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Hvordan smitter Pancreas Disease (PD)?Konferanse på Færøyene
27. Februar 2009Cato Lyngøy, Marine Harvest Norway
203/03/2009
Agenda
• Spredningsmønster i Norge• Horisontal spredningspotensiale testet i
modeller• Vertikal smitterisiko
303/03/2009
1995
403/03/2009
1996
503/03/2009
1997
603/03/2009
1998
703/03/2009
1999
803/03/2009
2000
903/03/2009
2001
1003/03/2009
2002
1103/03/2009
2003
1203/03/2009
2004
1303/03/2009
2005
1403/03/2009
2006
1503/03/2009
2007
1603/03/2009
20082008
1703/03/2009
0
20
40
60
80
100
120
1 2 3 4 5 6 7 8 9 10 11 12
2004
2005
2006
2007
2008
2008_d+s
PD-registreringer/diagnoser per måned – akkumulert per 31.12.2008Kilde : Veterinærinstituttet
1803/03/2009
Biophysical properties and practical adviceDavid Graham
Veterinary Sciences Division
7th Meeting of the PD(+)Tri-nation GroupNovember 2007
Bergen
This project (Grant Aid Agreement No. IND/04/12 is carried out with the support of the Marine Institute and the Marine RTDI Measure Productive Sector Operational
Programme, National Development Plan 2000 – 2006YOUR PLAN- YOUR FUTURE
1903/03/2009
Objectives and targets
• Objective 2. To determine the survival of SAV at different temperatures and in different waters under different organic loads• 20°C, 15°C, 10°C, 4°C• SW, estuarine, FW• ± organic loading• Sterile conditions
Source: David Graham, Veterinary Sciences Division
2003/03/2009
Non-sterile seawater
Reduced half life
Source: David Graham, Veterinary Sciences Division
2103/03/2009
1
10
100
1000
-10 0 10 20 30 40 50 60 70 80
Distance from farm, km
max
imum
pat
hoge
n co
ncen
tratio
n14.5 day half-life (and above) Tidal/turbulence
1cm/s2cm/s4cm/s8cm/s16cm/s
Simple hydrodynamic model
Source: David Graham, Veterinary Sciences Division
2203/03/2009
Dr. Grahams conclusions
1. Suggests that SAV could survive for extended periods in the aquatic environment
2. Viral survival was clearly temperature related- t1/2 most prolonged at low temperatures/no organic matter
3. Differences between sterile and non-sterile environments
4. Supports the hypothesis that virus could be transmitted between farms without direct or indirect human or animal intervention
Source: David Graham, Veterinary Sciences Division
Vertikal overføring?
Kilder:• Veterinærinstituttet Oslo, Dr. Edgar Brun• Veterinary Science Division, Northern Ireland, Dr. David Graham• Castric J et al• Universitet i Bergen, Dr. Are Nylund• Marine Harvest Norway v/ Dr. Olav Breck
2403/03/2009
PD Group at National Veterinary Insitute, Oslo
Mona Dverdal Jansen1, Marianne Sandberg1, Brit Gjerset2, Marit Wasmut2, Birgit Dannevig2, Hilde Sindre2, Anne Berit Olsen2, Olav Breck3, Torunn Taksdal2, Edgar Brun2
1Norwegian School of Veterinary Science2National Veterinary Institute
3Marine Harvest
Kilde: Veterinærinstituttet Oslo, Dr. Edgar Brun
2503/03/2009
Results & Conclusion • 48 hatcheries sampled• 50-50% within and outside PD zone• Followed up all fish groups in sea phase and through to harvest• Pooled 5 fish in one sample
• No detections of PD in freshwater phase• First detection of PD 2 months post transfer to sea
• Low prevalence in smolt in hatcheries • Low level of virus in smolt in hatcheries• Proportion of outbreaks due to fresh water infected smolt is low
Kilde: Veterinærinstituttet Oslo, Dr. Edgar Brun
2603/03/2009
Transmission of SAV in Ireland & Scotland
David Graham
Veterinary Sciences DivisionNorthern Ireland
7th Meeting of the PD(+) Tri-nation Group November 2007
Bergen
2703/03/2009
• Project (ST/05/01) :Site Investigations and Disease Management of the Pancreas Disease Virus in Irish Farmed Salmon
• Strategic Marine RTDI Programme 2005
This project (Grant Aid Agreement No. IND/04/12 is carried out with the support of the Marine Institute and the Marine RTDI Measure Productive Sector Operational
Programme, National Development Plan 2000 – 2006YOUR PLAN- YOUR FUTURE
Source: David Graham, Veterinary Sciences Division
2803/03/2009
E2 gene-based phylogenetic analysis
1
IRE 1045(96)IRE 02-194(11)
IRE 93-125IRE 03-123(3)IRE 05-238(9)IRE 06-227(20)IRE 05-27(13)IRE 05-190(38)IRE 05-118(5)IRE 04-183(A1)IRE 05-294(3)
