Bath, immersion/dip, injection
02.10.2018
Smitte/sykdom
Size/time
Challenge/disease
Imm
ersi
on
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IP-injection? Harvest
Imm
ersi
on
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First feeding
~1 gram Approx. 25 gram
Vibrio logeiVibrio splendidus
Atypical Aeromonas salmonicidaVibrio logei
Imm
ersi
on
3-5 gram
02.10.2018
Intraperitoneal injection (i.p)
Site of injection
• Site of injection is close to the
basis of the ventral fins posterior
to the urogenital papilla
• The dose is deposited in the buccal
cavity.
02.10.2018
Intramuscular injection (i.m)
Site of injection
• Half way between the basis of the
dorsal fin and the lateral line.
• Only done in trials!
• Experienced up to 20% with vaccine in the intestine
• Loss of herd immunity
• Disease, and lack of vaccine effect
• Need a solution
• Ongoing trial to check for safety – Trial ends on 20.Sep
Vaccine injected into the intestine
Challenges
02.10.2018
Change: administration, adjuvant, regime
In need of a solution!
02.10.2018
• Injection site (i.p, i.m)? Is it safe?
• Adjuvant (water, oil)
• A.salm needs oil adjuvant for duration of
immunity
• Two injections, different adjuvant?
• Water based i.m injection, small fish
• Oil based i.p injection, large fish
• Theory in lab (ILAB) vs reality at site!