Amoebic Gill Disease
The current state of
knowledge
Barbara Nowak
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Amoebic Gill Disease in
Atlantic salmon
Environment
Hatchery
Seawater cage aquaculture
Atlantic salmon
smolts
N. perurans.
Risk factors:
Salinity - ≥32ppt
Temperature >16°C
Other environmental factors
Fish maturation
Mortalities
ies alleviated
with freshwater
ater
bathing
However increase in
costs of production
AGD aetiology (what causes AGD?)
1986
- First description of AGD in Tasmania and USA
Neoparamoeba sp. (Roubal et al. 1988)
Neoparamoeba pemaquidensis (Kent et al. 1988)
1993 – Immunohistochemical test for N. pemaquidensis
N. pemaquidensis (Howard & Carson, 1993)
2000 –
Detailed study on Neoparamoeba morphology
N. pemaquidensis or N. aestuarina? (Dykova et al. 2000)
2004 –
Molecular assessment of Neoparamoeba taxonomy
N. pemaquidensis (Wong et al. 2004)
2005 –
Molecular assessment of Neoparamoeba taxonomy
N. pemaquidensis or N. branchiphila (Dykova et al. 2005)
- Both species isolated from AGD-affected fish
Methods of isolation and characterisation
of amoebae associated with AGD
A
B
C
D
D
D
MYA
marine yeast agar
Non-cultured gill derived
amoebae (primary isolates)
Clonal culture
N. pemaquidensis
N. branchiphila
X
• Morphologically similar to NCGD amoebae
• Not infectious when clonal cultured
• Not yet linked with gill tissues or primary isolates
Koch’s postulates fulfilled for N. perurans
as an aetiological agent of AGD
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Severity of AGD increases proportionally to
the number of amoebae present in water
(Morrison et al 2005)
Gross
lesions/gill
Total Gill Lesions
60
50
40
30
20
10
0
0
100
200
300
400
Inoculating amoebae (cells/l)
Inoculating amoebae/L
Fish inoculated with 10 cells/L
500
60
AGD case definition
•  Based on histology
Presence of lesions and neoparamoebae
new species or new location –
confirmation by ISH required
•  Based on PCR and gross lesions
Presence of lesions and positive PCR for
N. perurans
AGD - definition
•  Diagnostic criteria – histopathology gold
standard
•  Case definition – presence of pathogen
and lesion
•  Quality control
The pathogen
Neoparamoeba perurans
Amebozoa
Dactylopodida
Vexiliferidae
The pathogen
Neoparamoeba perurans
Attached and floating state
Trophozoite the only stage known
-  The only marine amoebae which have
cysts are Acanthoamoebidae and
Heteroamoeba
-  The only marine amoebae which are
known to have flagellate stage are
Vahlkampifidae
Amphizoic – free living but
capable of colonizing a host and
becoming parasitic – facultative
parasite
•  Confirmed presence:
- water
- sediments
- copepods
- isopods
The pathogen
Neoparamoeba perurans
Neoparamoeba pemaquidensis and
Neoparamoeba branchiphila noninfectious in numerous experimental
challenge trials
Neoparamoeba perurans the only species
pathogenic to salmon
Cell shape plasticity and size differences (Dyková
et al 2005)
Validating ISH probes
ISH probes
Representative Neoparamoeba strains
N. pemaquidensis
non-cultured,,
gill-derived
amoebae
(NCGD)
N. branchiphila
non-cultured, gillderived amoebae
18S rRNA universal
(NCGD)
Application of ISH probes to AGD-affected Atlantic
salmon gill tissues
Probes
Tank-based AGD-affected
Atlantic salmon (n=3)
noncultured,
gill-derived
amoebae
(NCGD)
Farm-based AGD-affected
Atlantic salmon (n=4)
Young et al. (2008) Dis. Aquat. Org. 78, 217-223
Chinook salmon,
Oncorhynchus tshawytscha
Atlantic salmon,
Salmo salar
U.S.A
New Zealand
Atlantic salmon,
Salmo salar
Ireland
Atlantic salmon,
Salmo salar
Scotland
Turbot,
Psetta maxima
Spain
Atlantic salmon,
Salmo salar
Chile
Molecular detection of N. perurans
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PCR gill swabs vs gill score