IRE 06-182(55)IRE 05-184(5)IRE 2111-1IRE 06-43(4)
IRE 04-44(10)IRE 06-186(5)
SCO 06-139(33)IRE 91-115(A3)IRE 91-116(P1-6)SCO 03-209(3)
SCO 06-243(4)SCO 06-17(9)
SCO 07-02SCO 06-93(37)SCO 06-41(54)SCO 06-241(1)
SCO 04-224(17)SCO 06-267(9)SCO 05-124(5)SCO 05-310(10)
SCO 05-310(18)FRA EE37SPA 04-08(6)SPA 04-08(18)
FRA S49pFRA VF03-P4
SCO 02-85(9)SCO 06-119(7)GB 02-67(18)GB 04-212(5)
ITA 04-198(29)ITA 05-105(12)ITA 04-198(22)
SCO 06-290(6)SCO 06-290(8)
NOR PD97-N3NOR SavH20-03
NOR SavH10-02NOR SavSF21-03NOR 04 170(6)NOR 04-170(8)NOR 07-170(3)NOR 04-170(7)
VI
I
IV
II
III
V
97.0
97.5
53.7
73.1
92.7
87.0
93.0
87.1
59.0
99.7
10099.7
94.873.1
96.0
100
Source: David Graham, Veterinary Sciences Division
Source: David Graham, Veterinary Sciences Division
1
F02 9F06 182 55F05 184 5F06 43 4F07 110 5
F02 194 11F07 77 1
F93 125F 2111 1F05 190 38F03 123 3F05 238 9F06 227 20F05 27 13F05 118 5F04 183 A1F05 294 3F05 294 29F02 73 8
F06 173 3F07 160 12
F07 220F07 105 11
F06 17 9F07 02
F06 93 37F06 41 54F06 241 1
F05 310 10F05 310 18F04 224 17F06 267 9F05 124 5F07 80 3F04 44 10
F06 186 5F07 216 8
F91 115 A3F91 116 P1 6
F03 209 3F06 243 4
F06 139 33F07 66 11
F07 75 1S49pEE37VF03 P4
F05 105 12F02 85 9F02 67 18F06 119 7F04 212 5
F04 08 6F04 08 18F04 198 29F04 198 22p4.2p
F06 290 6F06 290 8
PD97 N3SavSF21 03F04 170 6F04 170 8F04 170 3F04 170 7
SavH20 03SavH10 02
100100
100
100
100
80.1
64.6
99.5
59
74
99.2
71.3
62.678.999
75.1
93.8
94.6
90.3
51.4
III
VI
I
V
IV
II
3003/03/2009
Transmission from carriers?
Millstone site06GS0
Glinsk site06GS1 Transferred June 2007
Cranford site06Gs0F06-186 5
Aug 06 F07-216 8July 07
S0 Population
0
10
20
30
40
50
60
70
80
90
100
0 54 74 157 201 227 241 270
Day
% p
ositi
v
294 315 359
e
% viraemic
% sera PCR pos
% seropos
% Ht PCR pos
% gill PCR pos
% kid PCR pos
Source: David Graham, Veterinary Sciences Division
3103/03/2009
Conclusions
• Geographical clusters• Ireland
• Kilkieran Bay (SAV1)• Mannin Bay (SAV1), long study• Mulroy Bay (SAV4), long study
• Scotland• Western Isles, long study
• Local horizontal cycling of specific SAV strains• Overlapping populations of infected or recovered fish
• Carrier state• (local reservoir)• Fish source vs virus strain does not support vertical introduction
• Co-ordinated local management?
Source: David Graham, Veterinary Sciences Division
3203/03/2009
A study on possible vertical transmission of SAV in Atlantic salmon
O. Breck1, N. Santi2, R.T. Kongtorp3, V. Aspehaug4, Anne Stene4 , P.A. Andreassen5,
C. Wallace6,R.S. Olsen7, T. Lyngstad3 & M. Sandberg3
9th PD Trination seminar
04 - 06 Feb, 2009
1. Marine Harvest Norway2. Aqua Gen3. National Veterinary Institute4. Patogen analyse5. Rauma Stamfisk6. Veso Vikan7. Salmo Breed
3303/03/2009
Content • Introduction
• Results from 3 studies on vertical transmission (MHN data)
• Screening of freshwater sites (MHN)
• Vertical trial
• Design
• Sampling
• Results
• Discussion
3403/03/2009
Introductory comments
any evidence of a vertical route ?
• Pattern of spread of disease within MHN does not reflect ova or smolt movements
stabile endemic “hot spots” within Hordaland for years smolt to other areas disease free
• Hordaland has delivered ova out of county for (20) years, eg. to Trøndelag, whilst PD for years remained a local problem
• PD endemic in Ireland for several years. MH brood fish sites undergone infection. SAV never found in freshwater.
3503/03/2009
Introductory comments
• Distribution of disease in Norway pattern of slow spread south and north out of early endemic areas (+ short lived ”out-layers”)
• Also examples of successful area fallowing..
• Pattern of disease spread within new regions (”domino effect”) suggests a horizontal route (eg. Hardangerfjorden, Romsdalsfjorden)
lack of historical evidence in MHN that a vertical route a majorcontributor, compared to horizontal route, based on disease distribution
3603/03/2009
However
• PD-virus found in 40-50% of smolt farms along Norwegian coast, incl. cases in fry in northern counties (Are Nylund, UoB)
• Cases of PD in northern counties, with links to smolt deliveries from western Norway (transport, wellboat, smolt..)