36
r = -0.9922
34
P<0.01
Cycle threshold (Ct)
32
30
28
26
24
22
20
1
2
3
4
Gross gill pathology score
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Water filtration –environmental detection
Host response
AGD related pathophysiology
•  Well documented AGD affected fish show lethargy and
respiratory distress
–  Mortality assumed to be respiratory failure
•  In vivo studies however have shown:
–  Differences in O2 uptake relatively minor
–  Higher CO2 tension and lower pH – respiratory acidosis
–  Lowered cardiac output and hypertensive
–  Hypertension attributed to elevated systemic resistance
–  Branchial resistance is not affected
AGD related pathophysiology
•  While AGD results in reduced gill surface area, respiratory
failure not supported by in vivo findings
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•  Defence of respiration – via filament and lamellae
recruitment
AGD related pathophysiology
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In vitro gill model for AGD
Chloride
cells
asAG-2
expressing
cells
(mucous
cells) are
abundant in
AGD
lesions
(Morrison and
Nowak 2008)
Inflammation and AGD
Leucocytes
(macrophage and neutrophil like)
MHC II bearing cells in AGD affected
gills (Morrison et al 2006)
-ve control
Characterisation of cells in AGD
lesions
• 
• 
• 
• 
• 
• 
• 
• 
PAS/Alcian Blue – positive
Anterior gradient protein - positive
NaKATPase - negative
PCNA - positive
Salmon Ig - negative
MHC-class II – some positive
T cell marker (CD3) – some positive
Interleukin 1 beta - positive
Immune response in AGD
•  Immune gene analysis in 5 challenge trials
•  Similar conditions
•  Consistent patterns
AGD – immune response
•  IL1 beta expression consistently
induced in gills of AGD affected
salmonids
•  IL1 beta expression restricted to
epithelium in gill lesions
•  Upregulation of IL1 beta but
downregulation of other genes in
pro-inflammatory pathway
•  Little inflammatory response
•  Inhibition of antigen processing
pathways
T cell receptor
signalling pathway
2
T cell receptor
signalling pathway
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Do immunostimulants reduce
AGD?
Compound
Lab or field
Effect
Levamisole
Lab, Field
NO
Beta glucans
Lab
NO
CpGs
Lab
YES
EcoActiva
Lab
NO
EcoBoost
Lab
NO
Survival of Atlantic salmon fed
experimental diets
60
*
*
Survival %
50
40
> 27 % increase in
relative percentage
survival
30
20
10
0
control diet
experimental experimental
diet 1
diet 2
(Log rank test 2=12.28, df=2,
p=0.002)
AGD severity
100
Infected filaments %
90
80
70
60
No diet effect
50
40
30
20
10
0
control diet
experimental experimental
diet 1
diet 2
AGD Immunity
Resistance to disease
Systemic response to AGD
•  Survival of
subpopulation
after initial
infection
(Bridle et al., 2005)
•  Survival of
infection and
resistance to
reinfection
(Findlay et al., 1995)
•  Against
amoeba
antigens
(Akhlaghi et al., 1996)
•  In fish after
initial infection
(Findlay et al., 1995; Vincent
et al., 2006)
•  Not always
protective,
high in
moribunds (Taylor
et al., 2010)
Mucosal response?
•  Not detected
after initial
infection in
cutaneous
(Vincent et al., 2006)
or gill mucus
(Akhlaghi et al, 1996).
AGD Vaccine history
Vaccine high priority
DNA
vaccines
(CSIRO)
Sonicated culture
Paramoeba sp
IP/FCA
No protection
(Akhlaghi et al., 1996)
Live and
sonicated
amoebae IP and
PA
No protection with cultured
or wildtype
(Zilberg et al., 2001)
Claudio Reyes/EFE
Approach to vaccine
development
Identification of MBP – like in N. perurans, similar to
pp
attachment factors of Acanthamoeba spp.
Acanthamoeba spp.
MBP surface protein
Virulence factor
If blocked no cytopathic effect
Blocking attachment of N. perurans to the gill reduce
severity of AGD ?
rotein
ein
Vaccine candidate recombinant MBP-like protein
Dependant on presence of Ab in gill mucus
Recombinant Protein
Injected fish
Serum and mucus
200 µg Ag/fish
fish
0
2
  0,
4, 8, 10 and 12
weeks post inoculum
4
6
8
Weeks
10
12
14
SERUM response against recombinant protein
and WT amoebae was present
ELISA
200
350
300
160
c
250
200
150
b
a
100
a
Antibody level (units)
Antibody level (units)
180
c
140
120
100
c
80
60
a
a
0
4
b
bc
10
12
40
50
20
0
0
0
4
8
Week
R protein
10
µg.mL-1
10
12
8
Week
WT
amoebae
2.4 µg.mL-1
SERUM response against recombinant protein
and WT amoebae was confirmed
ICC
Western blot
RProtein
0
8
10
12
WT amoebae
Weeks
0
8 10 12
Neg serum
Serum Wk 8
MUCUS response against recombinant
protein and WT amoebae was present
100
90
c
80
70
c
60
50
40
b
30
20
10
a
a
10
12
0
0
4
8
Week
R protein
10
µg.mL-1
Antibody level (units) per mg of mucus
proetin
Antibody level (units) per mg of mucus
protein
ELISA
100
90
c
80
70
c
60
50
40
b
30
20
10
a
a
10
12
0
0
4
8
Week
WT
amoebae
2.4 µg.mL
-1
Immune response to
recombinant protein
Systemic response
High responses in ELISA after week 8
Higher response against Recombinant protein than WT
amoebae
Confirmed by Western blot and ICC
Significant mucus response
Lower levels than serum
Earlier response than in serum (4 weeks)
Freshwater treatment
% #+ 0../,
Number of Paramoeba x1000
3500
3000
2500
2000
1500
1000
500
0
Before
Day 1
Day 3
Day 5
Time from freshwater bath
Day 10
Hydrogen peroxide
•  Assessment of H2O2 treatment efficacy in vivo
300 Atlantic salmon smolt @ 100g
4000L
to Neoparamoeba
Gill and blood Exposed
samples
collected: sp. for 21 days
Recirc sys
ammonia < 1 mg.l-1)
(temptreatment
15oC, pH 8.2,
-  Immediately pre
(n=10)
-  Immediately post treatment (n=10 per treatment)
-  7, 14 & 21 days post treatment (n = 10 per tank)
FW - 3 hour treatment
H2O2 – 15 mins @ 1250mg.l-1
Analysis
H2O2
FW
FW
H2O2
Histology
(%
of
lesion
affected
oC
12oC
18filaments)
12oC
18oC
Osmolality
MortalityFish
andfrom
health
indices split
at sampling
each treatment
into duplicate tanks (300L)
Tanks within one system, no added cells.
Re-infection over 21 days (15oC).
54
Hydrogen peroxide
Average percentage
± standard with
errorsN.perurans
(S.E.) of gill filaments
with hyperplastic
No re-infection
was evident
at
lesions
positive
to the presence
of Neoparamoeba
perurans
Plasma
osmolality
pre and immediately
post treatment
7, 14 & 21 days post treatment regardless of treatment
Morbidity post-treatment for AGD (percentage of fish in the treatment)
Treatment
Temp
p oC
% 24 h PT*
% Day
y2-7
% Day
y 7 - 14
% Day
y 14 - 21
Dam water
12
2
0
1.4
0
0
Dam water
18
8
0
5
1.5
0
Peroxide
Peroxi
xide
xi
de
12
2
5.0
1.5
3
0
Peroxide
Peroxi
xide
xi
de
18
8
4.2
2.9
0
0
H2O2 treated fish 7 days after bath
55
Hydrogen peroxide
Results summary:
•  N0 re-infection evident at 7, 14 & 21 days
post treatment in either freshwater or peroxide
treated fish
•  Pre-treatment disease severity was clinically
light (10% of lesion affected filaments)
Further research:
Determine efficacy under more advanced
disease scenarios
Investigate treatment efficacy under a
broadened range of exposure times and
concentrations
Examine physiological responses to H2O2
treatment
Controlled surface access
(Dempster et al 2011)
Current AGD research
•  Epidemiology – relationship between presence
in water and AGD severity
•  Immunostimulants/diets
•  Immune response (collaboration with Prof
Koppang)
•  Vaccine development
•  Treatments – in vitro screening
•  Management through behaviour manipulation
(collaboration with Dr Dempster)
•  Susceptibility of other species
•  Culture
•  In vitro model
Aquatic Animal Health
Research Group, NCMCRS,
UTAS
Acknowledgements
• 
• 
• 
• 
• 
• 
• 
• 
• 
Europharma Lofoten Seminar 2013
FRDC, Seafood CRC
Salmon industry (TSGA, Saltas)
Skretting
Victoria Valdenegro, Stewart Dick, Dr Phil
Crosbie, Dr Mark Adams, Dr Melanie Leef , Dr
Stephen Hindrum, Mark Polinski, Karine Cadoret
(University of Tasmania)
Prof Ben Koop (University of Victoria, Canada)
Prof Chris Secombes (University of Aberdeen,
Scotland)
Prof Erling Koppang (Norwegian Veterinary
College, Norway)
Dr Tim Dempster, University of Melbourne
Any questions?