• Transmission experiments broodfish (rainbow trout, France): found PD/SD virus (in one case also disease) in offspring, after i.p. injection of broodfish
Industry cannot disregard a possible vertical /FW route
MHN: three studies on possible vertical transmission by
following offspring from SAV positive broodfish, by use of qPCR
collaboration with UoBergen, Patogen, VESO, Veterinary Institute (VI)
3703/03/2009
Three MHN studies on possible vertical transmission
•NRC-funded project with 2002G broodfish (autumn 2004) (UoB)• high ct, low prevalence of SAV+ brood stock• ova –> smolt• 2/50 eyed ova and 4/170 fry SAV +, 0/630 parr-smolt
•Trial with early spawners, 2004G, autumn 05 (UoB)• ova from SAV positive broodfish, 6 months post disease, • ova 1/220 , eyed ova 1/220 and fry 2/430
• Trial with same broodfish population at natural maturation autumn 06 • fry of positive parents 0/103 including stress test (handling, salinity)
10/1824 (0,55%) SAV +, ova and early fryct values 36 – 43, most > 38
What is the biological significance of these findings ?
3803/03/2009
Screening of FW sites 2007
• in co-operation with Patogen
• PCR-method comparable with UoBergen, run to ct 45
• 15 freshwater sites from Agder to Nordland
• 26 groups of 07 S0 and 08 S1 screened (30-60 per group) for SAV
• Different broodstock background
• Findings
0/1223 samples SAV-3 positive
4/1223 SAV 1 pos PD-vaccine
3903/03/2009
• Main objectives
1. Does vertical transmission occur ?
2. If yes, is this ”true” or ”false” (egg associated) ?
inclusion of different disinfection regimes
3. Can a latent infection only be detected at certain ontogeneticdevelopment stages (shocking, startfeeding, handling stress andsmoltfication)?
testing at different stages and followed throughout smoltification
Vertical transmisson trial 07 – 09
4003/03/2009
Vertical transmisson trial 07 - 09
• Broodstock population with clinical PD summer 07 (20 % losses), high prevalence of SAV (82%) and ct-values 30-37
• Population culled, except 50 ♀ and 10 ♂
• Stripping December 07, wanted to establish ova groups from 4 female with lowest ct and positive eggs. Each family divided into 4 disinfection regimes. In addition ova from other broodstock source for pos and neg control
• Six ova groups established : 1. un-disinfected 2. disinfected once 3. double disinfected 4. double dis + formalin bath 3x/wk, 5. neg controls 6. positive control (5 min exposure of ova to 50 ml SAV, prior to fertilisation)
• Study presence of virus/disease in broodfish, ova and off spring(qPCR, serology, histology, in situ PCR …)
4103/03/2009
PatoGen SAV Real-time PCR ProtocolSalmonid Alphavirus • Hodneland, K. and C. Endresen. 2006. Sensitive and specific
detection of salmonid alphavirus using real-time PCR (TaqMan).J. Virol Methods 131: 184 - 92
• Sensitive and specific assay to detect SAV
• Detect <0.1 TCID50 - virus particles• Detect 1 – 10 plasmid copies • 35 positive compared to 29 positives in standard RT- PCR
4203/03/2009
Date Day Group/tank Episode Live Dead # sampl sum
13.12.07 2 PC/K19/PC/K20 2 d post fert/challenge x 150 x 2 30031.12.07 20 DR1/K4 mortality x 78 7812-25.01.08 32-45 PC/K19/K20 mortality x 58+53 11130.01.08 30 DR1/K4/DR4/
K16/PC/K19/PC/K20
At shocking x 30x4 12009.02.08 60 DR1/K1 mortality x 80 8013.02.08 64 DR1/K4/DR4/K16/
PC/K19/K20After shocking x 15x4 60
21-23.05.08 162-164 DR1/K1/DR1/K4/PC/19/PC20
Fry 1 wk post stress x 75+75+60+60+ 270
21-27.05.08 162-166 PC/19/PC/20 mortality x 8+28 3603-06.06.08 175-178 DR1/DR2/DR3/PC mortality x 6+1+22
+13 4208-19.11.08 333-344 NC/PC mortality x 12+13 2519.01.09 DR1 Finish at smoltification x Heart x 300
Pseudobr x 300 600
1350 372 1722 1722
Overview PCR analysis Feb 09
ALL NEGATIVE
4303/03/2009
KonklusjonerPD smitter alt overveiendehorisontalt I sjøfasen - mellomfiskepopulasjoner, enten via frievannmasser, via overføring avlevende matriale, eller biologiskevektorer.
Den vertikale ruten skal ikkefullstendig utelukkes, men flertalletav forskere mener at dennesmitteveien har forsvinnende litenbetydning i praktisk oppdrett.
Av forskere som støtter vertikaloverføring av PD, understrekes detat risikoen er betydelig mindre ennved f.eks. ILA
